CN1563387A - Earthworm fibrinolytic enzyme gene and genetic engineering strain, and construction and application thereof - Google Patents

Abstract

The invention discloses an earthworm plasmin gene and a genetic engineering strain as well as construction and application thereof, wherein the plasmin gene consists of 747 nucleotides and encodes 246 amino acids, the gene expresses active genetic engineering plasmin protein in pichia pastoris, the activity unit of fermentation broth exceeds 3,390,000U/L, and the genetic engineering plasmin protein has wide application in the research and development fields of medical, sanitary and health-care medicines.

Description

A kind of earthworm fibrinolysin gene and engineering strain and structure and application

Technical field

The present invention relates to a kind of nucleotide sequence of Lumbricidae powder Lumbricus earthworm fibrinolysin gene, the preparation method who also relates to this gene simultaneously the invention still further relates to a kind of structure and the application of genetically engineered plasmin in drug development research of expressing the pichia pastoris gene engineering bacterial strain of this gene.

Background technology

Cardiovascular and cerebrovascular diseases such as acute myocardial infarction, cerebral infarction, pulmonary infarction, peripheral arterial thrombosis and dvt are one of principal diseases of harm humans life and health.According to World Health Organization's statistics, annual nearly 1,200 ten thousand people in the whole world die from heart trouble and cerebral apoplexy.In China, along with rapid development of economy, the raising of people's living standard, the sickness rate of cardiovascular and cerebrovascular diseases and case fatality rate thereof also improve just year by year.Therefore, develop and develop efficient, special, safety, the little thrombolytic agent of untoward reaction, be worldwide in recent years heat subject always.

Earthworm is commonly called as earthworm, and at the applicating history in existing thousands of years of China, but heat-clearing, flat liver, Zhichuan, vein relaxing cure mainly illnesss such as high fanatical hot-tempered, convulsion with spasms.Nineteen eighty-three, Japan H.Mihara found that first Lumbricidae (Lumbrici dae) earthworm aqueous extract has direct solution fibrin and Profibrinolysin activation, its activeconstituents is called plasmin (earthworm fibrinolytic enzyme, EFE), claim Lumbrukinase again.The pharmacological mechanism of plasmin thrombolysis mainly contains: 1) have special avidity with scleroproein, not only the scleroproein of Profibrinolysin is rich in hydrolysis, can also hydrolysis do not contain the scleroproein of Profibrinolysin, scleroproein is degraded rapidly, can also hydrolysis of fibrin former.2) similar tPA, plasminogen activation and bring into play indirect action, experiment in vitro shows that plasmin has hormesis to the release of cattle blood vessel endotheliocyte tPA.3) hydrolysis factor I has suppressed the adhesion of thrombocyte on its surface.

Result of study shows: the intravital plasmin of earthworm has various ingredients, only has been separated at least 6 kinds of different components in the positive earthworm of powder (Lumbricus rubellus).The cDNA sequence of from earthworm, having separated 6 coding fibrinolytic enzyme genes now abroad.Domesticly also from earthworms such as Eisenia foetida, Lumbricidae Bimostos, be cloned into the earthworm fibrinolysin gene, but do not seen that the report that separates fibrinolytic enzyme gene and carry out clonal expression from the positive earthworm of powder is arranged.

At present existing multiple earthworm fibrinolysin preparation is applied to clinical, is mainly various capsule preparations, all extracts from earthworm enteric cavity and body fluid,, hundred Austria multiple, Bo Luoke as general grace, and thrombolysis capsules etc. are used in the cardiovascular and cerebrovascular diseases aspect more.Because plasmin can improve hemorheology, reducing platelet aggregation rate in addition, thus can be used in the some diseases relevant with hemorheology, as Meniere, sudden deafness, pulmonary heart disease etc.

Traditional earthworm fibrinolysin capsule preparations is applied to clinical certain curative effect is arranged, but also has obvious defects, at first is complicated component, can not intravenous injection, and the oral administration effect is slow, thereby can not be applied to treat the acute heart, cerebral vessels embolism disease; Secondly capsule complex manufacturing technology is produced the restriction that is subjected to vermiculture season, cycle, product production, quality instability; Separation and purification plasmin technology is loaded down with trivial details from polypide, may contain various micromolecule polypeptides or other chemical substance in the product, may cause the anaphylaxis of body.Use engineered method then can obtain plasmins in a large number by simple fermentation, separation purifying technique is simple, and the product purity height is at the bottom of the cost.Therefore, the genetically engineered plasmin has purposes widely in the research and development field of medical treatment, health, health-related medicine, can be used for preparing the water injection that is used for the treatment of the embolic cardiovascular disorder, medicinal preparation for oral administration etc., also can be used for producing healthcare products with prevention thrombus disease, useful cardiovascular health.

Aspect the genetically engineered drug exploitation, most widely used expression system general, the technology comparative maturity has at present: intestinal bacteria system, yeast expression system and expressing cho cell system.

Escherichia expression system is present general, the most sophisticated most widely used expression system, be suitable for expressing the better simply protein of non-glycosylated protein matter and secondary structure, expression product generally is to form inclusion body in tenuigenin, product need just have activity through renaturation, the carrier that also has can be secreted target protein in intercellular substance, or in tenuigenin solubility expression.Its maximum shortcoming is processing and the modification (such as glycosylation) after can not expressing, and this course of processing is that a lot of protein acquisition functions are necessary.

The expressing cho cell system: this is to make up and express the relatively large expression system of difficulty at present, be fit to express glycosylated protein and the complicated protein of space structure, expression product generally is present in the tenuigenin, if when gene constructed, before target protein, add a segment signal peptide, expression product can be secreted into outside the born of the same parents, become activated albumen by cell translation back packing, but expression amount is low, cost is high, purification difficult makes it be difficult to use in suitability for industrialized production.

Summary of the invention

The object of the present invention is to provide a kind of Lumbricidae powder Lumbricus earthworm fibrinolysin gene, the protein of this genes encoding has thrombolytic effect.

Another object of the present invention is to provide a kind of method for preparing Lumbricidae powder Lumbricus earthworm fibrinolysin gene, and this method is simple, and is easy to operate.

The further purpose of the present invention is to provide a kind of engineering strain, and this bacterial strain can be expressed earthworm fibrinolysin.The preparation method who also relates to this bacterial strain simultaneously is used to prepare genetically engineered plasmin albumen.

Purpose of the present invention further provides a kind of method for preparing the genetically engineered Pichi strain, and this engineering strain can be used for expressing the earthworm fibrinolysin gene, obtains genetically engineered plasmin albumen.

The invention still further relates to genetically engineered plasmin albumen in the application of treatment in the cardiovascular disease medicine, such medicine has thrombolytic effect and to the provide protection of cerebral ischemia.

One of technical essential of the present invention is nucleotide sequence of Lumbricidae powder Lumbricus earthworm fibrinolysin gene and preparation method thereof,

Nucleotide sequence of a kind of Lumbricidae powder Lumbricus earthworm fibrinolysin gene and preparation method thereof is provided in one embodiment of the invention, this method is to be template with the total RNA of Lumbricidae powder Lumbricus earthworm, use the specific oligonucleotide primer, obtain fibrinolytic enzyme gene cDNA through the reverse transcription PCR method.The positive earthworm of powder is purchased in Hua Zhong Agriculture University, and gene sequencing result shows that cDNA is made up of 747 Nucleotide, and 5 ' end to 3 ' terminal sequence is:

Table 1: sequence homology SEQ ID NO.: 1 AB002794, U46118RNU46118U19482AF026216AB002739, AB002730, AB002728, AF058796, AB002777, AF020187, AF009411, AB015609, AF006627, M95076, U43527, AB002741, U25846, AF006628, AF019043, D83352, U10355, M99575, U48288, AF090440, AB007504, U82480, Y15794, AJ005572, AF029349, L10111, S80963, U38894, L41731, AF022733, AJ225108, Y11879, AF001688, U33214, Z97178, AF009413, AF019907, AF016371, X71980, AF001522, M77169, AF023132, D83476, AJ009675, U90554, AF069324, AF048691, Z34799, AF004947, U60149, AF022732, AF019887, L02937, U55848, AF011331, AF045770, AF043533, X69524, M32882, U08214, U50847, AF017364, U35364, Y14339, AJ005969, U90555, U44430, U74296, AF001501, AJ223316, D78609, U41060, Z54362, AF034387, U62398, AB005545, AB002533, Y13865, AF009959, L02938, U10555, AL010246, AC003041, U37699, S75970, AF071010, Z81311, AB003681, X16353, AB002731, X53081, D63450, D10911, D90115, X97065, Z82275, AB016891, Y09455, X77990,, W58357, W07820, AA188593, W81046, AA858164, AI018124, AI139112, AA769634, W00437, D80849, AA448160, N98650, T31293, H15307, H51146, N29314, AA770301, AA187822, AA978299, T31823, AA936410, AA993194, N41386, W45601, W81099, C05691, D60153, AA780119, AA929004, R68608, Z43271, N37024, AA974718, AA928663, T30120, AA936583, H51109, N20251, W92917, T36035, AA480197, Z24908, N79450, AA872019, AA902275, N59810, R13443, AA740162, AA937759, N72168, AA970708, T36257, AA433833, H15700, D51185, AA249138, R37356, W93028, Z28641, AA371494, N72757, AA719126, N56164, T31790, D57384, Z41959, N58984, AA847848, AA587009, Z39343, AA654834, AA505490, Z38243, D20344, AA252395, AA025593, H70133, R09101, AA159862, AI032981, Z28355, C75020, AI139642, D82421, AI126922, C75170, AI016032, C18748, C75478, AI129334, C75472, AA310765, D63057, C05952, AA357303, C16591, D82132, L48852, D82799, C75176, AI124552, AA669404, U30155, C75118, AA374918, C75108, C05853, U30151, D57346, AI127548, AA918527, AA317816, AA573490, R21699, AA917928, R36311, AA361522, AA701252, AI085492, H44387, AA156256, AA587935, AA976510, AA515269, W73374, T27986, N34493, AA737770, N32609, N32612, H64420,, AA415243, AA413717, AA117350, AA242502, AA117343, AA545256, AA795651, AA106372, U31322, AA681967, AA221922, AA600546, AA050610, AU018628, C80932, AA920654, AA863834, AI099036, AA183239, AI115182, AA590910, W65628, AA162291,, AA109440, AI052952, AA999324, AI105714, AI026280, AI072678, AA964820, AA754198, AA944557, AI045710, C94989, AA471630, AA933231, AA509077, AA257557, AA509328, AA109365, AA963207, AA435473, AA999306, AA406684, AI105662, AA509174, AI108597, AA109374, AA925182, AA471671, AA088161, AA752812, AA626989, AA406924, AA840999, AA509033, C94899, AA406875, AA842672, AA123619, AA072471, AA509339, AA933108, AI110259, AA842135, N99262, AA661430, AA842512, AA180648, AI066048, AA559515, AA406761, AA109423, AF074736, AA257676, D87312, AA257488, AA114390, AI087701, W06665, W32852, AA123649, AA842505, AA509330, AA908025, AA257279, AI105717, AA406673, AA627018, AA406850, AA161668, AA114453, AA109372, AA257572, AA803997, AA406943, AA257495, AA842019, AA661368, AI082949, AA842888, W06539, AA118223, AA406986, AA471717, AI096212, AA257707, AA257649, AI053126, AA508950, AA713366, W69049, AA965381, AA208680, AI057997, AA161707, AI058071, AA675858, AA257513, AI083337, AA406908, AI083256, AA659956, AA842532, AA754036, AA753165, W18199, AI109620, AI058022, AI064026, AA471431, AA032116, AA754544, AI108146, C42045, AA843025, AI109103, AA509237, AA161620, AA257665, AA471605, W84932, AA841358, AA843040, AA471695, AA003471, AA840977, AA509267, AA841711, AA471488, AA257699, N98079, AA454318, W04102, AA842874, N99754, AA509307, AA751845, AA661358, AA508954, AA406746, AA842720, W23363, AA508951, AI068913, AA417420, AI108220, AA508946, AA471497, AA842911, AA842501, AA123614, AA509218, AA547812, AA752986, AA752003, AA406839, AA508993, AA161747, AA509008, AA180623, AA508986, AA842627, AA753129, AA509214, AA406733, AI105737, N74818, AA753300, AA430818, AA417415, AA114426, R46936, AA979824, AA246112, AA753138, AI058077, AA842464, AA966639, AA842265, AA509025, AA601853, W15128, AA660039, AA842275, AA454424, AA180651, AA842423, AA257507, AI063375, AA180692, AA051990, W15094, AA180620, W00308, AA406729, AA257712, AA257445, AA433148, C48534, AA495533, C08939, AA406845, AA933362, AA509242, AA842023, W29144, AI087490, AA842237, AA601823, W06538, AI087739, AI087524, AA842164, AA842642, AA471553, AA842419, AI113700, AA675813, AI083003, AI021727, AI083274, AI083309, W32823, AA253962, AA114520, AA430792, C47230, AA842674, AI053141, D75937, AA843034, W68979, AI105681, W91819, AA756971, AA509104, AA111828, AI109720, AI110161, AA933311, AA180602, AA842093, R86419, AI058057, AA186285, AA406891, AI105725, AA406888, AA649397, N94700, W51718, AA547916, AA840646, U74116, AA257756, AI082996, W91818, N74830, AA123585, AA118224, AA454446, AA841367, AA509109, AA842387, AI052833, AA751998, AA123634, AA257327, AA803962, AA842493, AA471448, AA406716, AA841361, AA840661, AA713447, AA406754, AA257695, AA841403, AA751834, AA406947, AI096182, AI105734, W15132, W59918, AA089352, AA180566, AA257427, AA257522, AA406980, AA471469, AA509264, AA509032, AA749469, AA752028, AA754167, AA754646, AA842335, AA842574, AI065957, AI065970, AI096289, AA109379, AA109472, AA114484, AA161689, AA430830, AA406948, AA433246, AA454371, AA471492, AA471703, AA752086, AA842624, AI096185, R47079, R86415, AA161711, AA161655, AA180549, AA257424, AA257437, AA257749, AA433393, AA471419, AA471602, AA430922, AA661401, AA752034, AA752066, AA842384, AI082936, AI083329, AI105677, Z33912, AA109362, AA109417, AA051807, AA430797, AA406799, AA430806, AA495548, AA661116, AI082951, AI105685, AI114069, R47062, W59884, AA109292, AA751829, AA754172, AA842257, AI082934, AI082958, AA180706, AA471516, AA508962, AA751563, AA751816, AA753137, AA753167, AA840972, AA842079, AA874756, AI105522, AI108824, AA180588, AA406676, AA471392, AA509142, AA840909, AA842175, AI066854, R47172, AA161565, AA257643, AA753161, AA471447, AA680450, AA752897, AA753237, AA842230, AA842544, AA161635, AA454428, AA508933, AA509309, C41215, AA842585, D73786, D75990, D75808, AA627049, N74809, AA257682, AA257517, AA257668, AA471470, AA509251, AA509051, AA990913, C39627, C41200, C42101, C46071, C48450, AA840970, AA842156, AA430902, AA842216, AA007706, AA406765, AA180574, AA842310, AI067585, AI077003, AA756933, AA114501, AA406690, AA454440, AA661371, AA056797, AA114372, AA180676, AI082955, AA406735, AA675746, AA752005, W29142, AA109261, AA842401, AA842602, AA246079, AI043420, AA626993, AA123655, AA257716, AA406931, AA791379, AI105668, AA842538, R47669, AA088150, AA109308, AA738555, AA123638, AA180582, AA123597, AA471535, AA180728, AA161741, AA186201, AA430817, AA433170, AA454407, AA471686, AA161596, AA509110, AA675874, AA471540, Z26577, AA753093, AI066829, D37716, AA627017, N99292, AA508936, AA257656, AA406768, AA471587, H39287, AA842431, AA161715, AA509204, AA979935 SEQ ID NO.: 2, AF086243, AE001154, X62889,, W67765, W67764, AA947751, AI141491, W76469, AA906091, AA872676, AA349825, H77545, H91001, W72232, AA948309, AA361403, R57582, AA337188, AA215714, AA481093, T75310, AA976452, , AI120744, AA462558, AI158491, AA014020, AA285990, AA051044, AA473453, AA896862, W56907, AA218305, AA020167, W85164, AA017810, W30604, AA541978, W20894, AA023164, AA760425, AA024084, AA840044,, AA231755, T20644, C45833, T24185, C68940, D37618, AA964657, AI011924 SEQ ID NO.: 3, L08501, Z97205, Z48950, AF010400, AC005162, L08502, D50608, , AA707653, AA861639, AA292496, AA702524, AI097367, AI138504, AI147933, AI141836, AI075247, N63868, W88668, AA703146, AA625621, AA292247, AI032848, AA005331, AA699781, AA427941, D31111, D31113, N92013, AA884207, AA044752, R70900, R16693, AA906542, R35112, AA481286, D80100, AA235512, AA960995, AA867982, D59403, W88874, AA558590, R10048, AA719917, H43573, R49500, AA744780, AA047136, AA789101, AA906332, AA747301, AA830606, AA434559, AA236698, AA328889, N984 69, W17299, W46605, AA258082, AI078045, R11930, W74577, AA703312, AI082727, R45189, N76461, AA010500, W63646, W38891, AA490651, AA558805, W87891, AA160849, AA618177, AA776126, AA161281,, AA822308, AA499768, AA028780, AA183100, AA276783, AA120227, AA200285, AA212541, AA116265, AA821616, AA265629, AA108594, W61565, AA030311, W42275, AA419903, AA268027, AA028211, AA518504, AA125394,, C92235, C92002, C29917, AI145662, C65317, AA901847, AI137443, Z14719, D35515, R90625, H76970, SEQ ID NO.: 4, AL031178, Y11905, AF031904, M34309, M29366, L33953, L33952, L33956, L33954, L33957, U41289, X13369, AL022072, AJ223074, AF078695, AC004683, X17267, AF025526, AF071798, AC005274, AB004537, X68248, AF035537, AF056116, AB009052, AL023286, AF058701, L20725, AF001308, U22438,, AA211485, AA579574, AI078750, AA568661, AA604128, H49462, AA767424, H97012, AA565823, H49463, AA827171, T87152, F22114, AA748475, T31504, Z42997, T89930, R02581, H75949, T87057, AA322268, R02700, AA005034, AA576177, AI014302, AA348159, T83607, AA322497, AA211532, AA019517, R27657, AA026869, N67589, T83782, AA129383, Z39829, AA814308, AA425564, H67997, AA004420, AA644513, AA007691, T62593, AA333601, H75334, AI057250, T62521, N73865, AA004558, AA004484, AA007690, AA910241, AA456251, AA004568, AA341266, T28757, AA132360, AA781316, AA456942, AA782765, AA662593, AA461351, AA885220, N33840, AA843737, T62232, AA608559, AA643270, R44662, AI074863, AI051088, N63305, W22455, AA085886, W31918, AA047466, AA178965, AA369890, AA515015, N79466, AA768162, AA864694, AA811390, AA834531, AA293263, AA768335, AA749083, T57520, AA292001, AI085512, AA969032, AI027062, AA595663, AA827242, AA744475,, AA472485, AA097011, AA959170, AA647546, AA986669, AI006628, W12604, AI122356, AA571721, AA433697, AA880171, AA0154 63, AA116290, C87721, AA437983, AA990395, AA265925, W14785, W89262, AA059703, AA413613, AI020231, AA667024, AA407526, AA221491, AA222523, C76436, AA409700, W35766,, AA859485, H33704, T46512, C25899, T41882, C26586, C92302, AA660448, C94493, C25527, D35045, D33727, F14543, D37473, T45969, AA040979, D68274, T38420, AA228607, AA660541, T38283, AA842575, C71889, T38745, D68465, T46732, AA598393, C23775, C64798, AA042714, F19972, C65873, N97974, AA114425, AA900456 SEQ ID NO.: 5, U29344, S80437, M76767, X62889, M84761, J03514, X62888, X13415, X13135, U05714, AC004013, U58675, S47635, Z81533, AC005250, AC003661, AB008567, Y13444, X96401, AF026487, AF026488,, AA781445, AI037943, AI129371, C15883, AT073336, AA904077, AA569042, AI039428, AA568701, AA058907, AA911112, AA234022, N95359, AA565390, AA082427, AA588430, D60358, AA045488, AA084417, N72089, AI143390, R51974, AA081439, AA635907, T47621, AA579930, AA995057, AA827039, AA872490, AA588319, AA069032, AA062768, AA534011, W96404, H06082, AA938900, AA971262, AA836547, AA251544, AA250742, AA830405, AA906492, AA102653, R38286, AA258075, H83302, H38522, R61787, R33742, R24094, AI052406, AA931452, R23634, R39640, AA974568, AI028383, AA312451, AA148800, AI138982, R36172, AA926921,, AA472563, AA914598, AA423256, AA476186, AA041992, W84938, AA797706, AA709956, AA544361, AI006075, AA717202, AA008602, AA033399, AA472306, AI037430, AI152943, AA475582, AA471742, AA530292, AA277496, AA718588, AA823112, AA822771, AA710973, AA981780, AA718846, AA797557, AA510746, AI036049, AA543891, AA606513, AA981758, AA822268, AA413161, AA472201, AA646527, AA213036, AA168254, AA718549, AA458342, AA119418, AA879925, AA050917, AA718814, AA793638, AA177849, AA575627, AA537367, AI121788, AA203946, AA413157, AA517432, AA867736, AI037420, W30436,, Z71851, AA957415, AI112847, AA955881, AA963915, AI043663, C95061, AI009894, AA957229, AI044678 SEQ ID N0.: 6, AC002426, AC004674, AC004675, AC004006, L12157, U20839, U46028, U20835, AC004058, X67115, X76266, M25485, U59759, AC005172, U70848, AF042274, X14724, L31840, L41679,, W52480, AA863014, W56770, AA286755, AA164604, W52777, AA814246, AA765427, AA873647, AA360577, AI139274, AA770312, AA732557, AA568651, AA164603, W56724, AA194905, AA865009, R16884, R08110, F07672, AA577790, T11467, AA502489, AA366688, AA480628, AA516318, AA090005, AA081908, F01265, AA780686,, AA466811, AA465808, AA197646, AI157174, AA153086, AA197667, AA066612, AA058086, AA024186, AA110104, AA880419, AA870161, AA153880, AA072995,, AI031052, AI043934, AA849320, D15181, U95093, AI096188, T09655, C62926, AA996434, AA925202, AA817271, AA901051, R03660, AA948980, AA658709, AA899446, AA957150, AA800160, T76196, AA891787 SEQ ID NO.: 7, X63547, X63546, AF017306, U44839, U20657, AF017305, AJ001589, L21998, D21270, Z48245, U63834, Z68006, L04573, D63819, AC005266, AF025468, M94131,, AI056961, C15588, AA373847, AA887911, N87070, R39133, AA226825, AA353972, AA325352, R88378, AA490675, AA456219, AA701415, AA046611, AA456224,, AA065652, AA221447, AA710221, AA717401, AA254049, AA041745, AA510261, AA170745, AA656404, AA572506, AA174539, AA217630,, AI134284, C58457, C11202, AA413362, AA728532 SE ID NO.: 8, AB016886, Z92811, AL021481, U52078, L46702, U23515, AC005555, Z84814, Z93016, D21138, X57513, L07144, D83711, M28488, U15974, U52513, AF026939, L19120, J05258, Z13985, Z70691, U59227,, AA190526, AA764854, AI014655, H88220, AA622877, N32046, H89609, AA682362, AA464420, AA375477, AA284905, AA257109, N80276, H89373, N92393, AA884334, AA778708, AA766209, AA535677, W46414, AA770266, AA983635, AA721113, H88219, R87349, AA628091,, AI116513, AA183589, AI035517, AA209952, AA184622, AI097904, AA016440, AA162370, AA939521, AA162376, AA718152, AA048154, AA253815, AA795350,, AA996981, C44448, C41783, C44695, C44039, C62421, C47063, C45014, D36613, C60872, C50136, C69177, AI055708, D27648, C65648, AI114022, AI064663, AI107213, AA395461, AA800126, AA891487, T01611, C27943, AI113482, C83830 SEQ ID NO.: 9, L43510, U71249, L11275, X73541, Z28317, AB008270, X75652, U05987, U85262, U24189, Z46676, X70823, AC004981, D11079, X56121, M58053, U33636, Z92773, U23168, D31662,, R15557, F01629, N71722, AA2525 48, AA806751, T59557, AA612671, AA329585, AA295 675, AA166990, AA128100, H46363, AA125810,, AA396888, AA408999, AA270873, AA144722, AA863954, W10303,, C38016, N96746, Z37622, ​​AA395862, C38756, F15295, N99294, W68877, C44749, C24349, F15569, AA113611, AA689147, N96676, C73598, F15564, L46559, W06235, T09718, H77154, T14151, AI082914, AA650815, AA728021, AA072559, C92112, T46743, C61743, AA550223, C12798, AA275531, AA681003, T75878, AI100047, T75882, AA848187, T44109, AA542686, H21339 SEQ ID NO.: 10, Z81595, U41372, U01317, AE000696, AC002057, X66250, L11665, D13438, U29377, Z50028, U40953, AA114228, AI025080, R80188, W28745, AA381819, AA381991, Z44165, H75915, AI124743, H08139, AA375957, AA381750, AA166751, R42511, T26985,, AA471592, AA661387, AA606231, D35147 SEQ ID NO.: 11, X76498, Z97632, AC004232, AC005184, AC002545, AC003991. Equivalents ...

Wherein 3-740 position Nucleotide is the mature peptide sequence of genes encoding, i.e. plasmin protein sequence, and the 741-743 position is terminator codon TAA.The protein amino acid sequence of this genes encoding is:

The aminoacid sequence of Lumbricidae powder Lumbricus earthworm fibrinolysin (SEQ ID NO:2) MELPPGTKIVGGIEARPYEFPWQVSVRRKSSDSHFCGGSIINDRWVVCAAHCMQGE APALVSLVVGEHDRSAASTVRQTHDVDSIFVHEDYNTNTLENDVSVIKTSVAITFD INVGPICAPDPANDYVYRKSQCSGWGTINSGGICCPNVLRYVTLNDTTNQYCEDVY PLNSIYDDMICASDNTGGNDRDSCQGDSGGPLSVKDGSGIFSLIGIVSWGIGCASG YPGVYSRVGFHAAWITDIITNN

The plasmin molecular weight of albumen that the present invention relates to is 26.4kD, and iso-electric point is 4.5, is rich in glycine, Serine, Xie Ansuan and aspartic acid.Its biochemical characteristic also has: 1. thermostability: 60 ℃ of heating of plasmin are after 30 minutes, and enzymic activity does not obviously reduce, and more than 65 ℃, enzymic activity begins to descend, and 30 minutes enzymic activitys of 85 ℃ of heating completely lose; 2. acid acceptance: plasmin does not have considerable change in pH1～11 enzymic activity, and pH is lower than 1 or be higher than at 11 o'clock, and enzymic activity obviously descends, and best fibrinolytic reaction pH value a wider range is between 7～10.

The fibrinolytic enzyme gene that the present invention relates to can also obtain by the following method: (1) subclone: with gene fragment involved in the present invention is template, be template perhaps with the recombinant plasmid that contains gene fragment involved in the present invention, be template perhaps with the engineering strain DNA that contains gene fragment involved in the present invention, by conventional molecular biology working method (as PCR), subclone obtains corresponding gene fragment, these fragments can comprise gene order involved in the present invention whole or part, these fragments can be on bacterium, yeast, animal and plant cells or other eucaryon, express in the prokaryotic organism, obtain protein or polypeptide fragment, these protein or polypeptide fragment have identical biological function with protein involved in the present invention; (2) synthetic: according to amino acid involved in the present invention or nucleotide sequence, can be by the method synthetic DNA fragment of chemosynthesis, these fragments can comprise gene order involved in the present invention whole or part, can be cloned in the corresponding carrier and express, expressed protein or polypeptide have identical biological function with protein involved in the present invention.

Gene fragment involved in the present invention can be modified, and such as some nucleotide sequence of mutator gene, but does not influence its encoded protein matter aminoacid sequence; Can increase, lack amino acid involved in the present invention or nucleotide sequence, these are modified can not influence proteinic biochemical characteristic, higher structure and proteinic biological function, such as: can add secreting signal peptide at protein or polypeptide N-end or C-end, can also add that suitable joint (as 6 Histidines) is convenient to proteins extraction, purifying.

Gene involved in the present invention with can be cloned among the corresponding host after carrier is connected so that the preservation of gene, amplification and expression.Be applied to cloning vector of the present invention include but are not limited to following carrier: pBR322, pBR325, pACYC177, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/-, pQE, pGEM-T, pET serial carrier." the molecular cloning laboratory manual " that carrier feature and cloning process can be write referring to cold spring harbor laboratory.---carrier system includes but are not limited to following system: bacterium---the phage system that is applied to host of the present invention, bacterium---plasmid vector system, yeast---yeast vector system, Mammals---viral system (vaccinia virus, adenovirus etc.), insect cell---rhabdovirus system.

A kind of preparation of Lumbricidae powder Lumbricus earthworm fibrinolysin gene is provided in one embodiment of the invention and has been cloned into the method for intestinal bacteria (E.coli), this method comprises: amplimer is synthetic, the total RNA of earthworm extracts, the reverse transcription of mRNA, the pcr amplification of goal gene, the recovery of target gene fragment, target gene fragment are cloned into carrier, earthworm fibrinolysin cDNA sequencing.

(1) amplimer is synthetic: according to the earthworm fibrinolysin cDNA sequences Design PCR oligonucleotide amplimer of having delivered among the GeneBank.Long 18 Nucleotide: the ACATGGAACTTCCTCCCG of upstream primer (P1), long 19 Nucleotide: the ATCACCAACAACTAAACCG of downstream primer (P2), primer is through the PACE purifying.

(2) the total RNA of earthworm extracts: get 3～5 earthworm adults, after the aqua sterilisa rinsing of handling with DEPC, be placed in the plate that is covered with the filter paper that wets hunger and spend the night, make it tell intravital to the greatest extent silt as much as possible.After cleaning up with DEPC water once more, in liquid nitrogen, be ground into powder, extract total RNA according to the product description of the RNeasyR Mini Kit of QIAGEN company.The RNA solution that extracts is stored in-80 ℃ with standby.

(3) the synthetic cDNA article one chain of reverse transcription: the ReverseTranscription Reaction test kit specification sheets according to Promega company carries out, and is synthetic cDNA first chain of primer with Oligo (dT) 20.Reaction conditions is: 42 ℃ 1 hour; 95 ℃ 5 minutes; 3 ℃ 5 minutes.

(4) pcr amplification of goal gene: carry out the PCR reaction according to the PCR reaction system parameter that provides in the Reverse Transcription Reaction test kit.With cDNA article one chain is that template is carried out pcr amplification, and its reaction parameter is: 95 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 20 circulations 2 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 10 circulations 2 minutes; 72 ℃ were extended 10 minutes again.

(5) recovery of target gene fragment: reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim target gene fragment.

(6) target gene fragment is cloned into carrier: the PCR product of recovery is connected with the T carrier, connect product transformed competence colibacillus cell intestinal bacteria (E.coli) JM109, picking colony rapid extraction plasmid carries out enzyme and cuts evaluation, resulting positive colony is to comprise the intestinal bacteria that the present invention relates to gene, is used for the propagation and the preservation of gene.

(7) earthworm fibrinolysin cDNA sequencing

Extract the plasmid DNA dideoxy method and measure nucleotide sequence, sequencing primer is the M13 promoter primer.The sequencing result of Lumbricidae powder Lumbricus earthworm fibrinolysin gene nucleotide: long 747 Nucleotide of earthworm fibrinolysin cDNA sequence, adenosine (A), cytidine(C (C), the fast content of holding in the mouth or the eyes nucleosides (G) and thymidine (T) of bird are respectively: A-185, C-197, G-189; T-176.Gene mature peptide sequence is a 3-740 position Nucleotide, 246 amino acid of encoding, and all the other nucleotide sequences are non-coding region.This protein cDNA mature peptide terminator codon is positioned at gene 741-743 position, and termination codon is TAA.

Two of technical essential of the present invention is to express the structure and the expression of the pichia pastoris gene engineering bacterial strain of fibrinolytic enzyme gene, and this bacterial strain can be expressed fibrinolytic enzyme gene, obtains genetically engineered plasmin albumen and is used to prepare the medicine for the treatment of cardiovascular disorder.

Fibrinolytic enzyme gene involved in the present invention can be expressed in including but are not limited to following carrier-host system, obtains engineered protein: intestinal bacteria system, yeast expression system, expressing cho cell system, baculovirus expression system, filamentous fungus expression system, genus bacillus expression system.

Finishing red (Pichia) yeast expression system is a kind of eukaryotic expression system efficiently that development in recent years is got up, compare with other expression system, it has the following advantages: (1) expression amount height, because pichia spp fast growth, and the expression vector use is strong promoter---alcohol oxidase promoter, the exogenous protein expression amount is very high.(2) good stability does not exist because the expression vector of this system is not plasmid with self-replicating in yeast, but by stable being incorporated on the yeast chromosomal of homologous recombination, so the engineering strain that makes up is very stable.(3) purifying is easy, and is easy and simple to handle.Expression product generally is secreted into outside the born of the same parents, and directly testing goal albumen from nutrient solution does not need renaturation, and can directly extract albumen from supernatant.(4) active high.The largest benefit of foreign protein secreting, expressing in pichia spp can be carried out the protein translation post-treatment exactly, comprises being shaped as of disulfide linkage, protein folding and glycosylation etc.Compare with some other eukaryotic expression systems such as cereuisiae fermentum, pichia spp more approaches mammiferous glycosylation to the foreign protein glycosylation, so the glycoprotein of Pichia anomala expression is considered to the human body that is used for preferably.(5) be convenient to suitability for industrialized production.That pichia spp has is ready-made, sophisticated fermentation technique carries out enlarged culturing, and pichia yeast expression system can utilize methyl alcohol to grow as sole carbon source, and large scale fermentation is convenient for production, and cost is low, is easy to realize industrialization.

The earthworm fibrinolysin cDNA that the present invention relates to derives from the positive earthworm of powder (Lumbricusrubellus), and this fragment has 741bp, 246 amino acid of encoding.The present invention expresses this gene in Yeast system, obtain the genetically engineered plasmin by the method production of fermenting, and detected this proteic biological activity by the fibrin plate method.The research of domestic existing earthworm fibrinolysin aspect expression in escherichia coli at present, but its expression in yeast does not appear in the newspapers.

Yeast expression vector includes but are not limited to following carrier: pPIC6, pPIC6 α, pPICZ, pPIC α Z, pPIC9k, pPIC3.5k, pPAO815, pGAPZ, pGAP α Z; Pichiapastoris expression strain includes but are not limited to following bacterial strain: GS115, KM71, KM71H, X-33, SMD1168, SMD1168H.

Provide a kind of in one embodiment of the invention and made up engineering strain, in pichia spp, expressed the method for earthworm fibrinolysin gene, this method is to express the earthworm fibrinolysin gene with pPIC6 α A carrier (Invitrogen company) in pichia spp X-33, pichia spp Pichiapastoris X-33 (pPIC6 α A-EFE), this bacterial strain is in China typical culture collection center (CCTCC) preservation, be numbered: M204005, the preparation method of this bacterial strain are the pcr amplification of (1) earthworm fibrinolysin gene; (2) the PCR product cloning is to the T carrier; (3) structure of shuttle expression plasmid; (4) structure of the Yeast gene engineering bacterial strain of expression fibrinolytic enzyme gene.

(1) pcr amplification of earthworm fibrinolysin gene: with the T vector plasmid that contains fibrinolytic enzyme gene is template, according to the design of the cloning site on EFE mature peptide and expression vector pPIC6 α A primer, and in upstream primer, add 6 successive Histidine encoding sequences, add enteropeptidase cleavage site sequence afterwards, and designed a pair of earthworm fibrinolysin pcr amplification primer.Therefore, the expression product of this project bacterial strain is a fusion rotein, at the proteic N end of plasmin 6 successive Histidines are arranged, be used for the separation and purification of expression product, there is amino acid in the non-natural that can cut fusion rotein N end after purified product cuts through enteropeptidase, obtains forming identical engineered protein with natural plasmin.Provide a pair of primer sequence in one embodiment of the invention, upstream primer P1:C GAATTCCACCACCACCACCACCACGGTGGTGGTAGCAGCGACGACAAG ATGGAACTTCCTCCCGGAACA (the underscore place is restriction endonuclease EcoRI site).Downstream primer P2:G TCTAGATTAGTTGTTGGTGATGATGTCGGTGATCCATGCAGCAT (the underscore place is restriction endonuclease XbaI site).

(2) the PCR product cloning is to the T carrier: reclaim the gene fragment in the PCR product, and clone the carrier in pGEM-T, (pGEM-T-EFE2) changes in the e. coli jm109 with this recombinant vectors.

(3) structure of shuttle expression plasmid: extract recombinant vectors pGEM-T-EFE2, with EcoRI and XbaI double digestion to obtain purpose fragment EFE; Same enzyme is cut pPIC6 α A empty carrier and make it dephosphorylation under the CIAP effect.With the EFE that obtains behind linearizing pPIC6 α A empty carrier behind the dephosphorylation and the double digestion under the effect of T4DNA ligase enzyme 4 ℃ spend the night, to obtain recombinant expression vector pPIC6 α A-EFE, this recombinant vectors is changed in the e. coli jm109 increases.

(4) structure of the Yeast gene engineering bacterial strain of expression fibrinolytic enzyme gene;

A. linearization of shuttle expression plasmid: extract recombinant plasmid pPIC6 α A-EFE, be cut into linearity with the SacI enzyme, enzyme is cut empty plasmid pPIC6 α A in contrast equally.

B. transform: method is with reference to Invitrogen company expression vector specification sheets: utilize biochemical method that linearizing recombinant plasmid pPIC6 α A-EFE and empty plasmid pPIC6 α A are transformed the X-33 yeast strain, on the YPD that contains blasticidin (Blasticidin) 300mg/ml (1% yeast extract, 2% peptone, 2% glucose) solid medium, cultivated 2-3 days, up to there being single bacterium colony to occur.

C screens positive transformant: picking 10-20 single bacterium colony in the YPD liquid nutrient medium 30 ℃, 250rpm shaking table were cultivated until logarithmic growth late period, extracted the PCR that genome carries out recon and identified.The universal primer that primer provides for Invitrogen company expression vector, its sequence is upstream: GACTGGTTCCAATTGACAAGC, downstream: GCAAATGGCATTCTGACATCC, the positive colony pichia pastoris engineered strain called after Pichia pastorisX-33 (pPIC6 α A-EFE) that identifies, this bacterial strain at China's typical culture collection center deposit number is: CCTCC M204005.

The gene A OX1 and the AOX2 of two coding alcohol oxidases are arranged in pichia spp, can utilize methyl alcohol to grow fast, and when having other carbon source in the substratum, the AOX gene is not expressed as sole carbon source.In pichia pastoris engineered strain, foreign gene is cloned into the control of AOX1 promotor down, must also be that sole carbon source is realized efficiently expressing of foreign protein simultaneously with methyl alcohol as inductor therefore, when abduction delivering, should replenish methyl alcohol to keep its content of 0.5%.

A kind of method of pichia pastoris engineered strain abduction delivering is provided in one embodiment of the invention, the substratum that can be used for the pichia pastoris engineered strain abduction delivering includes but are not limited to following substratum: MGY, MM, BMM, BMMY, the collocation method of various substratum is the method that present technique field personnel are familiar with very much, can obtain detailed information by the reference pertinent literature.

Proteinic separation and purifying have several different methods, and its principle is with itself and other protein separation according to the physics of target protein and chemical property.Can select centrifuging, ultrafiltration process, gel-filtration chromatography as size and albumen shape according to protein molecular weight, difference according to protein solubility can be selected ammonium sulfate precipitation method or acetone precipitation, different choice isoelectric focusing electrophoresis method or isoelectric precipitation method according to iso-electric point, can select hydrophobic interaction chromatography method, reverse hplc method according to the hydrophobicity difference, select the immobilized metal affinity chromatography method according to the binding ability of protein and metal ion.

A kind of expression in pichia spp X-33, separation, purifying earthworm fibrinolysin gene are provided in one embodiment of the invention, have obtained the method for engineered protein, this method comprises: the abduction delivering of (1) pichia pastoris engineered strain; (2) separation of genetically engineered plasmin, purifying; (3) proteic functional study of genetically engineered plasmin and determination of activity.

(1) abduction delivering of pichia pastoris engineered strain

The pichia pastoris engineered strain derivational expression method is as follows: single bacterium colony of picking reorganization bacterium is to the 20mlYPD liquid nutrient medium, and 30 ℃, 250rpm cultivation activate; 30 ℃ of the bacterium liquid switching 500ml MGY liquid nutrient mediums, the 250rpm that get after 16 hours after 10ml activates cultivate enrichment; The bacterium liquid of enrichment after 24 hours is through centrifugal collection thalline (1500g, 10 minutes, 4 ℃); The thalline of collecting is carried out abduction delivering with being resuspended in 1000ml MM liquid nutrient medium, and it is 0.5% that every 24h adds methyl alcohol to final concentration; Induce centrifugal collection fermented liquid supernatant behind the 96h (30000g, 20 minutes, 4 ℃);

(2) separation of genetically engineered plasmin, purifying

The method that can be used for the separation of genetically engineered plasmin, purifying includes but are not limited to following method: Ni2+ column chromatography, ion exchange chromatography, affinity chromatography, gel-filtration chromatography, electrophoresis absorption method, ultrafiltration process, ammonium sulfate precipitation method etc.

The method of a kind of separation, purifying gene engineering plasmin is provided in an embodiment of the present invention, be divided into 3 steps, it at first is the Ni2+ column chromatography purification, genetically engineered plasmin albumen has 6 successive histidine residues, they can combine with Ni2+ engineered protein is combined on the fixed metal ion post, and other foreign protein can not combine with the metal ion post and directly eluted, thereby target protein matter eluted the purpose that reaches protein purification with suitable elutriant at last.Ni2+ column chromatography purification product adds the interior enteropeptidase of interior enteropeptidase of highly purified ox or reorganization, carries out DeR, can cut the alpha-non-natural amino acid residue in the fusion rotein, obtains forming identical engineered protein with native protein amino acid.At last the enteropeptidase degradation samples is directly added in the QAE-Tyoypearl 550C chromatography column, because the difference of different proteins iso-electric point is the separation and purification of genetically engineered plasmin, purified product is verified through the SDS-PAGE electrophoretic analysis.

(3) proteic functional study of genetically engineered plasmin and determination of activity

The current national standard of still not having the earthworm fibrinolysin determination of activity, however as a kind of new medicine, need set up the method for a cover activity and titration.Simultaneously this measuring method should be both accurately and reliably, and is simple and easy to do again.Current measuring method can only be with reference to the activity determination method of other thrombolytic drug.Such as the bubble rise method of urokinase activity and arginine esterase method (TAME) method of mensuration Ahylysantinfarctase, but all inapplicable mensuration earthworm fibrinolysin of these two kinds of methods, because plasmin has the effect of plasminogen activator and proteolytic ferment, when thrombus, can bring into play this two kinds of functions, and the antiplasmin activity of this enzyme is significantly greater than kinase activity.

The fibrin plate method can be expressed the ability of plasmin thrombolysis objectively.Contain proplasmin in the fibrin plate, under the zymogenesis of plasmin, change Tryptase into.Answer the lyase effect of plasmin in addition, make fibrinogen generation hydrolytic action, become soluble small molecular peptide and amino acid, the application of sample place is dull and stereotyped to form translucent solusphere thereby make, and determines the activity unit of enzyme according to the size of circle.Within the specific limits, dull and stereotyped solusphere size and concentration journey linear dependence, the activity of therefore measuring fibrin plate can be regarded the dual function of plasmin and activating enzyme as.This method has simple to operate, and dissolving circle is clear, be easy to observe advantages such as good reproducibility.

Use with the proteic functional study of genetically engineered plasmin of gene involved in the present invention and the method for determination of activity and include but are not limited to following method: fibrin plate method, bubble rise method, arginine esterase method.

The method that provides a kind of fibrin plate method to measure the biologic activity of genetically engineered plasmin in one embodiment of the invention, by determination of activity, the activity unit of engineering strain fermented liquid of the present invention surpasses 3,390,000U/L.

Three of technical essential of the present invention is the application of genetically engineered plasmin albumen in the treatment cardiovascular and cerebrovascular diseases.

Engineered protein involved in the present invention has purposes widely in the research and development field of medical treatment, health, health-related medicine, can be used for preparing freeze-dried, the aqueous injection, tablet, suppository, sprays of treatment embolic cardiovascular disorder etc., also can be used for producing healthcare products with prevention thrombus disease, useful cardiovascular health.

Compare with traditional earthworm fibrinolysin capsule preparations, genetically engineered drug involved in the present invention has following advantage: the effect of (1) earthworm fibrinolysin capsule preparations oral administration is slow, can not intravenous injection, can not be applied to treat the acute heart, cerebral vessels embolism disease; (2) capsule complex manufacturing technology needs artificial a large amount of breeding earthworm, and separation and purification plasmin technology is loaded down with trivial details from polypide, may contain various micromolecule polypeptides or other chemical substance in the product, may cause the anaphylaxis of body.Use engineered method then can obtain plasmins in a large number by simple fermentation, separation purifying technique is simple, and the product purity height is at the bottom of the cost.

Compare with other thrombolysis class medicine (comprise biological extraction with genetically engineered drug), genetically engineered drug involved in the present invention has following advantage:

(1) streptokinase (streptokinase SK) and urokinase (urokinase UK): SK and UK belong to first-generation thrombolytic drug, and they can make it to be transformed into the plasmin with cellulolytic activity by direct or indirect plasminogen activation.SK is a kind of exogenous plasminogen activator that extracts from β type Hemolytic streptococcus nutrient solution, is indirect plasminogen activator.UK is the serine protease of separating from people's urine, also can be by obtaining in human embryonic kidney cell's nutrient solution.There are two unavoidable problem in these two kinds of enzymes in actual use: at first, their specificity of effect is poor, often cause the systematicness of fibrinolytic protein proenzyme to activate at thrombus, and cause digestion, increased the danger of internal hemorrhage in the therapeutic process the indifference of solidifying egg white; Secondly and substrate bonded avidity little, directly a little less than the thrombolysis effect.Using dosage is big when treatment, causes treating the cost costliness.SK is a kind of non-zymoprotein, and in use Zui Da problem is its antigenicity.More or less all contain anti-SK antibody in almost all people's the blood.The existence of antibody is " neutralization " medicine on the one hand, and drug effect is reduced, and may cause allergic reaction on the other hand.

(2) (the tissue type plasminogen activator of tissue-type plasminogen activator, tPA): tPA is a kind of serine protease that is mainly produced by vascular endothelial cell, the Profibrinolysin of energy catalysis non-activity becomes activated plasmin, makes thrombolysis.The plasma concentration of normal buman tPA is about 5ng/ml, 5 minutes transformation period.TPA uses it to come into one's own as s-generation thrombolytic drug to the specificity of the Profibrinolysin that is present in thrombus part, and in American-European developed country, the tPA that produces with gene engineering method has begun to be used for clinical, has shown certain superiority.But because the transformation period is short, need repeatedly a large amount of medications just can prove effective, increased systemic molten fibre thus and cause the possibility of internal hemorrhage, and limited the widely-used of it because cost is high.

(3) other thrombolytics: except UK, outside SK and the tPA, the snake venom antithrombotic enzyme in addition of research at present, single chain urokinase type plasminogen activator type plasminogen activator (pro-UK), acetylize Profibrinolysin streptokinase activity mixture (APSAC) etc.But there is bigger side effect in the application that these preparations have, and anaphylaxis can take place; The mechanism of action that has is not clear, uses complexity very much, and high to the product property requirement, what have costs an arm and a leg.

The thrombolysis class medicine of biological extraction needs charcoal absorption in process of production, mainly is to be used for decolouring, to adsorb thermal source, the removal of impurity and to help filter.Engineered protein purity height involved in the present invention, pure, the pyrogen-free of quality avoids taking off the pollution to environment of charcoal process and gac, and production cost is low, can be directly used in to produce water agent for injecting, pulvis.

The genetically engineered plasmin that the present invention produces can mix with acceptable carrier (as vehicle, weighting agent, absorption enhancer etc.) pharmaceutically, perhaps mix use with other medicines, can be made into required formulation according to the ordinary method of pharmaceutical field, can be by mode administrations such as oral, injections.That these formulations include but are not limited to is freeze-dried, aqueous injection, tablet, suppository, sprays.

Genetically engineered drug involved in the present invention can be applicable in the treatment or assisting therapy of cardiovascular and cerebrovascular diseases, these diseases include but are not limited to: ischemic encephalopathy, acute myocardial infarction merges hyperfibrinogenemia, coronary heart disease, the blood high viscosity syndrome that coronary heart disease causes, coronary heart disease and angina pectoris, unstable angina pectoris, Fibrinogen and platelet aggregation increase disease, genetically engineered drug involved in the present invention also can be applicable in the treatment and assisting therapy of the relevant some diseases of hemorheology, and these diseases include but are not limited to Meniere, sudden deafness, pulmonary heart disease, diabetes, retinal vein occlusion, nephrotic syndrome.

Engineered protein involved in the present invention can be used as the production that main active ingredient is applied to healthcare products, is used to prevent thrombus disease, promotes cardiovascular and cerebrovascular health.

Above-mentioned is medicine or other biological products of main active ingredient with the genetically engineered plasmin, its making processes or method are familiar with by this technical field personnel, production process should be carried out according to country relevant law, rules, comprise the Pharmacopoeia of the People's Republic of China (Chinese Pharmacopoeia Commission, 2000) and " Chinese biological goods rules " (the Chinese biological standard of articles council, 2000).

In an embodiment of the present invention; a kind of method for preparing genetically engineered plasmin thrombolytic drug preparation is provided; and in animal model, carried out thrombolysis and to the test of cerebral ischemia provide protection; the result shows that genetically engineered plasmin involved in the present invention has good thrombolytic effect; and the cerebral ischemia of experimental animal had provide protection, these can say the purposes of genetically engineered plasmin in cardiovascular and cerebrovascular diseases medicament research and development field that relates to as illustrations the present invention.

There is cardiovascular and cerebrovascular diseases people about 1,500 ten thousand in the whole world, about 2,000,000,000 dollars of the potential market of required thrombolytics, about 5,000 ten thousand people of all kinds of patients that available plasmin is treated, about 3,500,000,000 dollars of potential market, therefore, the genetically engineered plasmin has vast market prospect.

A kind of engineering strain, pichia spp Pichia pastoris X-33 (pPIC6 α A-EFE), depositary institution: Chinese typical culture collection center, preservation date: on January 12nd, 2004, deposit number: CCTCC No:M204005.

Description of drawings

The pcr amplification result of accompanying drawing 1 fibrinolytic enzyme gene

Swimming lane 1 is dna molecular amount standard (λ DNA/HindIII); Swimming lane 2 is reverse transcription synthetic cDNA article one chain; Swimming lane 3 is the pcr amplification product of fibrinolytic enzyme gene

The enzyme of accompanying drawing 2 recombinant plasmid pUCm-T-EFE is cut qualification result

Swimming lane 1 is dna molecular amount standard (λ DNA/HindIII); The pcr amplification product of swimming lane 2,3 fibrinolytic enzyme genes, swimming lane 4 are dna molecular amount standard (λ DNA/EcoRI+HindIII); Swimming lane 5 is the result of recombinant plasmid pUCm-T-EFE with NotI and SalI double digestion; Swimming lane 6 is the result of recombinant plasmid pUCm-T-EFE with the SalI single endonuclease digestion; Swimming lane 7 is the result of recombinant plasmid pUCm-T-EFE with the SphI single endonuclease digestion.

The enzyme of accompanying drawing 3 recombinant expression vector pPIC6 α A-EFE is cut qualification result

Swimming lane 1 is the PCR molecular weight standard; Swimming lane 2 is the PCR qualification result of recombinant expression vector; Swimming lane 3 is the result of recombinant expression vector with the EcoRI+XbaI double digestion; Swimming lane 4 is the result of recombinant expression vector with the SacI single endonuclease digestion; Swimming lane 5 is dna molecular amount standard (λ DNA/EcoRI+HindIII)

SDS-PAGE electrophoresis detection result behind the accompanying drawing 4 genetically engineered plasmin purifying

Swimming lane 1 is the small molecular weight protein standard; Swimming lane 2 is genetic engineering bacterium Pichiapastoris X-33 (pPIC6 α A-EFE) supernatant, i.e. the genetically engineered plasmin albumen behind the purifying.

Accompanying drawing 5 fibrin plate methods are measured the biologic activity of plasmin

A is the genetically engineered plasmin albumen behind the purifying; B is 10ul plasmin standard substance (10000U/ml); C is 10ul contrast bacterium Pichia pastoris X-33 (pPIC6 α A) supernatant purifying after product.

Embodiment

The invention will be further described below in conjunction with drawings and Examples, but not as the restriction to interest field of the present invention.

Substratum among all embodiment and molecular biology working method are familiar with by these those skilled in the art, can be with reference to " molecular cloning " (laboratory manuals such as Sambrook, the cold spring port, 1989) reach " fine works molecular biology experiment guide " (work such as U.S./F. Ao Sibai, Yan Ziying etc. translate, Beijing, Science Press, 1998).

The gene PCR amplification and the clone of embodiment 1. earthworm fibrinolysins

(1) amplimer is synthetic

According to the earthworm fibrinolysin cDNA sequences Design PCR oligonucleotide amplimer of having delivered among the GeneBank.Long 18 the Nucleotide ACATGGAACTTCCTCCCG of upstream primer, long 19 the Nucleotide ATCACCAACAACTAAACCG of downstream primer.Primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized, through the PAGE purifying.

(2) the total RNA of earthworm extracts

Get 3～5 earthworm adults, after the aqua sterilisa rinsing of handling with DEPC, be placed in the plate that is covered with wet filter paper hunger and spend the night, make it tell intravital to the greatest extent silt as much as possible.After cleaning up with DEPC water once more, in liquid nitrogen, be ground into powder, extract total RNA according to the product description of the RNeasyRMini Kit of QIAGEN company.The RNA solution that extracts is stored in-80 ℃, with standby.

(3) the synthetic cDNA article one chain of reverse transcription

Reverse Transcription Reaction test kit specification sheets according to Promega company carries out, and is synthetic cDNA first chain of primer with Oligo (dT) 20.The concrete operations step is as follows: following reagent is added one in the PCR reaction tubes that DEPC soaks and sterilising treatment is crossed: 25mMMgCl ₂12 μ l; Damping fluid 6 μ l; 10mM dNTP 6 μ l; Recombinant RNasinRibonuclease Inhibitor 2 μ l; AVM ThermoScript II 4 μ l; Oligo (dT) 206 μ l; The total RNA 15 μ l of earthworm; Sterilization distilled water 9 μ l.Reaction conditions is: 42 ℃ 1 hour; 95 ℃ 5 minutes; 3 ℃ 5 minutes.

(4) pcr amplification of goal gene

Get 8 μ L reverse transcription reaction products, carry out the PCR reaction according to the PCR reaction system parameter that provides in the Reverse Transcription Reaction test kit.Reactive component is: damping fluid 2.5 μ l; 25mM MgCl ₂1.5 μ l; 10mM dNTP 2 μ l; Upstream primer 1 μ l; Downstream primer 1 μ l; MRNA reverse transcription product 15 μ l; RT-PCR enzyme mixture 2 μ l; Sterilization distilled water 5 μ l.Its reaction parameter is: 95 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 20 circulations 2 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 10 circulations 2 minutes; 72 ℃ were extended 10 minutes again.1% agarose detects PCR result, and electrophoresis result as shown in Figure 1.

(5) recovery of target gene fragment

Reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim target gene fragment, its process is as follows: get 30 μ lPCR reaction product and add 1.4mlPB liquid (test kit provides), mixture moved in the adsorption column centrifugal 15 seconds, abandoned waste liquid; In adsorption column, add 400 μ lPB liquid, leave standstill 1 minute after, centrifugal 15 seconds, abandon waste liquid; Add 500 μ lW1 liquid (test kit provides) again, centrifugal 15 seconds, abandon waste liquid, repeat once; In adsorption column, add 300 μ l T1 liquid (test kit provides), leave standstill 1 minute after, centrifugal 30 seconds, collect centrifugate, be stored in-20 ℃.

(6) target gene fragment is cloned into carrier

Target gene fragment is cloned into the pUCm-T carrier, and this carrier is given birth to worker bio-engineering corporation available from Shanghai.Reactive component is as follows: PCR product 5 μ l; PUCm-T carrier 1 μ l; 10 * T4DNA ligase enzyme damping fluid, 1 μ l; T4DNA ligase enzyme 1 μ l; Deionized water 2 μ l; Cumulative volume is 10 μ l, and 4 ℃ of connections are spent the night.

The preparation of competent cell: the single JM109 bacterium colony of picking, being inoculated in 3ml does not contain in the LB substratum of penbritin, 37 ℃ of overnight incubation, get above-mentioned bacterium liquid next day is inoculated in the 50mlLB nutrient solution in proportion at 1: 100,37 ℃ of vibrations 3 hours treat that bacterium liquid OD value is at 0.6 o'clock, centrifugal 8 minutes of 4 ℃ of 5000rpm, abandon supernatant, precipitation 0.1M CaCl ₂Suspend, 5000rpm is centrifugal 8 minutes again, abandons supernatant, and precipitation is with an amount of 0.1M CaCl ₂Resuspended, in the rearmounted ice bath of packing 6 hours standby.

Connect the conversion of product: get above-mentioned connection product 5 μ l and added 100 μ l competent cell ice baths 60 minutes, 42 ℃ of heat-shockeds 90 seconds, put ice bath again 2 minutes, the LB substratum 0.9ml that adds no penbritin, cultivated 1 hour for 37 ℃, getting 200 μ l bacterium liquid adds to be coated with behind 40 μ l 20mg/ml 5-bromo-4 chloro-, 3 indoles (D-galactoside (X-gal)) and 4 μ l 0.1M isopropylthiogalactoside (IPTG) mixings and contains penbritin LB plate, cultivated 16 hours for 37 ℃, picking white colony rapid extraction plasmid carries out enzyme and cuts evaluation, and enzyme is cut product and detected through 1% agarose.The result as shown in Figure 2, its positive colony called after pUCm-T-EFE.

The quick a small amount of preparation of plasmid DNA: picking list colony inoculation contains the LB substratum of penbritin in 4ml, and 37 ℃ of shaking culture are spent the night.Bacterium liquid adds in the 1.5ml centrifuge tube, centrifugal 8 minutes of 5000rpm, abandon supernatant, precipitation is suspended in 15 μ l suspension (50mM glucose, 25mMTris-HCl, 10mMEDTA, PH8.0), add 200 μ l lysate (0.2MNaOH, 1%SDS), put ice bath after 5 minutes, add 150 μ l neutralizer (3.0MKAc, pH4.8), ice bath 10 minutes, centrifugal 10 minutes of 12000rpm abandons precipitation, on reset and add that equal-volume phenol/the chloroform extracting once, on reset and add 2 times of volume dehydrated alcohols, centrifugal 10 minutes of 12000rpm, throw out is washed 1 time with 70% ethanol, vacuum is drained and is added 20 μ l TE (10mMTris-HCl, 1mM EDTA, PH8.0 and 1 μ l RNaseA 10mg/ml) ,-20 ℃ are frozen standby.

(7) earthworm fibrinolysin cDNA sequencing

Extract the plasmid DNA dideoxy method and measure nucleotide sequence, sequencing is given birth to worker's biotechnology company limited by Shanghai and is finished, and sequencing primer is the M13 promoter primer.

Measurement result: long 747 Nucleotide of earthworm fibrinolysin cDNA sequence, the content of adenosine (A), cytidine(C (C), guanosine-(G) and thymidine (T) is respectively: A-185, C-197, G-189; T-176 (sequence No.1), 246 amino acid (seeing sequence No.2) of encoding.Gene mature peptide sequence is a 3-740 position Nucleotide, and all the other nucleotide sequences are non-coding region.This protein cDNA mature peptide terminator codon is positioned at gene 741-743 position, and termination codon is TAA.

(8) modification of earthworm fibrinolysin gene and transformation

Gene fragment involved in the present invention can be modified, and such as some nucleotide sequence of mutator gene, but does not influence its encoded protein matter aminoacid sequence, Can increase, lack amino acid involved in the present invention or nucleotide sequence, these are modified can not influence proteinic biochemical characteristic, higher structure and proteinic biological function, such as: can add secreting signal peptide at protein or polypeptide N-end or C-end, can also add that suitable joint (as 6 Histidines) is convenient to proteins extraction , purifying. can manually synthesize all or part of nucleotide sequence, synthetic nucleotide sequence sequence can be variant with sequence provided by the invention, but the present invention encodes a protein involved in the same protein sequence, which may be synthetic sequences are:...

Wherein m represents a or c; R represents a or g; W represents a or t; S represents c or g; Y represents c or t; K represents g or t; V represents a or c or g; H represents a or c or t; D represents a or g or t; B represents c or g or t; N represents g or a or t or c.

Embodiment 2. expresses the structure of the pichia pastoris gene engineering bacterial strain of fibrinolytic enzyme gene

(1) pcr amplification of earthworm fibrinolysin gene

With the pUCm-T-EFE plasmid is template, according to the design of the cloning site on EFE mature peptide and expression vector pPIC6 α A primer, upstream primer P1:C GAATTCCACCACCACCACCACCACGGTGGTGGTGACGACGACGACAAGATGGAACTTCCTCC CGGAACA (the underscore place is restriction endonuclease EcoRI site).Downstream primer P2:G TCTAGATTAGTTGTTGGTGATGATGTCGGTGATCCATGCAGCAT (the underscore place is restriction endonuclease XbaI site).Primer is synthetic by Shanghai Sangon bio-engineering corporation, and synthetic product is through the PAGE purifying.Reactive component is: damping fluid 2.5 μ l; 25mM MgCl2 1.5 μ l; 10mM dNTP 2 μ l; Upstream primer 1 μ l; Downstream primer 1 μ l; PUCm-T-EFE plasmid 1 μ l; LA Taq archaeal dna polymerase 2 μ l; Sterilization distilled water 19 μ l.Its reaction parameter is: 95 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 30 circulations 2 minutes; 72 ℃ were extended 10 minutes again.

(2) the PCR product cloning is to the T carrier

Reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim target gene fragment, extension amplification outcome obtains recombinant vectors pGEM-T-EFE2 in pGEM-T carrier (Promega company), and this recombinant vectors is changed in the e. coli jm109.

(3) structure of shuttle expression plasmid

Extract recombinant vectors pGEM-T-EFE2, with EcoRI and XbaI double digestion to obtain purpose fragment EFE; Same enzyme is cut pPIC6 α A empty carrier and make it dephosphorylation under the CIAP effect.With the EFE that obtains behind linearizing pPIC6 α A empty carrier behind the dephosphorylation and the double digestion under the effect of T4DNA ligase enzyme 4 ℃ spend the night, to obtain recombinant expression vector pPIC6 α A-EFE, this recombinant vectors is changed in the e. coli jm109 increases.Picking white colony rapid extraction plasmid carries out enzyme and cuts evaluation, and enzyme is cut product through 1% agarose detected result as shown in Figure 3.

(4) transform pichia spp X-33

Method is with reference to Invitrogen company expression vector specification sheets (Pichia expressionvectors for selection on blasticidin and purification of recombinant proteinsmanual).

The single bacterium colony of picking pichia spp X-33 is in 5 milliliters of YPD substratum of inoculation, 30 ℃ of 250rpm shaking table overnight incubation, by 1% inoculum size inoculation 200mlYPD liquid nutrient medium, 30 ℃ of 250rpm shaking tables were cultivated 3-5 hour, cell concentration reaches 0.8-1.0, centrifugal 10 minutes of 1500g, precipitation is resuspended with 10ml sterilization distilled water, centrifugal 10 minutes of 1500g, precipitation is resuspended among the LiCl of 0.4ml 100mM and is transferred in the 1.5ml centrifuge tube of sterilization, and centrifugal 15 seconds of 12000g abandons supernatant, the LiCl that adds 0.1 6ml 0.1M is resuspended, and is standby.

Extract recombinant plasmid pPIC6 α A-EFE, be cut into linearity with the SacI enzyme, same enzyme is cut empty plasmid pPIC6 α A in contrast.

Get the above-mentioned competent cell of 50 μ l, centrifugal 15 seconds of 12000g carefully draws supernatant with pipettor, adds following component in order successively: 240 μ l 50%PEG; 36 μ l 1M LiCl; 25 μ l 2mg/ml salmon sperm dnas; 50 μ l (5-10 μ g) linearization recombinant plasmid pPIC6aA-EFE.With linearization empty plasmid pPIC6 α A in contrast.Concuss made complete mixing in one minute, 30 ℃ left standstill 30 minutes, 42 ℃ heat shock 20-25 minute, centrifugal 15 minutes of 6000-8000rpm, precipitation is resuspended with the 1mlYPD substratum, cultivates 1-4 hour, and gets 25-100 μ l figure and be distributed in the YPD solid medium that contains 300 μ g/ml blasticidins (Blasticidin) for 30 ℃, cultivated 2-3 days for 30 ℃, up to there being single bacterium colony to occur.

(5) screening positive transformant

Picking 10-20 single bacterium colony in the YPD liquid nutrient medium 30 ℃, 250rpm shaking table were cultivated until logarithmic growth late period, extracted the PCR that genome carries out recon and identified.

The pastoris genomic dna preparation method: it is centrifugal 10 minutes to get 1.5ml nutrient solution 4000r/ minute.Thalline is with the liquid nitrogen broken wall of milling.Add 7mL DNA damping fluid (100mmol/L Tris--HCl, pH8.0,10mmol/L EDTA, 1%SDS) mixing, 65 ℃ of insulation 1h.10000r/ minute centrifugal 15 minutes.Supernatant phenol extracting 2 times, each 5000r/ minute centrifugal 7 minutes.Use the chloroform extracting again 1 time, 5000r/ minute centrifugal 7 minutes.(add the 0.3mol/L sodium-acetate, pH5.2), 10000r/ minute centrifugal 10 minutes with 2.5 times of ethanol sedimentations.70% washing with alcohol, hair dryer dries up, and adds TE or aqueous suspension.

The PCR authentication method: the universal primer that primer provides for Invitrogen company expression vector, its sequence are upstream: GACTGGTTCCAATTGACAAGC; Downstream: GCAAATGGCATTC TGACATCC.PCR carries out according to a conventional method, and amplification condition is: 94 ℃ 45 seconds, 54 ℃ 1 minute, 72 ℃ 1 minute, 40 circulations.Whether amplified production is correct with detecting its size with 0.8% agarose, and the result as shown in Figure 3.

The positive colony called after Pichia pastoris X-33 (pPIC6 α A-EFE) that is obtained, this bacterial strain at China's typical culture collection center deposit number is: CCTCC M204005.

The proteic expression of embodiment 3. genetically engineered plasmins, separation, purifying;

(1) fermenting process of pichia pastoris engineered strain X-33 (pPIC6 α A-EFE):

A. single bacterium colony of picking reorganization bacterium is gone into 20mlYPD liquid nutrient medium (1% yeast extract, 2% peptone, 2% glucose), and 30 ℃, 250rpm are cultivated and activated;

B.16 30 ℃ of the bacterium liquid switching 500ml MGY liquid nutrient medium liquid nutrient mediums, the 250rpm that get after individual hour after 10ml activates cultivate enrichment;

C. the bacterium liquid of enrichment after 24 hours is through centrifugal collection thalline (1500g, 10 minutes, 4 ℃);

D. the thalline of collecting is carried out abduction delivering with being resuspended in 1000ml MM liquid nutrient medium, it is 0.5% that every 24h adds methyl alcohol to final concentration;

E. induce centrifugal collection fermented liquid supernatant behind the 96h (30000g, 20 minutes, 4 ℃);

(2) sample preparation and Ni2+ column chromatography process:

Fermented liquid supernatant can dialysis tubing gets the column chromatography sample through following steps;

A.PEG20000 (Macrogol 2000 0) buries dialysis tubing and concentrates, and concentrates about 10 times;

B. distilled water is cleaned the dialysis tubing outside surface, the dialysis tubing that concentrated broth is housed is immersed in the 1 * BINDING BUFFER (5mMimidazole+0.5M NaCl+20mM TrisHCl PH=7.9) that equates with the original fermented solution volume spends the night, with balance fermented liquid PH, ionic concn;

C. with step a, be concentrated into volume and be about 1/10 of original fermented solution;

D. in dialysis tubing, add 1 * BINDING BUFFER, with 4 times of the diluted samples that concentrated;

E. with step c;

F. from dialysis tubing, take out concentrated broth, and the remaining sample in the dialysis tubing is cleaned, collect with a small amount of 1 * BINDING BUFFER;

G. with concentrated broth high speed centrifugation (15000RPM, 15 minutes), the supernatant after centrifugal filters the sample that obtains treating column chromatography with 0.45um millipore filtration (MILLIPORE company).

Annotate: above step is all carried out at 4 ℃.

The Ni2+ column chromatography:

The a.Ni2+ post is with 10 1 * BINDING BUFFER, 10 sterile water wash post beds, replenish Ni2+ with 10 1 * CHARGE BUFFER (5Mm NiSO4), use 10 1 * BINDING BUFFER to clean the post bed again, prepare beginning adsorption chromatography (1 promptly is the volume of medium in the post);

B. the sample that concentrated passes through the Ni2+ post in a looping fashion with peristaltic pump, makes to have 6 * HIS sample and fully combine with Ni2+, and collect and the reacted sample of Ni2+ (leakage liquid) from the Ni2+ column outlet back of spending the night;

C. make 20 1 * BINDING BUFFER flow through the Ni2+ post, stay small amount of sample in the post with flushing;

D. use on the imidazoles eluant solution Ni2+ post of 5mM-100mM non-target protein (wash-out bed number and elute soln concentration are looked total protein content and non-target protein and Ni2+ binding ability and changed flexibly) to annotate: if by last surface compositions, then wash-out foreign protein solution is the 60mM imidazoles.

E. use 1 * ELUTE BUFFER wash-out target protein, substep is collected, and per 1 (actual in column volume) volume is a pipe, the distribution of SDS-PAGE check target protein in elutriant.The Ni2+ post continues wash-out with 1 * ELUTE BUFFER (1M imidazole+0.5M NaCl+20mMTrisHCl PH=7.9), wash whole albumen in the post bed off, use the Ni2+ ion on 1 * STRIPBUFFER (100Mm EDTA+0.5M NaCl+20mM TrisHCl PH=7.9) flush away pillar again.

(3) enteropeptidase degraded (Enterokinase Cleavage) in the ox

The plasmin of Ni2+ column purification after 6-8 hour, is added enteropeptidase (Sigma company) in the highly purified ox with 1000 times of volume buffer A (25mMHepes/pH8.0,5mM EDTA) dialysis, carry out DeR, reaction conditions is as follows:

1 part of enzyme: 2000 parts of substrates (weight ratio)

37 ℃ of reactions add the P-Aminobenzawidine termination reaction after 15-20 hour

(4) QAE-Toyopearl 550C anion-exchange resin column chromatography

The preparation of pillar: behind the mixed with resin homogeneous, take out about 50 milliliters resin and add buffer A (25mM Hepes/pH8.0,5mM EDTA); After the resin precipitated, remove supernatant liquor, add the equal-volume buffer A, behind the mixed with resin homogeneous, natural sedimentation repeats 3 times like this.Fix a chromatography column and (after 1.5 * 30cm), block column outlet, in the post of subsequently resin of buffer A processing being packed into.Open then below the pillar after the outlet, make that damping fluid oozes pillar in the resin, use 200 milliliters of buffer A then, under 4 ℃ of following conditions, continue the balance chromatography column.

The preparation of sample:, in the semi-permeable membranes dialysis tubing of packing into,,, carry out dialyzed overnight 4 ℃ of following conditions with 2000 milliliters buffer A with the sample liquid of 10 milliliters of Ni2+ affinitive layer purifications.

Purge process: (temperature operation below 4 ℃): dialyzed sample is added chromatography column, collect effluent liquid; Buffer A with 3 * column volume (150 milliliters) is washed pillar; With buffer A and buffer B (25mM Hepes/pH8.0,300mM NaCl, 5mM EDTA), form device, the NaCl concentration gradient buffer solution elution chromatography column of preparation by a linear gradient.(200 milliliters of buffer A; 200 milliliters of buffer B); Elution samples is collected with every part 1 ml volumes step by step with the automatic sample collector.With OD280nm UV-light monitoring albumen elution peak.After confirming that with SDS-PAGE main protein peak sample hose contains plasmin, these samples are mixed.

(5) the SDS-PAGE electrophoretic analysis of purified product

A. recording of gel: with reference to referring to " molecular cloning " (laboratory manuals such as Sambrook, the cold spring port, 1989) method, preparation 8% separation gel solution 15ml adds catalyzer TEMED (N after mixing each composition successively, N, N ', N ' Tetramethyl Ethylene Diamine), mixing is opened encapsulating immediately, front cover layer 0.1%SDS (sodium dialkyl sulfate) covers liquid level, 37 ℃ of following polyase 13s 0 minute.Liquid after the polymerization fully on the emptying gel is in kind irritated and is concentrated glue, inserts comb, avoids producing bubble.

B. go up sample and electrophoresis: induce at yeast to add equal-volume 2 * sds gel sample loading buffer in the fermented liquid, in 100 ℃ of heating sex change in 3-5 minute, application of sample in order, in all no wells, add isopyknic sample loading buffer at last, advance separation gel with 100V electrophoresis to bromjophenol blue, improve voltage to 150V, finish electrophoresis (needing 4hr approximately) when bromjophenol blue arrives precontract 1cm place, bottom.

C. gel-colored: referring to " protein purification and identification experiment guide " (work such as U.S. D.R. Ma Xieke, the thick plinth of Zhu etc. is translated Science Press, 2002).Under the room temperature, gel slowly shakes on rotation platform, and carries out the following step.

Gel is soaked 2 times each 15 minutes in 50% formaldehyde solution; In 5% formaldehyde, soaked 10 minutes; Wash rapidly 3 times with distilled water; In 10 μ mol/L dithiothreitol (DTT) (DTT) solution, soaked 20 minutes; At 0.1%AgNO ₃(0.1%, advance to soak 20 minutes in w/v); Wash fast 1 time with distilled water earlier, use developing solution (15g yellow soda ash is dissolved in the 500ml water, with preceding adding 250 μ l 37% (w/v) formaldehyde solutions) to wash fast then 2 times, be no more than 15 seconds at every turn; In developing solution, soaked several minutes, until showing protein belt; Add solid citric acid (every 200ml developing solution adds about 10g citric acid), stop to develop; Cover gel with aluminium foil and continue to soak 10 minutes, thoroughly clean with distilled water then.

D. electrophoresis result: as shown in Figure 4.

Embodiment proteic functional study of 4. genetically engineered plasmins and determination of activity

Adopt the fibrin plate method to measure the biologic activity of plasmin, by determination of activity, the activity unit of fermented liquid surpasses 3E+06U/L.Fig. 5 is for surveying periodic fibrinolytic circle, and detection method can be with reference to the method [Hao Suli, Shen Jia, the research of Lumbrukinase biological activity assay method, 1996 the 10th the 6th phases of volume of Chinese Pharmaceutical Affairs] of Hao Suli etc., and concrete operation method is as follows.

(1) bed board: (it is an amount of to take by weighing Fibrinogen to get scleroproein stoste.The solution that contains the 4.2mg coagulable protein in being made into every milliliter with the Tris-HCl damping fluid) 12.7ml, proplasmin liquid (getting the solution that proplasmin contains lu in being made into every milliliter with the Tris-HCl damping fluid) 0.67ml, agar solution (take by weighing agar 0.8g and add Tris-HCl damping fluid 100ml, heating for dissolving) 13.4ml. zymoplasm liquid (get zymoplasm and be made into the solution that contains 70B.PU with the Tris-HCl damping fluid) 0.67ml.Mixing. in the synthetic glass dish of the diameter of falling people 9cm.Room temperature is placed half an hour.Coagulating good plate does not evenly have bubble, is creamy white.

(2) point sample: it is an amount of to take by weighing the plasmin standard substance.Contain 2200,1100,550,270 four concentration in being made into every milliliter with the Tris-HCI damping fluid.Draw 10 μ l reference liquid points on plate with microsyringe.Should leave a determining deviation between point and point, cover glass then and pull.Putting 37 ℃ of constant temperature and cultivated 18 hours, remove lid, with the perpendicular diameter of its solusphere of vernier caliper measurement, is ordinate with two diameter products.Standard substance unit is an abscissa, mapping drawing standard curve or ask regression equation on log-log graph paper.The unit of trial-product is taken in the standard curve range.With the vertical two diameter products of trial-product solusphere, push away at plasmin mark and to look into unit of activity on the curve.

(3) detected result: two diameter products are ordinate, and standard substance unit is an abscissa, and regression equation is lgY=-1.573+0.544 lgX, and Y is the perpendicular diameter product of solusphere, and unit is cm ², X is an activity unit.The result shows that the activity unit of fermented liquid is 3.39E+06U/L.

Embodiment 5. genetically engineered plasmins prepare thrombolysis class medicine

With the plasmin of purifying damping fluid (23 gram glycine/liter, 1.6 gram Sodium phosphate dibasics/liter, 0.55 gram SODIUM PHOSPHATE, MONOBASIC/liter, pH7.0) after the dialysis, contain 1 milligram of concentration to every milliliter with ultrafiltration and concentration, sterilize with the membrane filtration in 0.22 μ m aperture.Then, about 1 ml sample liquid (about 300000 unit of activity) bottling, finished product is made in lyophilize.During use, sample is first-selected with 1 milliliter of medical water dissolution, lives and the injection patient as biometric.

Genetically engineered plasmin sample specification is: 300000U genetically engineered plasmin; The 23mg glycine; 1.6mg Sodium phosphate dibasic; 0.55mg SODIUM PHOSPHATE, MONOBASIC.Be equipped with 5 milliliters of medical grade water in addition.

The application of embodiment 6. genetically engineered plasmin protein formulations---mouse anti-freezing experiment

Method is carried out [Chen Jiankang, Wang Lei, Li Ke, Guo Feng, Yang Jun, the anticoagulant and thrombolytic effect of ginkgolic flavone glycoside and genetically engineered plasmin, heart journal 2001,13 (4): 308-309] with reference to the document of Chen Jiankang etc.

Select 30 of Male Kunming strain mice, be divided into 3 groups at random, body weight 20 ± 2g.Successive administration 3 days, each group is injected 5000U/kg genetically engineered plasmin respectively, 0.2mg acetylsalicylic acid, control group injecting normal saline 0.5mL.2 hours begin to test after the medication in the 3rd day.1cm is cut off in the mouse tail tip, and 10s inhales serum deprivation with filter paper at interval, must not push the record mouse bleeding time.

Experimental result (seeing Table 1) shows that genetically engineered plasmin group and acetylsalicylic acid group bleeding time all prolong, and genetically engineered plasmin medication group and control group relatively have the significance meaning, illustrates that the genetically engineered plasmin has good blood coagulation resisting function.

Table 1 experiment mice afterbody bleeding time synopsis

Group	The example number	The average bleeding time (second)
			Control group	??????10	?????89±27
Genetically engineered plasmin group	??????10	?????237±46
			The acetylsalicylic acid group	??????10	?????198±32

(comparing ρ＜0.05 with control group)

The application of embodiment 7. genetically engineered plasmin protein formulations---to the provide protection of rat experiment cerebral ischemia

(1) preparation of the inaccessible model of intraluminal middle cerebral artery occlusion in rats (MCAO)

The method reference literature carries out [Tamura A, Graham DI, McCulloch J, Teasdale GM., Focal cerebral ischaemia in the rat:l.Description of technique and earlyneuropathological consequences following middle cerebral artery occlusion.J CerebBlood Flow Metab.1981; 1 (1): 53-60.] [Feng Yipu, intercalation beautiful spring-time, mouse transience MCAO model, Zhang Juntian, modern pharmacology experimental technique, combined publication society of China Concord Medical Science University of Beijing Medical University, 1998].

With 10% Chloral Hydrate (300mg/kg) ip anesthetized rat, face upward the position and fix neck median incision, expose right carotid, free successively by the crotch head-end, ligation is also cut off artery and superior thyroid artery under the occipital bone, in ligation of external carotid artery far-end and cut-out, free standby.Separate internal carotid artery, make a call to one with silk thread at the external carotid artery root and release, folder closes arteria carotis communis and internal carotid artery, and, slowly go into the cranium direction and advance through external carotid artery trunk otch with fine rule to internal carotid artery, be mark with the arteria carotis communis crotch, advance 20 millimeter.Tightening the external carotid artery root releases.After one hour, carefully extract fine rule, tighten the artery stump,, finish the arteria cerebri media embolism with surgery non-traumatic sewing thread skin suture.The postoperative room temperature is controlled at 22 ± 1 ℃ in the art.

Male Wister rat (50 ± 50g), be divided into 4 groups at random, promptly sham operated rats, model group, genetically engineered plasmin (2500U/kg) are organized, genetically engineered plasmin (5000U/kg) group.Wherein sham operated rats exposes left middle cerebral artery and left common carotid artery, not ligation.Each administration group is in preceding 10 minutes difference of ligation intravenous injection genetically engineered plasmin, and model group then gives physiological saline 0.5ml.

(2) the genetically engineered plasmin is to the influence of rats with cerebral ischemia survival time

Experimental result sees Table 2

Table 2 genetically engineered plasmin is to the influence of rats with cerebral ischemia survival time

Tahle?2?Effect?of?Lumbroinase?on?survival?time?in?ischemic?mice(x±S)

Divide into groups routine number (n) survival time (min)

Control group 15 9.5 ± 4.1

Sham operated rats 15 8.2 ± 5.4

The genetically engineered plasmin

15?????????????????11±8

(2500U/kg)

The genetically engineered plasmin

15?????????????????68±28 ²

(5000U/kg)

Compare ρ＜0.001 with control group

(3) the genetically engineered plasmin to rats with cerebral ischemia arteriae cerebri 24h after the influence of infarct size and brain water content, experimental result sees Table 3

Table 3 genetically engineered plasmin to the inaccessible 24h of mouse brain medium sized artery after the water content of infarct size and tissue

Table?3?Inhibitory?effect?of?lumbrokinase?on?infarction?area?and?water?content

in?brain?ofter?24h?MCAO?in?rats??(x±S)

Routine number (n) infarct size (1/2) water content (mL/g) of dividing into groups

Sham operated rats 8 1.22 ± 0.05 3.74 ± 0.09

Model group 8 7.17 ± 0.37 ^c4.99 ± 0.45 ^c

Genetically engineered fibrinolytic 8 5.77 ± 0.22 ^Bd4.77 ± 0.34

Enzyme (2500U/kg)

Genetically engineered fibrinolytic 8 4.07 ± 0.65 ^b4.41 ± 0.56 ^a

Enzyme (2500U/kg)

Compare a: ρ＜0.05, b: ρ＜0.01 with model group; Compare c: ρ＜0.001 with sham operated rats; Compare d: ρ＜0.05 with model+genetically engineered plasmin 2500U/kg and 5000U/kg group.

(4) the genetically engineered plasmin is to the influence of intraluminal middle cerebral artery occlusion in rats neurological dysfunction after inaccessible 24 hours

Above-mentioned 3 treated animals were observed the situation of its neurological dysfunction in 24 hours after the arteria cerebri media ligation, neurological dysfunction is divided into 5 grades: the I level is remembered 1 fen for the infringement of impassivity system function; The II level only has slight dysfunction, need not help though show as when walking, and gait is owed surely, and brain injury offside forelimb flexing when carrying tail is remembered 2 fens; The III level has the moderate dysfunction, shows as under helping to stand, and is not lasting, and brain injury offside forelimb flexing when carrying tail is turn-taked to the operation side, remembers 3 fens; The IV level is the severe dysfunction, shows as consciously to change, and is drowsiness, slow in reacting, can not stand, and remembers 4 fens; The V level is dead, remembers 5 fens.This method is a thrombolysis class medicine measuring method commonly used, can be with reference to [Lawner PM, Laurent JP, Simeone FA, Fink EA, Effect ofextracranial-intracranial bypass and pentobarbital on acute stroke in dogs.JNeurosurg.1982 Jan; 56 (1): 92-96.] [Diaz FG, Mastri AR, Ausman JI, Chou SN., JNeurosurg.Acute cerebral revascularization after regional cerebral ischemia in thedog., 1979 Nov; 51 (5): 644-653.].

Experimental result sees Table 4.

Table 4 genetically engineered plasmin to the inaccessible 24h of mouse brain medium sized artery after the influence of neurological dysfunction

Table?4??Influence?of?lumbrokinase?on?neurologic?deficits(ND)scores

produced?by?24h?MCAO?in?rats(x±S)

Divide into groups routine number (n) neurological dysfunction scoring

Model group 8 4.21 ± 0.3 ^d

Sham operated rats 8 1.45 ± 0.5

Genetically engineered fibrinolytic 8 2.16 ± 0.5 ^b

Enzyme (2500U/kg)

Genetically engineered fibrinolytic 8 1.67 ± 0.3 ^b

Enzyme (5000U/kg)

Compare b: ρ＜0.01 with model group; Compare d: ρ＜.001 with sham operated rats.

(5) the genetically engineered plasmin is to cerebral tissue Total antioxidant capacity, NO content and the active influence of NOS after inaccessible 24 hours of mouse brain medium sized artery

Experimental result sees Table 5.

Table 5 genetically engineered plasmin to the inaccessible 24h of mouse brain medium sized artery after the cerebral tissue Total antioxidant capacity, an oxygen

Change the influence of nitrogen content and nitric oxide synthase activity

Table?5?Effect?of?Lumbrokinase?on?T-AOC.NO?content?and?NOS?activity?after

24h?MCAO(n＝6，x±S))

Total antioxidant capacity NO NOS

Grouping

(kU/g)???????(μmol/g)??????(kU/g)

Normal control 3.17 ± 0.56 1.11 ± 0.20 0.39 ± 0.06

Model group 1.20 ± 0.15 ^d3.20 ± 0.54 ^d0.84 ± 0.79 ^d

Sham operated rats 3.16 ± 0.58 1.09 ± 0.20 0.41 ± 0.04

The genetically engineered plasmin

1.71±0.24 ^af??3.20±0.38????0.75±0.15

(2500U/kg)

The genetically engineered plasmin

2.20±0.18 ^be??2.80±0.35 ^a??0.74±0.09 ^a

(5000U/kg)

Compare d: ρ＜0.001 with sham operated rats; Compare a: ρ＜0.05, b: ρ＜0.01 with model group; Organize relatively with model+genetically engineered plasmin 5000U/kg, e: ρ＜0.05,, f: ρ＜0.01.

Above-mentioned experimental result shows the reduction that the genetically engineered plasmin can dwindle infarct size, alleviate cerebral tissue Total antioxidant capacity behind cerebral edema and the ischemic, lowers NO content, reduces the NOS activity, so cerebral ischemia is played a protective role.

Application---the thrombolytic effect of embodiment 8. genetically engineered plasmin protein formulations

Rabbit external jugular vein thrombus method is easy and simple to handle, does not need specific apparatus, all has the research department to adopt at home and abroad, is the feasible method of research medicine thrombus dissolving effect.This method can be with reference to [Zhao Weiming, Xu Chaoqian, He Shuzhuan, Wang Ling, the experimental study of genetically engineered plasmin thrombolytic effect: Harbin Medical University's journal, 2002 (36) 3:183-185] [Xu Shuyun, Bian Rulian, the Chen Xiu chief editor. pharmacological experimental methodology. Beijing: People's Health Publisher, 1982.] [Zhu Yan, what is fair and unbiased, Sun Kedi, Wang Shaofei, the genetically engineered plasmin is to the influence of experimental thrombosis, China Medicine University's journal, 2000,31 (1): 50～52.]

(1) makes neck artery-vein bypass experimental model

Experimental rabbit with 20% urethane 5ml/kg auricular vein injecting anesthetic, is carried on the back the position and fixed, peel off tracheae, insert a plastic casing (tracheal secretion can pass through this sleeve pipe sucking-off for a long time), separate left side external jugular vein and right carotid.Put into 4 trumpeter's art silk threads of a segment length 6cm in three sections polyethylene tube stage casings, be full of heparin-saline solution (50U/ml) in the polyethylene tube: after inserting left external jugular vein with an end of polyethylene tube, accurately inject heparin-saline solution (50U/ml) anti-freezing by polyethylene tube, and then the other end of polyethylene tube inserted right common carotid artery, open bulldog clamp, blood flow in the polyethylene tube from right common carotid artery, return left external jugular vein, form the bypass of neck artery-vein, after keeping 3h, get blood, middle Herba Clinopodii takes out silk thread rapidly and weighs.

(2) experiment grouping and route of administration

36 of rabbit, body weight 2.5 scholar 0.2kg are divided into 6 groups at random, are respectively dosage group in model group, physiological saline control group, genetically engineered plasmin small dose group, the genetically engineered plasmin, the heavy dose of group of genetically engineered plasmin, urokinase positive controls.

It is as follows that each organizes concrete route of administration: model group: the neck artery-vein bypass model of manufacturing; Physiological saline control group: auricular vein equal-volume injecting normal saline; Genetically engineered plasmin small dose group: auricular vein injection genetically engineered plasmin 1250U/kg; Dosage group in the genetically engineered plasmin: auricular vein injection genetically engineered plasmin 2500U/kg; The heavy dose of group of genetically engineered plasmin: auricular vein injection genetically engineered plasmin 5000U/kg; Urokinase positive controls: auricular vein injection urokinase 2 * 10 ⁴U/kg.

Medication: after the neck artery-vein bypass modelling, physiological saline control group, genetically engineered plasmin small dose group, middle dosage group, heavy dose of group and urokinase positive controls are set up back 15min administration respectively at bypass, observe 3h.

(3) thrombolytic effect

Adopt rabbit artery-vein bypass thrombotic model, measure the thrombolytic effect (wet weight of thrombus, dry weight) of earthworm fibrinolysin; Heart extracting blood is measured the influence of earthworm fibrinolysin to plasma fibrin enzyme (FIB), blood plasma euglobulin lysis time (ELT), fibrin split product biochemical indicators such as (FDP).

Experimental result sees Table 6 and table 7.In this experiment in the genetically engineered plasmin dosage group and heavy dose of group ELT is significantly shortened, illustrate that the genetically engineered plasmin has activated fibrinolytic system.Genetically engineered plasmin small dose group, middle dosage group are compared with model group with heavy dose of group in this experiment can obviously increase FDP content, illustrates that the genetically engineered plasmin can make the fibrinolytic of blood system strengthen.In this experiment, FIB does not have obvious variation, shows that the genetically engineered plasmin is difficult for producing hemorrhage side reaction.

Table 6 genetically engineered plasmin is to the influence of ETL FDP numerical value and FIB content

ELT(min)????FDP(ng·L ^-1)

Group FIB (gL ^-1)

＜120????≥130????＜5????5-20????≥20

Model group 0 7.9 8.1 00 4.60 ± 2.89

Physiological saline group 0 7.9 8.1 00 5.90 ± 2.98

The genetically engineered plasmin is little by 0 7.8 4.9 0 2.5 5.34 ± 2.80 ^☆

Dosage group (1250U/kg)

In the genetically engineered plasmin

4.1 _·?4.1 ^·????1.1 ^·????4 ^·??????????3 ^·???????3.75±1.84 ^☆

Dosage group (2500U/kg)

The genetically engineered plasmin is big

5 ^·???3 ^·??????????1 ^·????????1 ^·??????????6 ^·???????3.00±1.93 ^☆

Dosage group (5000U/kg)

Urokinase group 5 3 0 1 7 2.60 ± 1.02 ^☆

ELT, FDP, compare with the physiological saline group through rank test all in routine number, are ρ＜0.01; FIB content is checked through q, and ☆ and physiological saline group be ρ＜0.05 relatively.

The influence that table 7 genetically engineered plasmin forms the rabbit thrombus in vivo (x ± S)

Group example dosage weight in wet base (ng) dry weight (ng) inhibiting rate

Number (%)

Model group 8 70.25 ± 2.04 22.01 ± 1.83

Heavily manage salt solution group 8 85.31 ± 3.61 28.25 ± 1.02

Genetically engineered fibrinolytic 8 1250U/kg 63.58 ± 9.60 ^{△ △}19.55 ± 3.67 ^{△ △}24.5

The enzyme small dose group

Genetically engineered fibrinolytic 8 2500U/kg 39.00 ± 5.9 ^{△ △ ☆}14.26 ± 1.05 ^{△ △}53.6

Dosage group in the enzyme ^☆

Genetically engineered fibrinolytic 8 5000U/kg 29.49 ± 4.58 ^{△ △}12.21 ± 2.01 ^{△ △}65.3

The heavy dose of group of enzyme

Urokinase group 82 * 10 ⁴U/kg21.11 ± 5.48 ^{△ △}8.91 ± 2.07 ^{△ △}75.1

Compare ρ＜0.01 with model group; △ △ and physiological saline group be ρ＜0.01 relatively; The large, medium and small dosage group of ☆ relatively; ρ＜0.05.

SEQUENCE?LISTING

NO：1

＜110〉Green Life Lab Co Ltd

＜120〉a kind of earthworm fibrinolysin gene and engineering strain and structure and application

＜130〉Lumbricidae powder Lumbricus earthworm (Lumbricus rubellus) fibrinolytic enzyme gene Nucleotide

Sequence

<160>??1

<170>??PatentIn?version?3.1

<210>??1

<211>??747

<212>??DNA

<213>??Lumbricus?rubellus

<220>

<221>??cDNA

<222>??(3)..(740)<223>

<400>??1

acatggaact?tcctcccgga?acaaaaattg?tcggaggaat?tgaagctaga?ccatacgagt?????60

tcccatggca?ggtgtccgtc?cgaaggaaat?cttccgattc?ccatttctgc?ggaggtagca????120

tcatcaacga?tcgttgggtt?gtctgcgctg?ctcactgcat?gcagggagag?gcccccgctc????180

tggtttcatt?ggtcgtgggt?gagcacgaca?ggagtgcagc?gagtacagta?cgtcagactc????240

atgacgttga?tagcatcttc?gttcacgagg?actacaacac?aaatacccta?gagaacgacg????300

tttctgtcat?caagacatct?gttgccatca?ctttcgacat?caacgttggt?ccaatctgcg????360

ccccagatcc?ggctaacgac?tacgtctacc?gtaagagcca?gtgttccgga?tggggaacta????420

tcaattcagg?tggaatctgc?tgtcccaacg?ttctgcgata?cgtgacgctg?aatgacacaa????480

ccaaccaata?ctgcgaagat?gtatacccac?taaattcaat?ctacgacgat?atgatttgcg????540

cgtcggacaa?cactgggggt?aacgacagag?actcctgcca?gggtgactcc?ggcggccctc????600

tgagcgtcaa?ggatggtagt?ggaatcttca?gcctgattgg?tattgtgtct?tggggaattg????660

gttgcgcttc?tggctatcca?ggagtctact?cccgcgtcgg?attccatgct?gcatggatca????720

ccgacatcat?caccaacaac?taaaccg????????????????????????????????????????747

SEQUENCE?LISTING

NO：2

＜110〉Green Life Lab Co Ltd

＜120〉a kind of earthworm fibrinolysin gene and engineering strain and structure thereof and application＜130 〉

Lumbricidae powder Lumbricus earthworm (Lumbricus rubellus) fibrinolytic enzyme gene aminoacid sequence

<160>??1

<170>??PatentIn?version?3.1

<210>??1

<211>??246

<212>??PRT<213>??Lumbricus?rubellus

<220><221>??PEPTIDE

<222>??(1)..(246)

<223>

<400>??1

Met?Glu?Leu?Pro?Pro?Gly?Thr?Lys?Ile?Val?Gly?Gly?Ile?Glu?Ala?Arg

1????????????????5??????????????????10??????????????????15

Pro?Tyr?Glu?Phe?Pro?Trp?Gln?Val?Ser?Val?Arg?Arg?Lys?Ser?Ser?Asp

20??????????????25??????????????????30

Ser?His?Phe?Cys?Gly?Gly?Ser?Ile?Ile?Asn?Asp?Arg?Trp?Val?Val?Cys

35??????????????????40??????????????????45

Ala?Ala?His?Cys?Met?Gln?Gly?Glu?Ala?Pro?Ala?Leu?Val?Ser?Leu?Val

50??????????????????55??????????????????60

Val?Gly?Glu?His?Asp?Arg?Ser?Ala?Ala?Ser?Thr?Val?Arg?Gln?Thr?His

65??????????????????70??????????????????75??????????????????80

Asp?Val?Asp?Ser?Ile?Phe?Val?His?Glu?Asp?Tyr?Asn?Thr?Asn?Thr?Leu

85??????????????????90??????????????????95

Glu?Asn?Asp?Val?Ser?Val?Ile?Lys?Thr?Ser?Val?Ala?Ile?Thr?Phe?Asp

100?????????????????105?????????????????110

Ile?Asn?Val?Gly?Pro?Ile?Cys?Ala?Pro?Asp?Pro?Ala?Asn?Asp?Tyr?Val

115?????????????????120?????????????????125

Tyr?Arg?Lys?Ser?Gln?Cys?Ser?Gly?Trp?Gly?Thr?Ile?Asn?Ser?Gly?Gly

130?????????????????135?????????????????140

Ile?Cys?Cys?Pro?Asn?Val?Leu?Arg?Tyr?Val?Thr?Leu?Asn?Asp?Thr?Thr

145?????????????????150?????????????????155?????????????????160

Asn?Gln?Tyr?Cys?Glu?Asp?Val?Tyr?Pro?Leu?Asn?Ser?Ile?Tyr?Asp?Asp

165?????????????????170?????????????????175

Met?Ile?Cys?Ala?Ser?Asp?Asn?Thr?Gly?Gly?Asn?Asp?Arg?Asp?Ser?Cys

180?????????????????185?????????????????190

Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ser?Val?Lys?Asp?Gly?Ser?Gly?Ile

195?????????????????200?????????????????205

Phe?Ser?Leu?Ile?Gly?Ile?Val?Ser?Trp?Gly?Ile?Gly?Cys?Ala?Ser?Gly

210?????????????????215?????????????????220

Tyr?Pro?Gly?Val?Tyr?Ser?Arg?Val?Gly?Phe?His?Ala?Ala?Trp?Ile?Thr

225?????????????????230?????????????????235?????????????????240

Asp?Ile?Ile?Thr?Asn?Asn

245

Claims (7)

1, a kind of earthworm fibrinolysin gene, it comprises the nucleotide sequence shown in the SEQ ID NO:1.

2, a kind of earthworm fibrinolysin albumen of the claim 1 of encoding, it comprises the aminoacid sequence shown in the SEQ ID NO:2.

3, a kind of method that realizes the described earthworm fibrinolysin gene of claim 1 comprises the following steps:

A, amplimer synthesize: according to the earthworm fibrinolysin cDNA sequences Design primer of having delivered among the GeneBank, long 18 Nucleotide: the ACATGGAACTTCCTCCCG of upstream primer P1, long 19 Nucleotide: the ATCACCAACAACTAAACCG. of downstream primer P2, primer is through the PAGE purifying;

The total RNA of B, earthworm extracts: get 3～5 earthworm adults, after the aqua sterilisa rinsing of handling with DEPC, be placed in the plate that is covered with the filter paper that wets hunger and spend the night, after cleaning up with DEPC water once more, be ground into powder in liquid nitrogen, extract total RNA and be stored in-80 ℃;

The synthetic cDNA article one chain of C, reverse transcription: with Oligo (dT) 20 be that primer synthesizes cDNA first chain, and reaction conditions is: 42 ℃ 1 hour; 95 ℃ 5 minutes; 3 ℃ 5 minutes;

The pcr amplification of D, goal gene: with cDNA article one chain is that template is carried out pcr amplification, and its reaction parameter is: 95 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 20 circulations 2 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 10 circulations 2 minutes; 72 ℃ were extended 10 minutes again;

The recovery of E, target gene fragment is reclaimed the test kit specification sheets according to the PCR product and is reclaimed target gene fragment;

F, target gene fragment are cloned into carrier: reclaim target gene fragment and be connected with the T carrier, connect product transformed competence colibacillus cell e. coli jm109, picking colony rapid extraction plasmid carries out enzyme and cuts evaluation, resulting positive colony is to comprise the intestinal bacteria that the present invention relates to gene, is used for the propagation and the preservation of gene.

4, a kind of engineering strain is characterized in that: pichia spp Pichia pastoris X-33 (pPIC6 α A-EFE) CCTCC No:M204005.

5, a kind of preparation method who realizes the engineering strain of claim 4 is characterized in that:

The pcr amplification of A, earthworm fibrinolysin gene: with the T vector plasmid that contains fibrinolytic enzyme gene is template, according to the design of the cloning site on EFE mature peptide and expression vector pPIC6 α A primer, and in upstream primer, add 6 successive Histidine encoding sequences, add enteropeptidase cleavage site sequence afterwards, and designed a pair of earthworm fibrinolysin pcr amplification primer;

B, PCR product cloning are to the T carrier: reclaim the gene fragment in the PCR product, and clone the carrier in pGEM-T, pGEM-T-EFE2 changes in the e. coli jm109 with this recombinant vectors;

The structure of C, shuttle expression plasmid: extract recombinant vectors pGEM-T-EFE2, with EcoR I and Xba I double digestion to obtain purpose fragment EFE; Same enzyme is cut pPIC6 α A empty carrier and make it dephosphorylation under the CIAP effect, with the EFE that obtains behind linearizing pPIC6 α A empty carrier behind the dephosphorylation and the double digestion under the effect of T4DNA ligase enzyme 4 ℃ spend the night, to obtain recombinant expression vector pPIC6 α A-EFE, this recombinant vectors is changed in the e. coli jm109 increase;

The structure of the Yeast gene engineering bacterial strain of D, expression fibrinolytic enzyme gene at first is linearization of shuttle expression plasmid, extracts recombinant plasmid pPIC6 α A-EFE, is cut into linearity with the SacI enzyme, and same enzyme is cut empty plasmid pPIC6 α A in contrast; Next is to transform, and utilizes biochemical method that linearizing recombinant plasmid pPIC6 α A-EFE and empty plasmid pPIC6 α A are transformed the X-33 yeast strain, cultivates 2-3 days containing on the YPD solid medium of blasticidin 300mg/ml, up to there being single bacterium colony to occur; The 3rd is the screening positive transformant, and picking 10-20 single bacterium colony in the YPD liquid nutrient medium 30 ℃, 250rpm shaking table were cultivated until logarithmic growth late period, extracts the PCR that genome carries out recon and identifies.

6, the preparation method of engineering strain according to claim 5 is characterized in that: the used PCR primer of earthworm fibrinolysin pcr amplification is: upstream primer: CGAATTCCACCACCACCACCACCACGGTGGTGGTGACGACGACGACAAGATGGAAC TTCCTCCCGGAACA; Downstream primer: GTCTAGATTAGTTGTTGGTGATGATGTCGGTGATCCATGCAGCAT.

7, the application of a kind of genetically engineered plasmin albumen in treatment cardiovascular disease medicine.

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