CN100460509C - Trichina paramyosin gene and application thereof - Google Patents
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- CN100460509C CN100460509C CNB2007100000181A CN200710000018A CN100460509C CN 100460509 C CN100460509 C CN 100460509C CN B2007100000181 A CNB2007100000181 A CN B2007100000181A CN 200710000018 A CN200710000018 A CN 200710000018A CN 100460509 C CN100460509 C CN 100460509C
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Abstract
The present invention relates to paramyosin gene of new trichina antigen, constitution of the vector containing the gene, prokaryon expression vector containing the gene and the expressed protein. The present invention also relates to the application of the gene and its coded protein. When the recombinant protein of trichina paramyosin gene is used in immunizing animal, high titer antibody and effective immunoprotection against attacting infection will generate.
Description
Invention field
The present invention is a kind of Trichinella spiralis neoantigen gene paramyosin gene, contains the structure and the prokaryotic expression carrier of the carrier of this gene, and expressed albumen reaches the antibody that is produced by protein immune animal.The invention still further relates to described gene and proteic application.
Background technology
Trichina(Trichinella spiralis) (Trichinel laspiralis, be called for short Trichinella spiralis), but infected person and more than 150 kind of animal, and its caused trichonematosis is a kind of infecting both domestic animals and human parasitosis, be global distribution, seriously threatened human health and livestock industry is caused the tremendous economic loss.Trichinella spiralis is summarized as follows the life history: when human or animal host eats raw or half a lifetime food contains the meat (pork, dog meats etc.) of Trichinella spiralis Nang Bao, in the capsule bag larva to overflow under gastric juice, intestinal juice effect and grow in small intestine be adult.Female, male worm After mating worm produces newborn larvae.Newborn larvae is invaded intestinal mucosa lymphatic vessel or vein, arrives host's voluntary muscle with lymph and circulation of blood and continues to grow.The pathogenic course of Trichinella spiralis was divided into for three phases: stage of invasion, larval migration and Nang Bao form the phase, and promptly each link in this worm life history all can make the host cause a disease.
The trichonematosis complicated clinical manifestation is various, only in time makes difficulty of accurate diagnosis according to clinical symptom, gives timely that treatment causes certain difficulty, and immunodiagnosis and prevention that therefore should disease become the task of top priority.Because pathogenic agent can not limit antigenic obtaining in the external cultivation of going down to posterity in a large number; The antigenic complicacy of Trichinella spiralis, diversity cause the present candidate antigens molecule that does not still have the antigen of good high sensitive high specific as diagnosis or protectiveness vaccine in addition.
Summary of the invention
The objective of the invention is to: seek and the new gene of clone's Trichinella spiralis antigen, the gene recombinant protein of this gene is carried out the research of immunologic function, for trichinous immunodiagnosis and immunoprophylaxis, treatment provide new candidate antigens molecule.
The invention provides isolating polynucleotide molecule, it comprises the nucleotide sequence of selecting from following group of nucleotide sequence:
(A) with sequence or SEQ ID NO:1 shown in the SEQ ID NO:1 in open reading frame (75-2732 position Nucleotide) at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% homologous nucleotide sequence;
(B) with sequence or SEQ ID NO:1 shown in the SEQ ID NO:1 in the nucleotide sequence that under medium stringent hybridization condition, preferred high stringent hybridization condition, can hybridize of the complementary sequence of open reading frame;
(C) with the protein of SEQ ID NO:1 coding identical sequence but because of the degeneracy of genetic code different nucleotide sequence on sequence;
(D) (A), (B) or (C) fragment of described nucleotide sequence; With
(E) with (A), (B), (C) or (D) described nucleotide sequence complementary nucleotide sequence.
Preferably, polynucleotide molecule coding of the present invention has immunogenic polypeptide.
In a specific embodiments, described polynucleotide molecule has open reading frame sequence shown in nucleotide sequence shown in the SEQ ID NO:1 or the SEQ ID NO:1 75-2732 position Nucleotide.
The present invention also provides isolated polypeptide, and it comprises the aminoacid sequence of selecting from following group of aminoacid sequence:
(A) with aminoacid sequence at least 70% shown in the SEQ ID NO:2, preferred at least 80%, more preferably at least 90%, especially at least 95% homologous aminoacid sequence;
(B) because the substituting of one or more (for example 1-25,1-20,1-15,1-10,1-5) amino-acid residue, disappearance or insert and with the different aminoacid sequence of sequence shown in the SEQ ID NO:2; With
(C) (A) or (B) immunogenic fragments of described aminoacid sequence.
Preferably, polypeptide of the present invention has immunogenicity.
In a specific embodiments, described polypeptide comprises aminoacid sequence shown in the SEQ ID NO:2.
The present invention also provides recombinant vectors, and it comprises polynucleotide molecule of the present invention.Preferred described recombinant vectors is an expression vector, and it comprises and can express and comprise polynucleotide molecule of the present invention.
The present invention also provides the host cell that contains recombinant expression vector of the present invention.
The present invention also provides the method for producing polypeptide, is included in to cultivate host cell of the present invention under the condition that can make described expression of polypeptides and reclaim expressed polypeptide.
The present invention also provides antibody, but its specificity is in conjunction with polypeptide of the present invention.Described antibody can be polyclone or monoclonal antibody.
The present invention also is provided for the pharmaceutical composition of the trichinzation of prevention or treatment animal, especially livestock and philtrum, contains polypeptide of the present invention or polynucleotide or antibody in the described pharmaceutical composition.
The present invention also is provided for diagnosing animal, especially the diagnostic kit of the trichinzation of livestock and philtrum, but polynucleotide molecule of the present invention wherein contained, can detect primer, polypeptide of the present invention or the antibody of the present invention of the nucleic acid probe specific amplification polynucleotide molecule of the present invention of polynucleotide molecule of the present invention.
But primer, polypeptide of the present invention or the antibody of the present invention that the invention provides polynucleotide molecule of the present invention, can detect the nucleic acid probe specific amplification polynucleotide molecule of the present invention of polynucleotide molecule of the present invention are used to diagnose animal in preparation, especially the purposes in the diagnostic reagent of the trichinzation of livestock and philtrum.
The present invention also provides polynucleotide molecule of the present invention or polypeptide or antibody to be used for preventing or treating the purposes of the medicine of people and animals' trichinzation in preparation.
The present invention also provides oligonucleotide, it contains in open reading frame sequence shown in nucleotide sequence shown in the SEQ ID NO:1 or the SEQID NO:1 75-2732 position Nucleotide or its complementary sequence at least 15, preferably at least 25, more preferably at least 50 Nucleotide or its similar sequences, and described similar sequences can be hybridized under the height stringent hybridization condition with nucleotide sequence shown in the SEQ ID NO:1 or its complementary sequence.Preferably, but described oligonucleotide is to detect the nucleic acid probe of polynucleotide molecule of the present invention or the primer of specific amplification polynucleotide molecule of the present invention.
The present invention also provides oligonucleotide of the present invention to be used to diagnose animal, especially the purposes of the trichinzation of livestock and philtrum.
The present invention also provides fusion rotein, and it comprises polypeptide of the present invention and another merges object.In a specific embodiments, described another merges liking beta-galactosidase enzymes, glutathione-S-transferase or polyhistidine etc.
Nucleotide sequence of the present invention can obtain from Trichinella spiralis cDNA gene library with immunoscreening.The invention still further relates to the varient of this sequence, for example disappearance of one or more bases, interpolation or replacement, but do not change coded proteinic function.Varient can be the varient that naturally occurring equivalent varient or non-natural exist.
Polypeptide of the present invention can be transformed in the suitable host cell by making up the protokaryon or the carrier for expression of eukaryon of said gene, and purified polypeptide prepares from culture.Polypeptide of the present invention comprises the polypeptide of sequence 2, and its derivative, fragment or the analogue with equivalent function.
As specific examples, the inventor utilizes Protocols in Molecular Biology, as: cDNA library immunoscreening, molecular cloning, polymerase chain reaction (PCR), determined dna sequence, Southern hybridization, Western blot hybridization method, the clone has identified the new gene paramyosin gene of Trichinella spiralis antigen; Utilize genetic expression and immunological technique to obtain the recombinant protein of this gene and the height immune serum of tiring; And this proteic cellular localization, immunogenicity and immune protective analyzed.
The technical indicator that this gene reaches is:
1. adopt cDNA library immunoscreening to obtain the full length cDNA sequence of this gene in the present invention.Total length 2996bp, initiator codon ATG are positioned at sequence 74bp place, and TGA is a terminator codon, and encoded protein matter contains 885 amino acid.
2. 885 of trichina paramyosin gene amino acid recombinant proteins obtain to express and purifying (Fig. 1) in the e. coli bl21 strain.
3. trichina paramyosin gene recombinant protein immune animal is obtained the anti-paramyosin immune serum of high titre.
4. the trichina paramyosin gene recombinant protein is discerned by the patients serum, sick porcine blood serum, the sick rabbit anteserum that infect Trichinella spiralis.And with paramyosin gene recombinant protein artificial immunization mouse serum react (Fig. 2).
5. behind the trichina paramyosin gene recombinant protein immune animal challenge infection is produced effective immune protective (table 1).
The present invention has found trichina paramyosin antigen gene full-length cDNA; Obtained the paramyosin gene recombinant protein of purifying and carried out the research of immunologic function, for trichinous immunoprophylaxis provides new protectiveness candidate antigens molecule.And also can be used to prepare diagnostic reagent.
Description of drawings
Fig. 1 SDS-PAGE electrophoresis: expression and the purifying of paramyosin gene recombinant protein in intestinal bacteria.Wherein, each swimming lane representative:
M. molecular weight of albumen Marker
1. empty carrier whole bacterial protein
2. without IPTG inductive whole bacterial protein
3.IPTG the whole bacterial protein after inducing
4. the paramyosin gene recombinant protein of purifying
The arrow indication is the recombinant protein band
Fig. 2 Western immunoblotting is identified the immunological characteristic of paramyosin gene recombinant protein.Wherein, each swimming lane representative:
1. the paramyosin gene recombinant protein of purifying and trichonematosis human serum reaction
2. the paramyosin gene recombinant protein of purifying reacts with the porcine blood serum that infects Trichinella spiralis
3. the paramyosin gene recombinant protein of purifying reacts with the rabbit anteserum that infects Trichinella spiralis
4. the paramyosin gene recombinant protein of purifying and this recombinant protein artificial immunization mouse sero-reaction.
Embodiment
Term used herein " polynucleotide molecule ", " polynucleotide sequence ", " encoding sequence ", " open reading frame (ORF) " etc. are meant strand or double-stranded DNA and RNA molecule, can comprise one or more protokaryon sequences, the cDNA sequence, the genomic dna sequence that comprises exon and intron, the DNA of chemosynthesis and RNA sequence, and justice and corresponding antisense strand are arranged.
The method of producing and operating polynucleotide molecule disclosed herein and oligonucleotide molecules is well known by persons skilled in the art, and can according to the recombinant technology of having described (referring to Maniatis etc., 1989,
Molecular cloning, laboratory manual, press of cold spring harbor laboratory, cold spring port, New York; Ausubel etc., 1989,
The molecular biology current techniques, Greene Publishing Associates﹠amp; Wiley Interscience, NY; Sambrook etc., 1989,
Molecular cloning, the laboratory hand Volume, the 2nd edition, press of cold spring harbor laboratory, cold spring port, New York; Innis etc. (volume), 1995,
The PCR strategy, Academic Press, Inc., San Diego; And Erlich (volume), 1992, round pcr, Oxford University Press, New York) finish.
The invention provides a kind of isolating polynucleotide molecule of the immunogenic polypeptide of encoding.In one embodiment, the isolating polynucleotide molecule of the present invention comprises the nucleotide sequence that is selected from the following member: the nucleotide sequence shown in the open reading frame (75-2732 position) among (1) SEQ ID NO:1 or the SEQ ID NO:1; (2) with SEQ ID NO:1 or SEQ ID NO:1 at least 70% identical, preferred at least 80% identical, more preferably at least 90% identical, the 95% identical nucleotide sequence most preferably of nucleotide sequence shown in the open reading frame; (3) under medium rigorous hybridization conditions (promptly at 0.5M NaHPO
4, in 7% sodium lauryl sulphate (SDS), 1mM EDTA in 65 ℃ with the DNA hybridization that is incorporated into filter membrane, and in 0.2xSSC/0.1%SDS in the condition of 42 ℃ of washings; Referring to (volumes) such as Ausubel, 1989,
The molecular biology current techniques, the 1st volume, GreenPublishing Associntes, Inc., and John Wiley ﹠amp; Sons, Inc., NY, P.2.10.3), the rigorous hybridization conditions of preferred heights is (promptly at 0.5M NaHPO
4, among 7%SDS, the 1mM EDTA in 65 ℃ with the DNA hybridization that is incorporated into filter membrane, and in 0.1x SSC/0.1% SDS in 68 ℃ of wash conditions; Consult Ausubel etc., 1989, above-mentioned document) nucleotide sequence that can hybridize with polynucleotide molecule down with open reading frame among SEQ ID NO:1 or the SEQ ID NO:1 or its complementary sequence; (4) with SEQ ID NO:1 or SEQ ID NO:1 in open reading frame coding identical sequence protein but because of the degeneracy of genetic code different nucleotide sequence on sequence; Perhaps arbitrary nucleotide sequence complementary nucleotide sequence in (5) and (1)-(4).
In one embodiment, polynucleotide molecule of the present invention comprises the nucleotide sequence of the trichina paramyosin of encoding.
In one embodiment, polynucleotide molecule of the present invention can be used for standard amplification technique amplification Trichinella spiralis specificity polynucleotide molecule, or as diagnostic reagent in order to detect by the existence of Trichinella spiralis specificity polynucleotide in the animal body fluid of trichinzation or the tissue sample.
The present invention further provides and comprising coding with a kind of separation polynucleotide molecule that comes from the nucleotide sequence of peptide more than trichina paramyosin of the present invention or the immunogenic polypeptide.Employed term " homology " is meant that polypeptide had the aminoacid sequence of trichina paramyosin or immunogenic polypeptide originally when mentioning the polypeptide that comes from trichina paramyosin or immunogenic polypeptide together herein, but (for example 1-25 is individual, 1-20 is individual for wherein one or more, 1-15,1-10,1-5) amino-acid residue is conservatively substituted by different amino-acid residues, and resulting polypeptide can be used for implementing the present invention.It is known in the art that conserved amino acid substitutes.Cause such alternate rule to comprise, M.D. described alternative rule such as (1978, national biomedical research fund, Washington, D.C., the 5th volume, supplementary issue 3) by Dayhof.More particularly, conserved amino acid substitutes and occurs in the amino acid family that is associated with its acidity, polarity or side chain size.Generally the amino acid of genetic coding can be divided into four groups: (1) acidic amino acid=aspartic acid, L-glutamic acid; (2) basic aminoacids=Methionin, arginine, Histidine; (3) nonpolar amino acid=L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane; (4) uncharged polare Aminosaeren=glycine, l-asparagine, glutamine, halfcystine, Serine, Threonine, tyrosine.Phenylalanine, tryptophane and tyrosine also common category are aromatic amino acid.One or more substituting in any particular group, for example substitute leucine or substitute Threonine with glutamic for aspartic acids or with Serine with Isoleucine or Xie Ansuan, or the amino-acid residue of being correlated with on any other amino-acid residue structure, similar acidity, polarity, side chain size are for example arranged, or have the amino-acid residue of similarity to substitute in its some combined aspects, generally function or the immunogenicity to polypeptide do not have too big influence.
Substantially homologous protein and polypeptide is characterized in that having one or more (for example 1-25 is individual, 1-20 is individual, and 1-15,1-10,1-5 is individual) amino acid replacement, deletion or interpolation.These change the less conversion of preferred influence, and promptly conserved amino acid substitutes (seeing Table 2) and other can not have a strong impact on folding and active the substituting of protein or polypeptide; Little deletion, normally about 30 the amino acid whose little deletions of 1-; And little amino or C-terminal extension, such as the N-terminal methionine residues, reach the little connection peptides of about 20-25 residue, or be convenient to the little extension (affinity labelling) of purifying, as polyhistidine bundle, albumin A (Nilsson etc., EMBO J.4:1075,1985; Nilsson etc., Enzymology method, 198:3,1991).The DNA of coding affinity labelling can buy from product vendor.
Said herein polypeptide " can be used for implementing the present invention " and is meant that this polypeptide can be used as diagnostic reagent, with detect recently by trichinzation or existing by Trichinella spiralis specific antibody in the animal blood of trichinzation or the serum sample; Perhaps can be used as antigen and produce Trichinella spiralis specific antibody (for example antibody of specific combination, neutrality antibody, therapeutic antibodies); Perhaps can be used as immunogen animal is carried out preventative immunity or therapeutic immunization, perhaps any purposes of this polypeptide mentioned in this article.
The present invention further provides with the above-mentioned polynucleotide molecule of the present invention and can hybridize, or with have the oligonucleotide molecules that can hybridize as the polynucleotide molecule of the nucleotide sequence of the complement of the above-mentioned polynucleotide molecule of the present invention.Such oligonucleotide molecules better is to be about 15nt at least, is more preferably and is about 25nt, and 50nt especially at least, and can be under the height stringent condition and one or more above-mentioned polynucleotide molecules hybridization.Said height stringent condition is to wash film in the 6XSSC/0.5% trisodium phosphate, and washes film temperature and be about 37 ℃ for being about 14 base persons, is about 17 base persons and is about 48 ℃, is about 20 base persons and is about 55 ℃, is about 23 base persons and is about 60 ℃.Other hybridization conditions than the long oligonucleotide molecule of the present invention can be determined according to standard technique by those skilled in the art.In a preferred embodiment, oligonucleotide molecules of the present invention is complementary to the above-mentioned polynucleotide molecule of a present invention part one of at least.
Oligonucleotide molecules of the present invention can be used for multiple purpose, comprising as the primer of amplification Trichinella spiralis specificity polynucleotide molecule being used for such as disease differential diagnosis, perhaps coding or as the antisense molecule of generegulation.With regard to the diagnosis aspect, can use the primer of suitable design to detect the existence of Trichinella spiralis specificity polynucleotide molecule in the samples such as animal tissues or body fluid.The generation of specific amplification products can be supported the diagnosis of trichinzation, and the product that lacks amplification then may not indicated not infection.For example in people's (1995, the source is the same) such as Innis and Erlicch (1992, the source is the same) editor's the aforementioned documents method that increases has been described, for example polymerase chain reaction (PCR) method.Other amplification techniques known in the art also can use, for example the ligase chain reaction method.Also can use the sequences Design of polynucleotide molecule disclosed herein to be used for separating homogenic primer from other kinds or the strain system of Trichinella spiralis.
The present invention further provides the cloning vector, the expression vector that comprise polynucleotide molecule of the present invention, comprise any said carrier by transformed host cell, and by its deutero-new strain system or clone.In a preferred embodiment, the invention provides the recombinant vectors that comprises a kind of polynucleotide molecule, said polynucleotide molecule has the nucleotide sequence of coding trichina paramyosin or immunogenic polypeptide.
Preferably, the building mode of recombinant vectors of the present invention, particularly expression vector can make polynucleotide molecule of the present invention encoding sequence with transcribe the one or more regulatory element operability ground required and be connected with translating encoding sequence, with the generation polypeptide.Term used herein " regulatory element " comprises but is not only limited to coding induction type and non-inducible promoter, enhanser, operon and the nucleotide sequence that can be used for driving and/or regulate other element that the polynucleotide encoding sequence expresses known in the art.In addition, the said encoding sequence of this paper and one or more regulatory elements " are connected to operability " and are meant that regulatory element can regulate and allow to transcribe encoding sequence effectively or translate its mRNA, or bring into play the function of two aspects.
The method that structure comprises the recombinant vectors of the specific coding sequence that is connected with suitable regulatory element operability ground is known, and can use these methods to realize the present invention.These methods comprise genetic recombination (as the above-mentioned document referring to people such as Maniatis (1989)) in extracorporeal recombination, synthetic technology and the body.
The various carriers that can be used for expressing trichina paramyosin of the present invention or immunogenic polypeptide encoding sequence of the present invention are known in the art, comprising the recombinant phage dna that contains the specific coding sequence, plasmid DNA and cosmid DNA expression vector.The plasmid that can contain polynucleotide molecule of the present invention through processing comprises pUC8, pUC9, pBR322 and pBR329 (BioradLaboratories, Richmond, CA), pPL and pKK223 (Pharmacia, Piscataway, NJ), pQE50 (Qiagen, Chatsworth, CA) and pGEM-T EASY (Promega, Madison, WI) etc.The typical carrier for expression of eukaryon that can contain polynucleotide molecule of the present invention through processing comprises moulting hormone induction type mammalian expression system (Invitrogen, Carlsbad, CA), based on system (Promega, Madison, the WI of cytomegalovirus promoter-enhanser; Stratagene, La Jolla, CA; Invitrogen) with based on expression system (Promega) of baculovirus etc.
The regulatory element of these and other carriers can have nothing in common with each other aspect its intensity and the specificity.Based on the host/vector system that is utilized, can use multiple suitable in the element any transcribed and translate.For example, when in the mammal cell line system, cloning, can use isolating promotor from the mammalian cell genome, as the mouse metallothionein promoter, or from grow in these intracellular viruses isolating promotor, as vaccinia virus 7.5K promotor or Moloney muroid sarcoma virus long terminal repeat.Can use the promotor that obtains with recombinant DNA or synthetic technology to transcribe the sequence that is inserted into.In addition, at specific elicitor, for example be suitable under the existence of the zinc of metallothionein promoter and cadmium ion, the expression that is started by some promotor can be enhanced.The non-limitative example of transcriptional regulatory district or promotor comprises β-gal promotor, T7 promotor, TAC promotor, trp and lac promotor, the trp-lac promoter, fusion etc. that are used for bacterium; Be used for zymic glycolytic ferment promotor, as ADH-and ADH-II promotor, GPK promotor, PGI promotor, TRP promotor etc.; And the SV40 that is used for mammalian cell early stage and late promoter, adenovirus major late promoter etc.The present invention further provides the polynucleotide molecule of the nucleotide sequence of the promotor that comprises trichina paramyosin gene, it is used in and expresses encoding sequence of the present invention in the Trichinella spiralis.
Encoding sequence for translation sufficiently is inserted into also needs the specificity start signal.These signals generally comprise ATG initiator codon and flanking sequence.Under the polynucleotide molecule of the present invention of initiator codon that will comprise himself and flanking sequence is inserted into situation in the suitable expression, other translation control signal may needn't be added.Yet, when only inserting a part of encoding sequence, may need to comprise the external source translation control signal of ATG initiator codon etc.Can be from various sources, promptly natural and synthetic source obtains these external source translation control signal and initiator codons.Moreover initiator codon must be consistent with the frame of coding region, meets the translation of frame ground to guarantee whole insertion fragment.
Also can make up the expression vector that expression is comprised the fused protein of protein of the present invention or polypeptide and fusion object.Such fused protein for example can be used for producing the proteic antiserum(antisera) of anti-Trichinella spiralis, the proteic biochemical property of research Trichinella spiralis, through engineering approaches and modifies Trichinella spiralis albumen that performance has different immunologys or functional property, helps to identify or Trichinella spiralis albumen that purification of Recombinant is expressed or improve its stability.Possible fusion protein expression vector comprises but is not only limited to the carrier of the sequence of having inserted coding beta galactosidase enzyme and trpE syzygy, maltose binding protein syzygy, glutathione-S-transferase syzygy and polyhistidine syzygy (carrier zone).The method that can be used for making up these and other Expression of Fusion Protein carriers of coding is known in the art.
Available fused protein helps purifying expressed protein.In a non-limiting embodiments, can use amylose resin purifying paramyosin or immunogenic polypeptide-maltose associativity fusion rotein, glutathione agarose bead purifying paramyosin or immunogenic polypeptide-glutathione-S-transferase fused protein can be used, nickelous resin purification paramyosin or immunogenic polypeptide-polyhistidine fused protein can be used.Perhaps, also can use the antibody of anti-carrier protein or peptide that fused protein is carried out affinitive layer purification.For example, the nucleotide sequence through engineering approaches of target epi-position of coding monoclonal antibody can be transferred in the expression vector that is connected with regulatory element operability ground, and be limited its position so that expressed epi-position is fused on the Trichinella spiralis albumen of the present invention.In a non-limiting embodiments, the available standards technology is inserted coding FLAG on certain point of amino that is equivalent to paramyosin or immunogenic polypeptide or C-terminal
TMIn the nucleotide sequence of epi-position mark (it is a wetting ability mark peptide) (Znternational Biotechnologies Inc.), can use commercially available anti-FLAG then
TMThe paramyosin that antibody test and affinity purification are expressed or immunogenic polypeptide-FLAG
TMEpi-position syzygy product.
Also can modify expression vector and make it to contain the polylinker sequence of encode specific protein enzyme cleavage site, thereby can handle and from the carrier zone or merge object and discharge the Trichinella spiralis albumen of having expressed through specific proteases.For example, the fusion rotein carrier can comprise the nucleotide sequence of coding zymoplasm or factor Xa cracking site.
The signal sequence that can use currently known methods will be arranged in Trichinella spiralis albumen coded sequence upstream and frame unanimity is worked into expression vector, to instruct expressed proteinic transportation and secretion.The limiting examples of signal sequence comprises the signal sequence of alpha factor, immunoglobulin (Ig), outer membrane protein, penicillinase and TXi Baoshouti etc.
In order to help to filter out host cell, can through engineering approaches modify the encoding sequence that carrier makes it further to comprise reporter gene product or other selection markers through recombinant vectors conversion of the present invention or transfection.Such encoding sequence better is connected with above-mentioned regulatory element operability ground.It is well known in the art being used to implement reporter gene of the present invention, comprises the gene of coding E.C. 2.3.1.28 (CAT), green fluorescent protein, firefly luciferase and human growth hormone etc.The nucleotide sequence of coding selection marker is known in the art, comprising that coding gives antibiotics resistance or metabolic antagonist resistance, or those nucleotide sequences of the gene product that auxotroph needs etc. are provided.The example of these sequences comprises the coding thymidine kinase activity, or those sequences of anti-methotrexate, penbritin, kantlex, paraxin, aminoglycoside or Totomycin etc.
The present invention further provides the transformed host cell that comprises polynucleotide molecule of the present invention or recombinant vectors, and by deutero-clone.Can be used for implementing host cell of the present invention can be eucaryon or prokaryotic cell prokaryocyte.Transformed host cell like this comprises but is not only limited to microorganism, for example use the bacterium of recombinant phage dna, plasmid DNA or the conversion of cosmid DNA carrier, the perhaps yeast that transforms with recombinant vectors, or zooblast, as insect cell with recombinant viral vector such as baculovirus infection, or with the mammalian cell of recombinant viral vector such as adenovirus or vaccinia virus infection etc.For example, can use coli strain, as DH5 α bacterial strain.Eukaryotic host cell comprises yeast cell, but also can effectively utilize mouse, mammalian cell such as hamster, ox, monkey or human cell line.The eukaryotic host cell that can be used for expressing recombinant protein of the present invention comprises Chinese hamster ovary (CHO) cell, NIH/3T3 etc.
Better be recombinant vectors of the present invention is transformed or transfection in one or more host cells of the basic homogeneous culture of cell.Generally can be according to known technique, for example protoplast transformation, calcium phosphate precipitation, calcium chloride processing, micro-injection, electroporation, contact through virus with reorganization infect, liposome-mediated technology such as transfection, the transfection of DEAE-dextran, transduction, joint or microparticle bombardment import carrier in the host cell.Can use standard method to select transformant, but for example express the method for the cell of selection marker relevant such as antibiotics resistance with recombinant expression vector by selection.
In case after expression vector imported host cell, the standard techniques such as pcr analysis that can use Southern hybridization analysis, restricted enzyme cutting analysis, comprise ThermoScript II PCR (rt-PCR) confirm whether polynucleotide molecule of the present invention is integrated and be retained in the host cell gene group or with the episome form and exist, perhaps use the protein of immunological detection detection of desired.A kind of evaluation in available following at least four kinds of general approach known in the art contains and/or expresses the host cell of polynucleotide molecule of the present invention: (i) DNA-DNA, DNA-RNA or RNA-sense-rna hybridization; (ii) detect the existence of " sign " gene function; The (iii) expression of detection specificity mRNA transcript in host cell is to estimate transcriptional level; Or (iv) detect existing of ripe polypeptide product with immunodetection known in the art.
In case with polynucleotide molecule of the present invention is stable import in the appropriate host cell after, can clonal expansion transformed host cells, and cultivate resulting cell helping maximum to produce under the condition of encoded polypeptide.Such condition generally comprises cultivates that cell transformed reaches high-density.When expression vector contains inducible promoter, can be as required, (as the carbohydrate analogue, for example isopropyl ss-D-thiogalactoside (IPTG), excess metabolism by product gather etc. suitably inductive condition with abduction delivering to utilize temperature change, nutrient substance to exhaust, add the gratuitous induction agent.
In the time of in polypeptide is retained in host cell, should gather in the crops and lysing cell, and under the extraction conditions that reduces protein degradation as far as possible known in the art, for example in 4 ℃ and/or have under the proteinase inhibitor existence condition, purifying or separated product basically from lysate.Can from host cell, secrete as polypeptide, then can collect the nutrient medium of having exhausted simply, and therefrom purifying or isolated polypeptide basically.
In case of necessity, can use standard method, comprise but be not only limited to ammonium sulfate precipitation, size fractionation separation, ion exchange chromatography, HPLC, density gradient centrifugation and affinity chromatography, purifying or isolated polypeptide basically from cell lysate or substratum.If polypeptide lacks biologic activity, then can do according to molecular size or with the reactivity of polypeptid specificity antibody, or detect it according to the existence of syzygy mark.Be used to implement when of the present invention, polypeptide can be to be secreted in the nutrient solution or to be present in not purified state in the cell lysate, but better is therefrom to obtain basic purifying or separation.Said herein polypeptide " basically purifying ", it is proteinic at least about 20% (weight) to be meant that this polypeptide constitutes in particular formulations.In addition, said herein polypeptide " separates ", refers to this polypeptide and constitutes in the particular formulations proteinic at least about 80% (weight).
Therefore, the invention provides purifying or isolating trichina paramyosin or immunogenic polypeptide basically by polynucleotide encoding of the present invention.In a preferred embodiment, trichina paramyosin or immunogenic polypeptide have the aminoacid sequence of SEQ ID NO:2.
The present invention further provides with coming from the polypeptide of described trichina paramyosin or immunogenic polypeptide, wherein term " homology " has above the implication with regard to polypeptide limited.
The present invention further provides the polypeptide of forming by the essential part of any aforementioned polypeptides of the present invention." essential part " of term used herein-polypeptide of the present invention or " peptide fragment " mean form to go up less than the complete amino acid sequence of corresponding overall length polypeptide but comprise its aminoacid sequence at least about 10%, preferably at least about 20% and can be used for implementing polypeptide of the present invention (" can be used for " has above the implication with regard to polypeptide limited).Particularly preferably be and have immunogenicity the peptide fragment of (promptly can induction of immunity reaction and produce the antibody of anti-corresponding overall length Trichinella spiralis polypeptide specifically).
The present invention further provides the fused protein that comprises any aforementioned polypeptides that merges with carrier known in the art or fusion object.
Polypeptide of the present invention can be used for multiple purpose, comprises as diagnostic reagent, for example with the Trichinella spiralis specific antibody in standard detection methods such as ELISA detection screening animal blood or the serum sample; Or it is as described below as the antigen that produces polyclone or monoclonal antibody, wherein can use described antibody as diagnostic reagent, for example with the Trichinella spiralis specific protein in the screening of standard techniques such as Western trace detection method zooblast, tissue or the humoral sample.
Can on protein level, modify any polypeptide of the present invention, to improve or to change its biology or amynologic characteristic.Can use known technology that polypeptide is carried out one or more chemically modifieds, to prepare its analogue.
Can on molecule, connect one or more chemical groups or another kind of protein such as serum albumin, keyhole worm relative hemocyanin or BSA, perhaps polyamino acid (as poly-lysine), or polysaccharide is (as Sepharose, agarose, or through modifying or not modified Mierocrystalline cellulose) first-class, with the derivative of preparation polypeptide of the present invention.The method of carrying out these ligations is known in the protein chemistry field.
The present invention further provides the separation antibody of anti-polypeptide of the present invention.In a preferred embodiment, can use currently known methods to produce the antibody of anti-trichina paramyosin or immunogenic polypeptide.Available part or purifying or isolating trichina paramyosin or immunogenic polypeptide recombinant protein basically, or its homologue described above, fusion rotein, essential part, analogue or derivative immunity are selected from various host animals such as pig, ox, horse, rabbit, goat, sheep or mouse.Can use adjuvant described below to improve the antibody generation.
Can from the serum of immunized animal, obtain and separate polyclonal antibody, and use ordinary method to test it antigenic specificity.Perhaps, also can use any technology preparation and the separation monoclonal antibody of producing antibody molecule by the continuous cell line of cultivating.These technology comprise but be not only limited to originally by Kohler and Milstein (nature, 1975,256:495-497) hybridoma technology of Miao Shuing, human B cell hybridoma technology (Kosbor etc., 1983, today immunology, 4:72; Cote etc., 1983, institute of NAS newspaper, 80:2026-2030), and the EBV hybridoma technology (Cole etc., 1985, monoclonal antibody and cancer therapy, Alan R.Liss, Inc., pp.77-86).Perhaps, can adopt the technology (as referring to United States Patent (USP) 4,946,778) of the manufacture order chain antibody of having stated to produce Trichinella spiralis antigen-specific single-chain antibody.
The antibody fragment that contains the specific binding site of polypeptide of the present invention is also included within the scope of the invention, and the preparation of available known technology.Such fragment comprises but is not only limited to the F (ab ') that can be produced by gastric pepsin digestion complete antibody molecule
2Fragment and can through the reduction F (ab ')
2Segmental disulphide bridges and the Fab fragment that produces.Perhaps, also can make up the Fab expression library (Huse etc., 1989, science 246:1275-1281), has required specific Fab fragment with rapid evaluation to Trichinella spiralis albumen.
It is known in the art producing with the technology of separating monoclonal antibody and antibody fragment, and is for example providing more detailed description: Harlow and Lane in the following document, 1988,
Antibody: experiment The chamber handbook, cold spring harbor laboratory and J.W.Cooding, 1986,
Monoclonal antibody: principle and reality Trample, Academic Press, London (it classifies this paper reference as).
The following example is just described for example, and is not used for limiting the scope of the invention.
Embodiment
The clone and the sequential analysis of embodiment 1 trichina paramyosin gene
1. the preparation of screening serum (artificial immunization rabbit anteserum and artificial challenge's rabbit anteserum)
Collect and separate from the Heilungkiang of pig body strain Trichinella spiralis (international standard worm strain coding ISS533) adult, the adult polypide is put in the mill, ice bath ground 15 minutes down fast.Liquid after grinding is moved into 10ml centrifuge tube, ultrasonic (on ice) 3 times, 3 minutes/time, interval 1 minute.The multigelation several, the centrifuging and taking supernatant promptly gets full worm soluble antigen.Immunity Japan large ear rabbit (1.6mg/ only) is strengthened weekly 1 time, strengthens 4 times, obtains the artificial immunization rabbit anteserum; Experimental rabbit is fed with 4000 Trichinella spiralis muscle larvaes, obtains artificial challenge's rabbit anteserum after 4 weeks.Measure through ELISA, the serum titer of artificial immunization rabbit anteserum and artificial challenge's rabbit anteserum all reaches more than 1: 4000.
2. immunoscreening expression library
Trichinella spiralis adult λ ZAP II cDNA expression library is available from Liu Mingyuan (reference " structure and the screening of Chinese Trichinella spiralis strain isolated adult and newborn larvae gene library ", Chinese animal doctor's journal, 1998,18 (2): 147-150.).
1 μ l Trichinella spiralis adult λ ZAP II cDNA expression library diluent adds among the 600 μ l host bacterium XL1-Blue, and 37 ℃, adsorb and added top-agar in 20 minutes, be inverted in the plaque that is cultured to the needle point size in 42 ℃ of incubators and grow.(Gelman company) is layered on the flat board nitrocellulose filter, is placed on 37 ℃ of incubator overnight incubation.At room temperature film was carried out mark in second day, and took off, immersed an anti-working fluid (the artificial immunization rabbit anteserum dilutes with 1:1000 with TBST) 30 minutes, added coliphage lysate (STRTAGENE company) in the serum from flat board.Film is immersed two anti-working fluid-1:7500 alkaline phosphatase-goat anti-rabbit iggs (Promega company), 30 minutes.Immerse among colour developing liquid NBT, the BCIP (Promega company), after the colour developing fully, add ddH
2O stops.Positive colony with the screening of 1:1000 artificial immunization rabbit anteserum obtains sieves as one anti-(diluting with 1:1000 with TBST) is multiple with artificial infected rabbits serum again, obtains 3 positive colonies.After PCR and Southern Blot evaluation, choose the Ts86 clone and carry out determined dna sequence.
3.Ts86cDNA sequence and amino acids coding analysis
Ts86cDNA total length 2996bp (seeing SEQ ID NO:1), open reading frame (75-2732 bit base, 2658b altogether), 885 amino acid (seeing SEQ ID NO:2) of encoding, its theoretical molecular is 102KD.Encoded protein is through homology analysis, and the paramyosin homology of discovery and Ancylostoma duodenale, Onchocerca caecutiens, Wuchereria malayi is respectively 82%, 82% and 79%, is inferred as trichina paramyosin gene.
Nucleotide sequence (SEQ ID NO:1) (underlined base is the initial final position of open reading frame):
GGCACGAGCAGATCTAAAAGGATCTGTTGTAAATTGATTCTCCATTCGACAATCGAAAAAAC
TACATCAGCACC
ATGTCTCTGTATCGCAGTCCCAGTGCGTCAGTGATGAGATCAGCAAGCAT
GCTCAGCCGAAGTGGCGGATTCGATGCTTACGGATTTGGAGGTTACGGTGCGCCAAGCCTCA
ACGTTGCCGACTTGGGTTCTTTGACCAGACTCGAGGATAAAATTCGCCTGCTTCAAGATGAT
TTGGAAACGGAAAGAGAATTGCGAAACCGAATTGAACGCGAACGTGCCGATTTGTCCTGCCA
ACTGATCAGCTTAACCGATCGATTGGAAGAGGCTGAAGGAACCACCGATGCCCAGATCGACG
CCAATCGAAAGCGTGAATCCGAATTGCAAAAGTTGAGAAAAATATTGGAAGATTCGCAATTG
GAAAGCGAAGATTCGCTGAACCAGCTGCGCAAGAAGCACCAAGAATCCCTTTTAGATTATCA
GCAGCAAATTGAACAACTTCAAAAGAAAAATAGCAAAATCGACAGAGAACGACAACGTTTGC
AGCATGAAGTCATTGAACTTACTGCCGGAATTGATCAGATGCAAAAAGACAAGCATGCCGCG
GAAAAAGCTGCCGAAAAGCACGAAGCGCATGCCAGAGAGCTTCAGAACAGAGTTGACGATCT
GGCAAAAAATTTGAACGACCTGGCCTCGCAGCGTCAACGTCTGCAACAGGAAAACAACGATT
TGATGAAAGAGTTGCACGATGTCAAAGTGCAAATGGAAAATATTCAACACGTCAAGACTCAA
CTTGCTCAACAGCTCGAAGAAGCACGTCGTCGACTCGAAGATGCGGAACGTGAACGTTCGCA
AATGCAAACCCAGTTGCATCAGATGCAGCTGGAATTGGATTCAATTCAAGGTGCGTTGGAAG
AGGAATCGTCCGCACGTGCCGAAGCAGAGCACAAATTGTCGTTGGCAAATACGGAAATTTCC
CAGTGGAAGAGCAAATTCGACGCCGAAGTTTCACTCCACCAAGAAGAAGTTGACGATCTGCG
TAAAAAAATGATCCAAAAACAAGCAGAATATGAGGAACAAATTGAAATTATGCTGCAAAAGA
TTTCCCAATTGGAAAAAGCGAAAAGCCGTCTGCAGTCGGAAGTTGAAGTTTTGATAGTTGAT
TTGGAAAAGGCTCAAAGCACCATTGCCATTTTGGAAAGACAAAAAGAACAGCTCGAAAGAAT
GGTCGCCGAGATGAAGACACGCCTGGACGAGGTGACACAAGAGCTCGAAGCTACGCAACGAG
AACTCAGAGCGACGCAAGCGGAATTGCAAAAGATGAAGCATCTTTATGAAAAAGCCGTCGAA
CAGAAAGAAGCTCTGGCTAGGGAGAACAAAAAATTGCAAGACGATTTGCATGAAGCAAAGGA
AGCTTTGGCCGACGCGAACAGAAAATTGCACGAGCTGGATTTGGAGAATGCACGTCTGGCCG
GTGAAATCAGAGAACTGCAAATCGCGCTCAAGGAAGCCGAAGCGGCTAGACGTGACGCGGAA
AGTCGCGCACAGCGTGCAGTAGCTGAATTGCAAGCTCTGCGCGTGGAAATGGAACGTCGTTT
ACAAGAAAAAGAAGAAGAAATGGAAGCATTGCGCAAAAATATGCAGTTCGAAATTGACCGAC
TGGCTGCAGCGTTGGCTGACGCTGAGGCTCGCATGAAGGCGGAAATTTCCCGTCTGAAGAAG
AAATACCAGGCGGAAATTGCCGAACTGGAAATGACCATCGACAATTTGAACCGGGCCAATTT
GGAAGCACAAAAGACGATCAAAAAACAAGCAGACCAACTGAGGGCTTTGCAAAGCAGCTTCG
AAGATTGCCAACGTCAACTGCAACAAACGCTCGACCAGTATGCAATTGCCCAGCGCAAGTTG
TCCGCTTTGTCGGCCGAATTGGAAGACTGCAAGAGTGCCTTGGACACGGCGATCCGGGTGCG
CAAACAAGCCGAAGCGGATCTGGAAGAAGCCCAGGGGAAAATTGCCGATTTGGTCAGCTTGA
ACAACAATTTGACCGCGATCAAGGGCAAATTGGAGACCGACTTGTCGACATGCCAAGCCGAT
TTGGACGAGACGACCAAAGAACTGCGAGCCGCCGACGACCGTGCCAACCGAGCTCAGCAGGA
TGCGGTCCGGGCTATGGAACAACTCCACGAAGAGCAGGAACATTCGATGAAAATCGACGCCA
TGCGCAAAGCGTTGGAAGAGCACGTCAAACAGTTGCAAGTGCAAATCCAAGAAGCCGAAGCG
GCAGCTCTGCTCGGTGGCAAGCGCGTCATTGCCAAACTCGAGACCAGGATTCGAGATTTGGA
AATGGCGTTGGATGAAGAATCGAGACGACACAAAGAAACCCAAGCCTGCTTGCGCAAAAAGG
ACCGTCGCGTCAAGGAAATGCAAATGCAAGTGGACGAGGAGCACAAAAATTTCGTCATGGCC
CAAGATACTGCCGAACGATTGGCCGAAAAATTGAACATCTACAAGCGACAGTTGACTGAAGC
GGAATCGTTGACAATGCAAAATTTGCAACGTGTCCGTCGTTACCAGCACGAATTGGAAGATG
CCGAAGGTCGAGCCGAACAAGCCGAAAGTAGCTTGCACTTGATCCGTGCCAAGCATCGTTCT
TCCGTTTCCATGGGAAAAAGTCTCTCTTCCAAGGTTTACGTGATGGAAGAAGGACATGAATA
T
TGATGAACAATACACTAATAATTAATTGCAGAAATTTATTTTTCTGGTGGAAGAGCAACCA
TGTTTTAGCTTCTTTCTTTCTCAATTTACTACCATAATAATAATAATATTATTAATATATTT
TTAATATACAATGATGATCAATTTTCTTTTTATTCAGAATGTAATCGCCAATTAGCTTTTTT
CTTTTTCTCTCTCAAACAACCAACCAATCATAATTCTCTTCTTCATTCACGCCAACAAAAAA
AAAAAAAAAAAAAAAAAAAA
Aminoacid sequence (SEQ ID NO:2) in the open reading frame:
MSLYRSPSASVMRSASMLSRSGGFDAYGFGGYGAPSLNVADLGSLTRLEDKIRLLQDDLETE
RELRNRIERERADLSCQLISLTDRLEEAEGTTDAQIDANRKRESELQKLRKILEDSQLESED
SLNQLRKKHQESLLDYQQQIEQLQKKNSKIDRERQRLQHEVIELTAGIDQMQKDKHAAEKAA
EKHEAHARELQNRVDDLAKNLNDLASQRQRLQQENNDLMKELHDVKVQMENIQHVKTQLAQQ
LEEARRRLEDAERERSQMQTQLHQMQLELDSIQGALEEESSARAEAEHKLSLANTEISQWKS
KFDAEVSLHQEEVDDLRKKMIQKQAEYEEQIEIMLQKISQLEKAKSRLQSEVEVLIVDLEKA
QSTIAILERQKEQLERMVAEMKTRLDEVTQELEATQRELRATQAELQKMKHLYEKAVEQKEA
LARENKKLQDDLHEAKEALADANRKLHELDLENARLAGEIRELQIALKEAEAARRDAESRAQ
RAVAELQALRVEMERRLQEKEEEMEALRKNMQFEIDRLAAALADAEARMKAEISRLKKKYQA
EIAELEMTIDNLNRANLEAQKTIKKQADQLRALQSSFEDCQRQLQQTLDQYAIAQRKLSALS
AELEDCKSALDTAIRVRKQAEADLEEAQGKIADLVSLNNNLTAIKGKLETDLSTCQADLDET
TKELRAADDRANRAQQDAVRAMEQLHEEQEHSMKIDAMRKALEEHVKQLQVQIQEAEAAALL
GGKRVIAKLETRIRDLEMALDEESRRHKETQACLRKKDRRVKEMQMQVDEEHKNFVMAQDTA
ERLAEKLNIYKRQLTEAESLTMQNLQRVRRYQHELEDAEGRAEQAESSLHLIRAKHRSSVSM
GKSLSSKVYVMEEGHEY
1. paramyosin gene prokaryotic expression and purifying
According to the Ts86cDNA sequence, design a pair of primer, upstream primer is that 5 ' CGGGATCCATGTCTCTGTATCGCAGTCCCAGT-3 ' (SEQ ID NO:3) (are total to 32nt, contain a BamHI restriction enzyme site), downstream primer is 5 ' CGGAATTCATATTCATGTCCTTCTTCCATCAC-3 ' (SEQ ID NO:4) (32nt contains an EcoRI restriction enzyme site altogether).The Ts86cDNA clone who obtains with immunity sieve storehouse is a template, carries out PCR, and product is subcloned on prokaryotic expression carrier PET-28a (+) (Novagen company) behind BamHI and EcoRI double digestion.Correct recombinant plasmid transformed e. coli bl21 strain (Novagen company), induced 3 hours through 37 ℃ of 150 rev/mins of 1.0mMIPTG, obtain paramyosin gene recombinant protein (whole open reading frame are totally 885 amino acid), recombinant protein makes purity higher (Fig. 1) through electroelution instrument (BIO-RAD company) wash-out behind His-binding affinity column (Novagen company) purifying.
2. the paramyosin gene recombinant protein of purifying added the equivalent incomplete Freund's adjuvant every 14 days with same dose antigen later on through skin multi-point injection immunity BALB/c mouse (20 μ g/ only), strengthened 2 times.Pluck eyeball after 5 weeks and get blood.ELISA measures antibody titer up to more than the 1:128000.
The immunological characteristic of embodiment 3 paramyosin gene recombinant proteins
Enzyme linked immunological absorption (ELISA) detects
With 1 μ g paramyosin gene recombinant protein (embodiment 2 preparations) is antigen coated 96 hole enzyme plates, one anti-is serum to be checked, two anti-corresponding (goat-anti people, goat-anti rabbit or goat-anti pigs) two for horseradish peroxidase-labeled are anti-, and normal human serum, normal rabbit serum, normal pig serum are respectively as negative control.According to test serum sample OD value greater than 2.1 times of positive judged results of person of negative control.Detect through ELISA, trichonematosis human serum (23 parts) positive rate is 86.96%, trichonematosis rabbit anteserum (2 parts) and trichonematosis porcine blood serum (4 parts) are all positive, the recombinant protein that this paramyosin gene is described has special antigenicity, is the diagnostic reagent with application potential.
2.Western engram analysis
The paramyosin gene expressing protein of purifying (embodiment 2 preparations) is (200ng) behind the SDS-PAGE electrophoresis, be transferred to pvdf membrane, detect with ECL chemical luminescence reagent kit (Amer sham company), respectively with patients serum 1:200, the serum 1:200 of infected pigs, infected rabbits serum 1:1000, paramyosin gene recombinant protein artificial immunization mouse serum (embodiment 2 preparations) 1:10000 as first antibody.The result shows that about 110KDa place all presents a tangible Western blot band (Fig. 2), shows that the paramyosin gene recombinant protein can be patients serum, the porcine blood serum that infects Trichinella spiralis that infects Trichinella spiralis, rabbit anteserum and the paramyosin gene recombinant protein artificial immunization mouse serum that infects Trichinella spiralis is discerned.
Protectiveness experiment in embodiment 4 animal bodies
30 of BALB/c mouse are divided into 2 groups at random, 15 every group.Paramyosin gene recombinant protein immune group adds Freund's complete adjuvant (FCA) subcutaneous injection of equivalent with 20 μ g/ antigen (embodiment 2 preparations) only.Added the equivalent incomplete Freund's adjuvant every 14 days with same dose antigen later on, strengthen 2 times.Last was injected back 10 days, and every mouse carries out challenge infection with 400 trichinzation larvas.The Tris-HCL of control group injection same dose accepts 400 trichinzation larvas simultaneously with immune group and attacks.After attacking 45 days, mouse is put to death, get whole muscle and collect larva, counting with gastric pepsin digestion.Through one-way analysis of variance, paramyosin gene recombinant protein immune group worm reduction rate (muscle larvae worm reduction rate %=(control group muscle larvae number-immune group muscle larvae number)/control group muscle larvae number * 100%) is 47%, compare with control group, significant difference (P＜0.01) (table 1) is arranged.
Because this experiment paramyosin gene recombinant protein produces effective worm reduction rate in animal body, can be used as the protectiveness candidate antigens in view of the above, is applied to trichinous immunoprophylaxis.
The protectiveness result of table 1 paramyosin gene recombinant protein immunity BALB/c mouse
All patents listed above, patent application and publication all are incorporated herein by reference document in full.
Scope of the present invention is not only limited to described particular, and these schemes just illustrate as the indivedual aspects of the present invention single and provide.The composition of functional equivalent and method are all within the scope of the present invention.Really, except the content of shown in this article and description, for the those skilled in the art that read foregoing, all be conspicuous concerning the various changes of the present invention.These changes will fall into the present invention and await the reply within the scope of claim.
Sequence table
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＜120〉trichina paramyosin gene and application thereof
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Claims (15)
1. isolating polynucleotide molecule, its nucleotide sequence is selected from:
(A) sequence of open reading frame among sequence shown in the SEQ ID NO:1 or the SEQ ID NO:1;
(B) with the protein of SEQ ID NO:1 coding identical sequence but because of the degeneracy of genetic code different nucleotide sequence on sequence; With
(C) with (A) or (B) described nucleotide sequence complementary nucleotide sequence.
2. polynucleotide molecule as claimed in claim 1 is characterised in that to have nucleotide sequence shown in the SEQ ID NO:1.
3. isolated polypeptide, its aminoacid sequence is shown in SEQ ID NO:2.
4. recombinant vectors wherein comprises claim 1 or 2 described polynucleotide molecules.
5. the recombinant vectors of claim 4, it is a recombinant expression vector.
6. the host cell that contains the recombinant vectors of claim 5.
7. produce the method for polypeptide, be included in and cultivate the described host cell of claim 6 under the condition that can make described expression of polypeptides and reclaim expressed polypeptide.
8. antibody, but its specificity is in conjunction with the described polypeptide of claim 3.
9. be used for preventing or treating the pharmaceutical composition of animal or human's trichinzation, contain the described polypeptide of claim 3 in the described pharmaceutical composition.
10. be used for diagnosing the diagnostic kit of animal or human's trichinzation, wherein contain claim 1 or 2 described polynucleotide molecules, the described polypeptide of claim 3 or the described antibody of claim 8.
11. claim 1 or 2 described polynucleotide molecules, the described polypeptide of claim 3 or the described antibody of claim 8 are used for diagnosing purposes in animal or human's the diagnostic reagent of trichinzation in preparation.
12. claim 1 or 2 described polynucleotide molecules or the described polypeptide of claim 3 or the described antibody of claim 8 are used for preventing or treating the purposes of the medicine of animal or human's trichinzation in preparation.
13. fusion rotein wherein comprises the described polypeptide of claim 3 and another merges object.
14. the fusion rotein of claim 13, wherein said fusion is to liking beta-galactosidase enzymes, glutathione-S-transferase or polyhistidine.
15. the purposes of the pharmaceutical composition of claim 9, the diagnostic kit of claim 10 or claim 11, wherein said animal is a livestock.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100000181A CN100460509C (en) | 2007-01-04 | 2007-01-04 | Trichina paramyosin gene and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100000181A CN100460509C (en) | 2007-01-04 | 2007-01-04 | Trichina paramyosin gene and application thereof |
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| CN100999737A CN100999737A (en) | 2007-07-18 |
| CN100460509C true CN100460509C (en) | 2009-02-11 |
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| CNB2007100000181A Expired - Fee Related CN100460509C (en) | 2007-01-04 | 2007-01-04 | Trichina paramyosin gene and application thereof |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103243105B (en) * | 2013-05-19 | 2018-11-02 | 中国农业科学院兰州兽医研究所 | A kind of Trichina recombinant protein and application |
| CN105820228B (en) * | 2015-01-09 | 2019-08-16 | 首都医科大学 | The restricted Th epitope P2 of trichina paramyosin H-2d, its composition and application |
| CN113447549B (en) * | 2021-06-22 | 2024-04-02 | 浙江理工大学 | Preparation method of immunosensor modified electrode for fox hair keratin detection |
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| US5958425A (en) * | 1988-09-13 | 1999-09-28 | Rhone Merieux | Viral vaccines |
| CN1159333C (en) * | 2002-07-25 | 2004-07-28 | 首都医科大学 | Trichina antigen gene Ts 87 and use thereof |
| CN1226413C (en) * | 2003-11-13 | 2005-11-09 | 首都医科大学 | Trichina TS88 gene, their protein products and uses |
| EP1705186A1 (en) * | 2005-03-22 | 2006-09-27 | Stiftung Tierärztliche Hochschule Hannover | Vaccine and antibody against Dictyocaulus viviparus |
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2007
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|---|---|---|---|---|
| US5958425A (en) * | 1988-09-13 | 1999-09-28 | Rhone Merieux | Viral vaccines |
| CN1159333C (en) * | 2002-07-25 | 2004-07-28 | 首都医科大学 | Trichina antigen gene Ts 87 and use thereof |
| CN1226413C (en) * | 2003-11-13 | 2005-11-09 | 首都医科大学 | Trichina TS88 gene, their protein products and uses |
| EP1705186A1 (en) * | 2005-03-22 | 2006-09-27 | Stiftung Tierärztliche Hochschule Hannover | Vaccine and antibody against Dictyocaulus viviparus |
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| 抗旋毛虫单克隆抗体细胞株的建立及其特性鉴定. 王晓光等.兽医大学学报，第9卷第2期. 1989 * |
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| CN100999737A (en) | 2007-07-18 |
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