CN102925468A

CN102925468A - Method for cloning and expressing carboxylesterase gene in mouse liver

Info

Publication number: CN102925468A
Application number: CN2012103667253A
Authority: CN
Inventors: 于源华; 周帅; 乔玉龙; 王保学; 张昊; 姚健; 王岩; 王维; 邬磊
Original assignee: CHANGCHUN UNIVERSITY OF SCIENCE AND TECHNOLOGY KANGYUAN BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: CHANGCHUN UNIVERSITY OF SCIENCE AND TECHNOLOGY KANGYUAN BIOLOGICAL TECHNOLOGY Co Ltd; Changchun University of Science and Technology
Priority date: 2012-09-28
Filing date: 2012-09-28
Publication date: 2013-02-13

Abstract

The invention discloses a method for cloning and expressing a carboxylesterase gene in a mouse liver, and belongs to the technical field of molecular biology. The expression of the gene is based on mouse liver total RNA as a template. The gene is proved to be Mceslf by the result of subcloning and sequencing of a DNA fragment which contains an EcoRI enzyme cutting site and an XhOI enzyme cutting site at terminals and is amplified by an RT-polymerase chain reaction (PCR) technology, and the result of sequencing of the DNA fragment by VectorNTI software and comparative analysis of the sequence with a corresponding NCBI sequence. Through the double digestion of the EcoRI and XhOI, the Mceslf is bonded onto the enzyme cutting site of an Escherichia coli high-efficiency expression vector under the action of T4DNAligase and recombinant plasmid PET-32alpha-Mceslf is constructed. The method has the advantages that the engineered bacteria constructed by the method and the recombinase produced thereby can be used to degrade the residual pesticide, so the environment can be improved; and compared with other degrading methods, the method has the advantages that the carboxylesterase is easy to make and low in cost and the degrading efficiency of the carboxylesterase is high.

Description

Mouse liver carboxylesterase gene clonal expression

Technical field

The invention belongs to technical field of molecular biology.

Background technology

Procaine esterase (Carboxylesterase, EC 3.1.1.1) belongs to the B-esterase, can catalytic hydrolysis contains aliphatics and the aromatic hydrocarbons organic compound of carboxylic acid ester groups.Procaine esterase is wide at the organic sphere distributed pole, from archeobacteria and eubacterium, to eukaryotic microorganisms, insect and even mammiferous various organizing active higher Procaine esterase is arranged all, and especially the enzymic activity in the liver is the highest.Procaine esterase can the catalysis ester the reaction with significant application value such as synthetic, the transesterify of hydrolysis, ester, because Procaine esterase has various application in the industry such as food, dyestuff, leather, paper, so Procaine esterase is being played the part of extremely important role at biological technical field.Be applied in addition containing in the industrial and agricultural wastewaters such as pharmacy, dyestuff, sterilant and sterilant the degraded of carboxylicesters base class toxic pollutants.

Be cloned into carboxylesterase gene from people, microorganism and insect at present, wherein obtain the enzyme of 259aa from achromobacter (Achromobacter pichaudii), its highest enzyme is lived and is 0.7U/ml; Obtain the enzyme of 29KDa from stearothermophilus ground bacillus (Geabacillusstearotheromphilus), its highest enzyme is lived and is 700U/ml; Obtain the enzyme of 45KDa from thermophilic brailer group bacterium (Dictyoglomus turgidum), its highest enzyme is lived and is 80U/mg; Obtain the enzyme of 100KDa from bollworm (Helicoverpa armigera), its highest enzyme is lived and is 0.32mmol/100ul; Obtain the enzyme of 60KDa from resistance mosquito (Culex quinquefasciatus), its highest enzyme is lived and is 135U/mg.All clone from sources such as prodenia litura (Spodoptera litura), plumbous blue or green streptomycete (Deduc Streptomyces lividans), wild mountain silkworm (Bombyx momdarina M), lucilia cuprina (Lucilia cuprina), Nilaparvata lugen (brown planthopper) (Nilaparvata lugens), rat (rat) and people's tire brains in addition and obtain corresponding Procaine esterase.

Yet the Procaine esterase report that the clone obtains from mouse liver is so far seldom used also seldom (13 pieces of research papers, NCBI PUBMID database retrieval) to its research abroad, domestic so far there are no research report.

Summary of the invention

The objective of the invention is: a kind of mouse liver carboxylesterase gene clonal expression is provided, it uses gene clone technology, successfully be cloned into mouse liver Procaine esterase (Mces1f) gene, in e. coli bl21, carried out the high efficient expression of this gene after being connected with expression vector PET-32a.And the agricultural chemicals such as mouse liver Procaine esterase (Mces1f) gene pairs Furadan, SevinCarbaryl, parathion-methyl, bifenthrin have Degradation.

Expression of the present invention is:

1. take the total RNA of mouse liver as template, by the RT-PCR technology terminal dna fragmentation that contains EcoR I and Xho I restriction enzyme site that increased, it subclone and order-checking have been carried out, with software Vector NTI mensuration sequence is compared with corresponding NCBI sequence (GenBank:BC013479.1), determine that this gene is Mces1f.

EcoR I initiator codon

GAATTC ATG TTCCTTAGCACTCTGTTCCTGGTGTCTCTAGCAACCTGTGTGATTTGCGGAAATCCCTCTTCACCACCTGTGGTAGACACTGCTCATGGTAAAGTCCTGGGGAAACACGTGAACGTAGAAGGATTTTCACAGCCTGTGGCCGTCTTCCTGGGAATCCCCTTTGCCAAGCCCCCTCTTGGATCCCTGAGGTTTGCTCCACCACAGCCTGCAGAGCCCTGGAGCTCAGTGAAGAATGCCACCACCTACCCACCTATGTGCTCCCAAGATGCAGCTAGAGGACAGGCGGTCAATGACCTCATAACCAATAGAAAGGAGAAAATCCATCTTGAATTTTCTGAAGATTGCCTGTACCTAAATATTTACACTCCTGCGGACTTTTCAAAGAACAGTAGGCTACCGGTGATGGTGTGGATCCATGGAGGTGGACTGAAGCTGGGTGGGGCATCAAGCTTTGATGGACGGGCTCTCTCTGCATACGAAAATGTGGTGGTGGTGGCCATTCAATATCGCCTGAGTATCTGGGGATTCTTCAGCACAGGGGATGAACACAGTCGGGGAAACTGGGGTCATTTGGACCAAGTGGCTGCTCTGCATTGGGTCCAGGACAACATTGCCAACTTTGGCGGGGACCCAGGCTCTGTGACCATCTTTGGAGAGTCAGCAGGAGGTTACAGTGTCTCAATTCTTATATTGTCCCCATTGTCCAAGAACCTCTTCCACAGTGCCATTTCTGAGAGTGGTGTGGCCTTCATTCCTGGAATGTTTACCAAAGATGTGAGGCCAATTACTGAGCAAATTGCTGTTACTGCTGGCTGTAAGACCACCACGTCTGCCGTCATTGTTCACTGCATGCGCCAGAAGACGGAGGAGGAGCTATTAGAGATCATGCATAAATTGAATCTGTATAAACTGAGTTTACAAGGAGATACCAAAAATAGCGACCAGTTCGTGACAAGTGTGCTTGATGGAGTGGTGCTACCAAAGGACCCCAAAGAGATCCTGGCTGAGAAGAACTTCAACACTGTGCCTTACATTGTGGGAATCAACAAGCAAGAATGTGGCTGGCTTCTGCCAACAATGACGGGATTTCTACCAGCTGATGTAAAATTGGACAAGAAGAAAGCCATTGCACTCCTGGAGCAATTTGCTTCCATGACTGGCATACCAGAGGATATTATTCCAGTTGCTGTTGAGAAGTACACAAAAGGTAGTGATGACCCTGATCAGATCAGAGAGGGAGTTCTCGACGCAATGGGGGATGTGGCATTTGGTGTTCCATCGGTGATTGTGTCCCGTGGCCACAGAGACACTGGAGCTCCCACCTACATGTATGAGTATCAATACTACCCAAGCTTCTCATCACCCCAAAGACCCAAGAATGTAGTAGGAGACCATGCAGATGATGTCTACTCTGTCTTCGGTGCTCCAATTTTAAGAGAGGGTGCCTCCGAAGAGGAGATCAATCTCAGCAAGATGGTGATGAAATCCTGGGCCAACTTTGCTCGGAATGGGAACCCTAATGGCAAAGGGCTGCCTCATTGGCCAAAGTATGATCAGAAAGAAGGATATCTTCATATTGGTGGCACCACCCAGCAAGCCCAG TGACTGAAGGAGGAGGAAGTGACTTTC

TGGACACAGTCCCTTGCCAAGAAACAACCCCAGCCATACCACAATGAGCTG TGA CTCGAG

Terminator codon Xho I

2. carry out double digestion (double digestion) by EcoR I and Xho I, make the Mces1f gene be connected to construction recombination plasmid PET-32a-Mces1f on the EcoR I of escherichia coli high-level expression carrier PET-32a and the Xho I restriction enzyme site through T4 dna ligase (T4 DNA ligase) effect, transform and express Host Strains BL21(DE3) (Studier F W, et al.J.Mol.Biol.1986,189:113), carry out the IPTG abduction delivering.Research finds that this enzyme forms (as follows) by 535 amino-acid residues, owing to selecting PET-32a as expression vector, SDS-PAGE shows that molecular weight is 76.9KD, and is the same with expection.

MFLSTLFLVSLATCVICGNPSSPPVVDTAHGKVLGKHVNVEGFSQPVAVFLGIPFAKPPLGSLRFAPPQPAEPWSSVKNATTYPPMCSQDAARGQAVNDLITNRKEKIHLEFSEDCLYLNIYTPADFSKNSRLPVMVWIHGGGLKLGGASSFDGRALSAYENVVVVAIQYRLSIWGFFSTGDEHSRGNWGHLDQVAALHWVQDNIANFGGDPGSVTIFGESAGGYSVSILILSPLSKNLFHSAISESGVAFIPGMFTKDVRPITEQIAVTAGCKTTTSAVIVHCMRQKTEEELLEIMHKLNLYKLSLQGDTKNSDQFVTSVLDGVVLPKDPKEILAEKNFNTVPYIVGINKQECGWLLPTMTGFLPADVKLDKKKAIALLEQFASMTGIPEDIIPVAVEKYTKGSDDPDQIREGVLDAMGDVAFGVPSVIVSRGHRDTGAPTYMYEYQYYPSFSSPQRPKNVVGDHADDVYSVFGAPILREGASEEEINLSKMVMKSWANFARNGNPNGKGLPHWPKYDQKEGYLHIGGTTQQAQ

3. adopt Ni-post affinity chromatography, the expression product of Mces1f has been carried out purifying, obtained the Mces1f of high purity (more than 90%).

4. adopt the Fast blue B method to record total soluble protein, the soluble recombinant protein of purifying, the enzyme activity concentration that the renaturing inclusion bodies recombinant protein of the total recombinant protein of inclusion body and purifying is corresponding is respectively 209U/ml, 537U/ml, 3122U/ml, 7727U/ml, and the enzyme specific activity is respectively 109.4U/mg, 257.1U/mg, 2584.4U/mg, 5396.0U/mg.

5. the expression product of Mces1f in the intestinal bacteria is done biodegradable determination of activity.Take Furadan, SevinCarbaryl, parathion-methyl and bifenthrin as substrate, (HPLC) carries out the determination of residual amount with high performance liquid chromatography, and research finds that this enzyme is all very strong to the biological activity of Furadan, SevinCarbaryl, parathion-methyl and bifenthrin.

The invention has the beneficial effects as follows: utilize the pesticide residue in the recombinase degraded environment of engineering bacteria that the present invention makes up and generation thereof, improve environment, to compare the enzyme making method simple with other degradation method for it, and production cost is low and degradation efficiency is high.

Use gene clone technology, successfully be cloned into mouse liver Procaine esterase (Mces1f) gene, in e. coli bl21, carried out the high efficient expression of this gene after being connected with expression vector PET-32a.Research finds that this enzyme is comprised of 535 amino-acid residues, SDS-PAGE shows that molecular weight is about 76.9KD, the maximum enzyme vigor concentration of gained enzyme is 7727U/ml, and the maximum enzyme specific activity is 5396U/mg, has cloned the Procaine esterase enzyme that obtains than all and has lived all high.Research finds that Mces1f has Degradation to agricultural chemicals such as Furadan, SevinCarbaryl, parathion-methyl, bifenthrins, and degradation efficiency is very high.

Description of drawings:

Fig. 1 Procaine esterase Mces1f gene agarose is identified

Line1:2000marker

Line2:Mces1f gene PCR product

The building process schematic diagram of Fig. 2 plasmid PET-32a-Mces1f

Fig. 3 SDS-PAGE detects Mces1f gene Expression in Escherichia coli

Line1:BL21(DE3)-pET-32a-Mces1f bacterium inclusion body total protein

Line2:BL21(DE3)-pET-32a-Mces1f bacterium total soluble protein

Line3: low molecular weight protein (LMWP) Marker

Line4:BL21(DE3) containing pET-32a-Mes1f induces

Line5:BL21(DE3) containing pET-32a-Mes1f does not induce

Line6:BL21(DE3) contain pET-32a: induce

Line7:BL21(DE3) containing pET-32a does not induce

Line8:BL21(DE3) induce

Line9:BL21(DE3) do not induce

Fig. 4 SDS-PAGE detects the Mces1f purification result

Line1:BL21(DE3)-pET-32a-Mces1f bacterium total protein

Line2:BL21(DE3)-pET-32a-Mces1f bacterium total soluble protein

Line3: the soluble M ces1f of ni-sepharose purification

Line4: low molecular weight protein (LMWP) Marker

Line5: the Mces1f inclusion body of ni-sepharose purification

Fig. 5 naphthyl alcohol typical curve

The blank group of Fig. 6 Mces1f degraded 50mg/L Furadan HPLC collection of illustrative plates

5 hours HPLC collection of illustrative plates of Fig. 7 Mces1f degraded 50mg/L Furadan

12 hours HPLC collection of illustrative plates of Fig. 8 Mces1f degraded 50mg/L Furadan

24 hours HPLC collection of illustrative plates of Fig. 9 Mces1f degraded 50mg/L Furadan

The blank group of Figure 10 Mces1f degraded 50mg/L SevinCarbaryl HPLC collection of illustrative plates

5 hours HPLC collection of illustrative plates of Figure 11 Mces1f degraded 50mg/L SevinCarbaryl

12 hours HPLC collection of illustrative plates of Figure 12 Mces1f degraded 50mg/L SevinCarbaryl

24 hours HPLC collection of illustrative plates of Figure 13 Mces1f degraded 50mg/L SevinCarbaryl

The blank group of Figure 14 Mces1f degraded 10mg/L parathion-methyl HPLC collection of illustrative plates

5 hours HPLC collection of illustrative plates of Figure 15 Mces1f degraded 10mg/L parathion-methyl

12 hours HPLC collection of illustrative plates of Figure 16 Mces1f degraded 10mg/L parathion-methyl

24 hours HPLC collection of illustrative plates of Figure 17 Mces1f degraded 10mg/L parathion-methyl

48 hours HPLC collection of illustrative plates of Figure 18 Mces1f degraded 10mg/L parathion-methyl

The blank group of Figure 19 Mces1f degraded 10mg/L bifenthrin HPLC collection of illustrative plates

5 hours HPLC collection of illustrative plates of Figure 20 Mces1f degraded 10mg/L bifenthrin

12 hours HPLC collection of illustrative plates of Figure 21 Mces1f degraded 10mg/L bifenthrin

24 hours HPLC collection of illustrative plates of Figure 22 Mces1f degraded 10mg/L bifenthrin

Embodiment:

Embodiment 1.Mces1f gene cloning and expression

1.Mces1f gene cloning

1.1 the extraction of the total RNA of mouse liver

Adopt TIAN GEN RNAprep pure total RNA from animal tissues to extract test kit and extract the total RNA of mouse liver.

1.2 by RT-PCR amplification Mces1f gene

1.2.1cDNA the first chain is synthetic

Utilize TINA GEN TIANScript cDNA the first chain synthetic agent box to synthesize cDNA the first chain, concrete steps are as follows:

1) in the centrifuge tube of the nuclease free of ice bath, add following reaction mixture:

10.5ul total RNA extracting solution;

2ul oligo（dT）15；

2ul dNTP（2.5mM each）；

2) 70 ℃ the heating 5min after rapidly at cooled on ice 2min.Add following each component behind the brief centrifugal collection reaction solution:

4ul 5XFirst-Strand Buffer (containing DTT); 0.5ulRNasin;

3) add 1ul(200U) TIANScript M-MLV, use the pipettor mixing gently;

4) 42 ℃ of temperature are bathed 50min;

5) 95 ℃ of heating 5min termination reactions;

6) use RNase-free-ddH ₂O is diluted to 50ul with reaction system, and this is cDNA the first chain.

1.2.2Mces1f the pcr amplification of gene fragment

Take 5 ＇-GGAATTCATGTTCCTTAGCACTCTGTTCCT-3 ＇ and 5 ＇-CTCGAGTCACAGCTCATTGTGGTATGG-3 ＇ as primer (it is synthetic that primer is given birth to the worker by Shanghai), take synthetic cDNA the first chain of 1.2.1 as template, carry out under the following conditions pcr amplification:

Reaction system:

Template 2ul, primer 1 1ul, primer 2 1ul, 10X PCR buffer 5ul, Taq polysaccharase 1ul, dNTP 4ul, distilled water 36ul.

The pcr amplification condition:

A. 94 ℃ of sex change 5min;

B. 94 ℃ of sex change 45s, 54 ℃ of annealing 2min, 72 ℃ are extended 2min, carry out 35 circulations;

C. 72 ℃ are extended 10min;

D. 4 ℃ of preservations are placed to room temperature, and 1% agarose gel electrophoresis detects.

1.2.3 the determination and analysis of sequence of Mces1f gene

1% agarose gel electrophoresis is detected the sizeable fragment that obtains, adopt sepharose to reclaim test kit (worker is given birth in Shanghai) and carry out the recovery of purpose fragment, detect concentration and the purity (such as Fig. 1) that reclaims fragment through 0.8% agarose gel electrophoresis, afterwards under the effect of T4 dna ligase, with pUCm-T carrier (worker is given birth in Shanghai), 22 ℃ connect 16h, construction recombination plasmid pUCm-T-Mces1f.Adopt CaCl ₂Transformed competence colibacillus bacillus coli DH 5 alpha (TaKaBa) carries out the amicillin resistance screening, and the single bacterium colony on the picking flat board carries out PCR and double digestion is identified, the subclone that preliminary evaluation is correct is sent to the living worker in Shanghai and carries out dna sequencing.

Sequencing result out after, utilize Vector nti 11.5.1 software that mensuration sequence is analyzed, find that the dna fragmentation of cloning is a complete open reading frame, its full length gene 1686bp, because causing extracting, sudden change stops 535 amino-acid residues of finally encoding.

1.3 the expression and purification of Mces1f

1.3.1 the structure of recombinant plasmid PET-32a-Mces1f (such as Fig. 2)

Plasmid pUCm-T-Mces1f cuts with EcoR I and Xho I enzyme, reclaim the small segment of 1698bp, be connected to the plasmid pET-32a(Novagen that cuts through EcoR I and Xho I enzyme) on, obtained correct recombinant plasmid, change this plasmid over to Host Strains BL21(DE3).

1.3.2 the abduction delivering of Mces1f gene in intestinal bacteria

The BL21(DE3 that will contain plasmid PET-32a-Mces1f) bacterial strain is seeded in (containing final concentration is the 100ug/ml penbritin) in the LB liquid nutrient medium according to the ratio of 1:50,37 ℃ of isothermal vibration shaking table overnight incubation (180rpm) are used as seed liquor.

Seed liquor is transferred to (containing final concentration is the 100ug/ml penbritin) in the fresh LB liquid nutrient medium according to the inoculum size of 1:20, and 37 degrees centigrade, the 180rpm shaking table is cultivated 2-3h.

Add IPTG(final concentration 1mM), continue shaking table and cultivate 5h.Centrifugal 10min under 10000rpm gets precipitation (thalline), and adding 1ml 8M urea is resuspended, and 4 ℃ of refrigerator overnight are got the expression that 10ml carries out 12%SDS-PAGE detected through gel electrophoresis target protein band.SDS-PAGE detected result (Fig. 3) shows the protein band that clauses and subclauses are arranged near 76.9KD, with the expection in the same size.

1.3.3 the separation and purification of Mces1f

Adopt the method abduction delivering engineering bacteria of 1.2.5, under 4 ℃ of conditions, 10000rpm, the centrifugal collection thalline of 10min; Bacterial sediment suspends with 0.2M phosphoric acid buffer (pH7.0), and add N,O-Diacetylmuramidase (100ug/ml) and carry out cell supersonic wave fragmentation (power 300W, ultrasonic 5 seconds, repeated 30 times at interval 9 seconds), 10000rpm, centrifugal 15min gets supernatant, obtains the enzyme storing solution.

The enzyme storing solution carries out Ni-post affinity chromatography.Wash with the 1mM of 10 times of column volumes and the imidazoles of 20mM respectively, use at last the imidazoles wash-out of 200mM, elutriant is used first 0.2M phosphoric acid buffer (pH7.0) dialysis, concentrates with sucrose again, detects purity with SDS-PAGE.The result shows, through above purge process, has obtained the Mces1f target protein (such as Fig. 4) of purity high (90%).

Adopt Fast blue B method (A study of Housefly esterases by means of a sensitive colorimeteric menthod

K.VAN ASPEREN 1962) enzymic activity of survey Mces1f.

2.1 required reagent

0.03M phosphate buffered saline buffer (PH7.0): Na ₂HPO ₄5.373g, NaH ₂PO ₄2.340g, dissolve respectively and be settled to 500ml, transfer PH to 7.0 after mixing;

10mM naphthyl alcohol standardized solution: claim the 1.441g naphthyl alcohol to be dissolved in the dehydrated alcohol, and be settled to 100ml;

0.03M α-naphthaleneaceticacidester solution: claim the 0.2793g α-naphthaleneaceticacidester to be dissolved in the dehydrated alcohol, and be settled to 50ml;

DBLS (Fast blue B developer): the 1g Fast blue B is dissolved in the dual water of 100ml, and 12.5g SDS is dissolved in the dual water of 250ml, filters stand-by after two kinds of solution are mixed;

Na ₂HPO ₄, NaH ₂PO ₄Be analytical pure with naphthyl alcohol, α-naphthaleneaceticacidester is available from Aladdin, and Fast blue B is available from Ourchem.

2.3 the preparation of naphthyl alcohol typical curve

Naphthyl alcohol standardized solution with 10mM is the different concns gradient with the dilution of 0.03M phosphate buffered saline buffer first, mix rear 27 ℃ of water-baths 10 minutes with 500ul DBLS, colorimetric under 600nm, OD600 value under the record concentration gradient, then take naphthyl alcohol concentration as X-coordinate, make naphthyl alcohol typical curve (such as Fig. 5) take the OD600 value as ordinate zou.

2.3 Mces1f enzyme assay

Adopt the Xylene Brilliant Cyanine G method to record the Mces1f total soluble protein, the solubility target protein of purifying, the total recombinant protein of inclusion body, the protein concentration of the renaturing inclusion bodies target protein of purifying is respectively: 1.911 mg/ml, 2.089 mg/ml, 1.208 mg/ml, 1.432mg/ml.

The enzyme that adopts the Fast blue B method to survey above four kinds of albumen is lived, specific practice is: 2.5ml α-naphthaleneaceticacidester (containing the 0.0003M α-naphthaleneaceticacidester) mixes rear 27 ℃ and reacts half an hour with the 500ul protein solution, adds 500ulDBLS after reaction finishes and places 27 ℃ of reactions to survey colorimetric under the 600nm after 10 minutes.Calculate corresponding naphthyl alcohol output according to typical curve.

Unit of enzyme activity's definition: catalysis produces the required enzyme amount of 1 μ M naphthyl alcohol and is defined as an enzyme unit, i.e. 1U=1mmolL-1min-1 alive in the unit time.

Concentration of enzymatic activity definition: the enzyme activity unit number that unit volume is contained, i.e. 1UL-1

Specific activity of enzyme definition: the enzyme activity unit number that unit mg enzyme has, i.e. 1Umg-1.

The amount that draws as calculated the generation α that albumen is corresponding in four-Ding naphthols is respectively 0.126 μ mol/L, 0.321 μ mol/L, 1.873 μ mol/L, 4.637 μ mol/L; Corresponding enzyme activity concentration is 209U/ml, 537U/ml, 3122U/ml, 7727U/ml; The enzyme specific activity is respectively 109.4U/mg, 257.1U/mg, 2584.4U/mg, 5396.0U/mg.

The acquisition of Mces1f and be used for degraded to agricultural chemicals such as Furadan, SevinCarbaryl, parathion-methyl and bifenthrins

3.1 the acquisition of Mces1f is with reference to the prepared enzyme storing solution of embodiment 1.3.3.

3.2 required reagent

Furadan mark product mother liquor: 1g/L; SevinCarbaryl mark product mother liquor: 4 g/L; Parathion-methyl mark product mother liquor: 1 g/L; Bifenthrin mark product mother liquor: 1 g/L;

Wherein Furadan, SevinCarbaryl, parathion-methyl are mark product (purity is more than 96%) available from China National Measuring Science Research Inst..Bifenthrin is mark product (purity is 96.6%) available from the Shanghai Pesticide Research Institute.

3.3 Mces1f is to the Degradation of the agricultural chemicals such as Furadan, SevinCarbaryl, parathion-methyl and bifenthrin

3.3.1 Mces1f is to the Degradation of Furadan

The enzyme liquid of 2ml mixes with 10ml Furadan (50mg/L) and is placed in 27 ℃ of constant-temperature shaking incubators, 200rpm reaction 5 hours, 12 hours, 24 hours, as blank, after the 0.22um filter filters, be C18 reverse-phase chromatographic column with the HPLC(chromatographic column with four reaction solutions with 2ml 0.03M phosphate buffered saline buffer (PH7.0) then; Moving phase is methanol/water=70/30; Flow velocity is 1.0ml/mim; The detection wavelength is 210nm; Sample size is 10ul; Column temperature is 30 ℃) detect the Furadan residual quantity, (as Fig. 6-Fig. 9) demonstration Mces1f has Degradation to Furadan, blank group retention time was 3.588 to the result in 0 hour, and peak area is 3053185; Retention time was 3.570 in 5 hours, and peak area is 2384557, and degradation rate is 21.90%; Retention time was 3.575 in 12 hours, and peak area is 2223100, and degradation rate is 27.19%; Retention time was 3.577 in 24 hours, and peak area is 2164976, and degradation rate is 29.09%.

3.3.2 Mces1f is to the Degradation of SevinCarbaryl

The enzyme liquid of 2ml mixes with 10ml SevinCarbaryl (50mg/L) and is placed in 27 ℃ of constant-temperature shaking incubators, 200rpm reaction 5 hours, 12 hours, 24 hours, as blank, after the 0.22um filter filters, be C18 reverse-phase chromatographic column with the HPLC(chromatographic column with four reaction solutions with 2ml 0.03M phosphate buffered saline buffer (PH7.0) then; Moving phase is methanol/water=60/40; Flow velocity is 1.0ml/mim; The detection wavelength is 220nm; Sample size is 10ul; Column temperature is 30 ℃) detect the SevinCarbaryl residual quantity, (as Figure 10-Figure 13) demonstration Mces1f has Degradation to SevinCarbaryl, blank group retention time was 6.156 to the result in 0 hour, and peak area is 8795249; Retention time was 6.155 in 5 hours, and peak area is 7769906, and degradation rate is 11.66%; Retention time was 6.160 in 12 hours, and peak area is 6593095, and degradation rate is 25.04%; Retention time was 6.148 in 24 hours, and peak area is 6270043, and degradation rate is 28.71%.

3.3.3 Mces1f is to the Degradation of parathion-methyl

The enzyme liquid of 2ml mixes with 10ml parathion-methyl (10mg/L) and is placed in 27 ℃ of constant-temperature shaking incubators, 200rpm reaction 5 hours, 12 hours, 24 hours and 48 hours, as blank, after the 0.22um filter filters, be C18 reverse-phase chromatographic column with the HPLC(chromatographic column with four reaction solutions with 2ml 0.03M phosphate buffered saline buffer (PH7.0) then; Moving phase is methanol/water=60/40; Flow velocity is 0.6ml/mim; The detection wavelength is 270nm; Sample size is 50ul; Column temperature is 30 ℃) detect the parathion-methyl residual quantity, (as Figure 14-Figure 18) demonstration Mces1f has Degradation to parathion-methyl, blank group retention time was 6.297 to the result in 0 hour, and peak area is 785679; Retention time was 6.184 in 5 hours, and peak area is 765334, and degradation rate is 2.59%; Retention time was 6.199 in 12 hours, and peak area is 756590, and degradation rate is 3.70%; Retention time was 6.196 in 24 hours, and peak area is 419315, and degradation rate is 46.6%; Retention time was 6.200 in 48 hours, and peak area is 228778, and degradation rate is 70.88%.

3.3.4 Mces1f is to the Degradation of bifenthrin

The enzyme liquid of 2ml mixes with 10ml bifenthrin (10mg/L) and is placed in 27 ℃ of constant-temperature shaking incubators, 200rpm reaction 5 hours, 12 hours, 24 hours, as blank, after the 0.22um filter filters, be C18 reverse-phase chromatographic column with the HPLC(chromatographic column with four reaction solutions with 2ml 0.03M phosphate buffered saline buffer (PH7.0) then; Moving phase is methanol/water=80/20; Flow velocity is 0.6ml/mim; The detection wavelength is 254nm; Sample size is 10ul; Column temperature is 30 ℃) detect the bifenthrin residual quantity, (as Figure 19-Figure 22) demonstration Mces1f has Degradation to bifenthrin, blank group retention time was 3.350 to the result in 0 hour, and peak area is 2960930; Retention time was 3.340 in 5 hours, and peak area is 2733521, and degradation rate is 7.68%; Retention time was 3.340 in 12 hours, and peak area is 1553286, and degradation rate is 47.54%; Retention time was 3.360 in 24 hours, and peak area is 656201, and degradation rate is 77.84%.

＜110〉Changchun University of Science and Technology

＜120〉mouse liver carboxylesterase gene clonal expression and application

〈160〉2

〈210〉1

〈211〉1698

〈212〉DNA

＜213〉mouse (Mures)

〈220〉

〈221〉CDS

〈222〉(7) ...（1611）

＜223〉the total 1698bp of this dna sequence dna, wherein the 1612-1614 position is terminator codon TAG, 7-1611 encoding mature, this maturation contains 535 amino acid.

〈400〉1

gaattc atg ttc ctt agc act ctg ttc ctg gtg tct cta gca acc tgt gtg att 54

Met Phe Leu Ser Thr Leu Phe Leu Val Ser Leu Ala Thr Cys Val Ile

1 5 10 15

tgc gga aat ccc tct tca cca cct gtg gta gac act gct cat ggt 99

Cys Gly Asn Pro Ser Ser Pro Pro Val Val Asp Thr Ala His Gly

20 25 30

aaa gtc ctg ggg aaa cac gtg aac gta gaa gga ttt tca cag cct 144

Lys Val Leu Gly Lys His Val Asn Val Glu Gly Phe Ser Gln Pro

35 40 45

gtg gcc gtc ttc ctg gga atc ccc ttt gcc aag ccc cct ctt gga 189

Val Ala Val Phe Leu Gly Ile Pro Phe Ala Lys Pro Pro Leu Gly

50 55 60

tcc ctg agg ttt gct cca cca cag cct gca gag ccc tgg agc tca 234

Ser Leu Arg Phe Ala Pro Pro Gln Pro Ala Glu Pro Trp Ser Ser

65 70 75

gtg aag aat gcc acc acc tac cca cct atg tgc tcc caa gat gca 279

Val Lys Asn Ala Thr Thr Tyr Pro Pro Met Cys Ser Gln Asp Ala

80 85 90

gct aga gga cag gcg gtc aat gac ctc ata acc aat aga aag gag 324

Ala Arg Gly Gln Ala Val Asn Asp Leu Ile Thr Asn Arg Lys Glu

95 100 105

aaa atc cat ctt gaa ttt tct gaa gat tgc ctg tac cta aat att 369

Lys Ile His Leu Glu Phe Ser Glu Asp Cys Leu Tyr Leu Asn Ile

110 115 120

tac act cct gcg gac ttt tca aag aac agt agg cta ccg gtg atg 414

Tyr Thr Pro Ala Asp Phe Ser Lys Asn Ser Arg Leu Pro Val Met

125 130 135

gtg tgg atc cat gga ggt gga ctg aag ctg ggt ggg gca tca agc 459

Val Trp Ile His Gly Gly Gly Leu Lys Leu Gly Gly Ala Ser Ser

140 145 150

ttt gat gga cgg gct ctc tct gca tac gaa aat gtg gtg gtg gtg 504

Phe Asp Gly Arg Ala Leu Ser Ala Tyr Glu Asn Val Val Val Val

155 160 165

gcc att caa tat cgc ctg agt atc tgg gga ttc ttc agc aca ggg 549

Ala Ile Gln Tyr Arg Leu Ser Ile Trp Gly Phe Phe Ser Thr Gly

170 175 180

gat gaa cac agt cgg gga aac tgg ggt cat ttg gac caa gtg gct 594

Asp Glu His Ser Arg Gly Asn Trp Gly His Leu Asp Gln Val Ala

185 190 195

gct ctg cat tgg gtc cag gac aac att gcc aac ttt ggc ggg gac 639

Ala Leu His Trp Val Gln Asp Asn Ile Ala Asn Phe Gly Gly Asp

200 205 210

cca ggc tct gtg acc atc ttt gga gag tca gca gga ggt tac agt 684

Pro Gly Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Gly Tyr Ser

215 220 225

gtc tca att ctt ata ttg tcc cca ttg tcc aag aac ctc ttc cac 729

Val Ser Ile Leu Ile Leu Ser Pro Leu Ser Lys Asn Leu Phe His

230 235 240

agt gcc att tct gag agt ggt gtg gcc ttc att cct gga atg ttt 774

Ser Ala Ile Ser Glu Ser Gly Val Ala Phe Ile Pro Gly Met Phe

245 250 255

acc aaa gat gtg agg cca att act gag caa att gct gtt act gct 819

Thr Lys Asp Val Arg Pro Ile Thr Glu Gln Ile Ala Val Thr Ala

260 265 270

ggc tgt aag acc acc acg tct gcc gtc att gtt cac tgc atg cgc 864

Gly Cys Lys Thr Thr Thr Ser Ala Val Ile Val His Cys Met Arg

275 280 285

cag aag acg gag gag gag cta tta gag atc atg cat aaa ttg aat 909

Gln Lys Thr Glu Glu Glu Leu Leu Glu Ile Met His Lys Leu Asn

290 295 300

ctg tat aaa ctg agt tta caa gga gat acc aaa aat agc gac cag 954

Leu Tyr Lys Leu Ser Leu Gln Gly Asp Thr Lys Asn Ser Asp Gln

305 310 315

ttc gtg aca agt gtg ctt gat gga gtg gtg cta cca aag gac ccc 999

Phe Val Thr Ser Val Leu Asp Gly Val Val Leu Pro Lys Asp Pro

320 325 330

aaa gag atc ctg gct gag aag aac ttc aac act gtg cct tac att 1044

Lys Glu Ile Leu Ala Glu Lys Asn Phe Asn Thr Val Pro Tyr Ile

335 340 345

gtg gga atc aac aag caa gaa tgt ggc tgg ctt ctg cca aca atg 1089

Val Gly Ile Asn Lys Gln Glu Cys Gly Trp Leu Leu Pro Thr Met

350 355 360

acg gga ttt cta cca gct gat gta aaa ttg gac aag aag aaa gcc 1134

Thr Gly Phe Leu Pro Ala Asp Val Lys Leu Asp Lys Lys Lys Ala

365 370 375

att gca ctc ctg gag caa ttt gct tcc atg act ggc ata cca gag 1179

Ile Ala Leu Leu Glu Gln Phe Ala Ser Met Thr Gly Ile Pro Glu

380 385 390

gat att att cca gtt gct gtt gag aag tac aca aaa ggt agt gat 1224

Asp Ile Ile Pro Val Ala Val Glu Lys Tyr Thr Lys Gly Ser Asp

395 400 405

gac cct gat cag atc aga gag gga gtt ctc gac gca atg ggg gat 1269

Asp Pro Asp Gln Ile Arg Glu Gly Val Leu Asp Ala Met Gly Asp

410 415 420

gtg gca ttt ggt gtt cca tcg gtg att gtg tcc cgt ggc cac aga 1314

Val Ala Phe Gly Val Pro Ser Val Ile Val Ser Arg Gly His Arg

425 430 435

gac act gga gct ccc acc tac atg tat gag tat caa tac tac cca 1359

Asp Thr Gly Ala Pro Thr Tyr Met Tyr Glu Tyr Gln Tyr Tyr Pro

440 445 450

agc ttc tca tca ccc caa aga ccc aag aat gta gta gga gac cat 1404

Ser Phe Ser Ser Pro Gln Arg Pro Lys Asn Val Val Gly Asp His

455 460 465

gca gat gat gtc tac tct gtc ttc ggt gct cca att tta aga gag 1449

Ala Asp Asp Val Tyr Ser Val Phe Gly Ala Pro Ile Leu Arg Glu

470 475 480

ggt gcc tcc gaa gag gag atc aat ctc agc aag atg gtg atg aaa 1494

Gly Ala Ser Glu Glu Glu Ile Asn Leu Ser Lys Met Val Met Lys

485 490 495

tcc tgg gcc aac ttt gct cgg aat ggg aac cct aat ggc aaa ggg 1539

Ser Trp Ala Asn Phe Ala Arg Asn Gly Asn Pro Asn Gly Lys Gly

500 505 510

ctg cct cat tgg cca aag tat gat cag aaa gaa gga tat ctt cat 1584

Leu Pro His Trp Pro Lys Tyr Asp Gln Lys Glu Gly Tyr Leu His

515 520 525

att ggt ggc acc acc cag caa gcc cag tgactgaaggaggaggaagtgactttct 1639

Ile Gly Gly Thr Thr Gln Gln Ala Gln ＊

530 535

ggacacagtcccttgccaagaaacaaccccagccataccacaatgagctgtgactcgag 1698

〈210〉2

〈211〉535

〈212〉PRT

＜213〉mouse (Mures)

〈220〉

〈222〉（1）...（535）

〈400〉2

Met Phe Leu Ser Thr Leu Phe Leu Val Ser Leu Ala Thr Cys Val

1 5 10 15

Ile Cys Gly Asn Pro Ser Ser Pro Pro Val Val Asp Thr Ala His

20 25 30

Gly Lys Val Leu Gly Lys His Val Asn Val Glu Gly Phe Ser Gln

35 40 45

Pro Val Ala Val Phe Leu Gly Ile Pro Phe Ala Lys Pro Pro Leu

50 55 60

Gly Ser Leu Arg Phe Ala Pro Pro Gln Pro Ala Glu Pro Trp Ser

65 70 75

Ser Val Lys Asn Ala Thr Thr Tyr Pro Pro Met Cys Ser Gln Asp

80 85 90

Ala Ala Arg Gly Gln Ala Val Asn Asp Leu Ile Thr Asn Arg Lys

95 100 105

Glu Lys Ile His Leu Glu Phe Ser Glu Asp Cys Leu Tyr Leu Asn

110 115 120

Ile Tyr Thr Pro Ala Asp Phe Ser Lys Asn Ser Arg Leu Pro Val

125 130 135

Met Val Trp Ile His Gly Gly Gly Leu Lys Leu Gly Gly Ala Ser

140 145 150

Ser Phe Asp Gly Arg Ala Leu Ser Ala Tyr Glu Asn Val Val Val

155 160 165

Val Ala Ile Gln Tyr Arg Leu Ser Ile Trp Gly Phe Phe Ser Thr

170 175 180

Gly Asp Glu His Ser Arg Gly Asn Trp Gly His Leu Asp Gln Val

185 190 195

Ala Ala Leu His Trp Val Gln Asp Asn Ile Ala Asn Phe Gly Gly

200 205 210

Asp Pro Gly Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Gly Tyr

215 220 225

Ser Val Ser Ile Leu Ile Leu Ser Pro Leu Ser Lys Asn Leu Phe

230 235 240

His Ser Ala Ile Ser Glu Ser Gly Val Ala Phe Ile Pro Gly Met

245 250 255

Phe Thr Lys Asp Val Arg Pro Ile Thr Glu Gln Ile Ala Val Thr

260 265 270

Ala Gly Cys Lys Thr Thr Thr Ser Ala Val Ile Val His Cys Met

275 280 285

Arg Gln Lys Thr Glu Glu Glu Leu Leu Glu Ile Met His Lys Leu

290 295 300

Asn Leu Tyr Lys Leu Ser Leu Gln Gly Asp Thr Lys Asn Ser Asp

305 310 315

Gln Phe Val Thr Ser Val Leu Asp Gly Val Val Leu Pro Lys Asp

320 325 330

Pro Lys Glu Ile Leu Ala Glu Lys Asn Phe Asn Thr Val Pro Tyr

335 340 345

Ile Val Gly Ile Asn Lys Gln Glu Cys Gly Trp Leu Leu Pro Thr

350 355 360

Met Thr Gly Phe Leu Pro Ala Asp Val Lys Leu Asp Lys Lys Lys

365 370 375

Ala Ile Ala Leu Leu Glu Gln Phe Ala Ser Met Thr Gly Ile Pro

380 385 390

Glu Asp Ile Ile Pro Val Ala Val Glu Lys Tyr Thr Lys Gly Ser

395 400 405

Asp Asp Pro Asp Gln Ile Arg Glu Gly Val Leu Asp Ala Met Gly

410 415 420

Asp Val Ala Phe Gly Val Pro Ser Val Ile Val Ser Arg Gly His

425 430 435

Arg Asp Thr Gly Ala Pro Thr Tyr Met Tyr Glu Tyr Gln Tyr Tyr

440 445 450

Pro Ser Phe Ser Ser Pro Gln Arg Pro Lys Asn Val Val Gly Asp

455 460 465

His Ala Asp Asp Val Tyr Ser Val Phe Gly Ala Pro Ile Leu Arg

470 475 480

Glu Gly Ala Ser Glu Glu Glu Ile Asn Leu Ser Lys Met Val Met

485 490 495

Lys Ser Trp Ala Asn Phe Ala Arg Asn Gly Asn Pro Asn Gly Lys

500 505 510

Gly Leu Pro His Trp Pro Lys Tyr Asp Gln Lys Glu Gly Tyr Leu

515 520 525

His Ile Gly Gly Thr Thr Gln Gln Ala Gln

530 535

Claims

1. mouse liver carboxylesterase gene clonal expression, its expression is:

(1). take the total RNA of mouse liver as template, by the RT-PCR technology terminal dna fragmentation that contains EcoR I and Xho I restriction enzyme site that increased, it subclone and order-checking have been carried out, with software Vector NTI mensuration sequence is compared with corresponding NCBI sequence, determine that this gene is Mces1f;

EcoR I initiator codon

TGGACACAGTCCCTTGCCAAGAAACAACCCCAGCCATACCACAATGAGCTG TGA CTCGAG

Terminator codon Xho I

(2). carry out double digestion by EcoR I and Xho I, make the Mces1f gene be connected to construction recombination plasmid PET-32a-Mces1f on the EcoR I of escherichia coli high-level expression carrier PET-32a and the Xho I restriction enzyme site through the effect of T4 dna ligase, transform and express Host Strains BL21, carry out the IPTG abduction delivering; This enzyme is comprised of 535 amino-acid residues, owing to selecting PET-32a as expression vector, SDS-PAGE shows that molecular weight is 76.9KD, and is the same with expection;

Adopt Ni-post affinity chromatography, the expression product of Mces1f has been carried out purifying, obtained the Mces1f of high purity more than 90%.

2. a kind of mouse liver carboxylesterase gene clonal expression as claimed in claim 1 is characterized in that: by the albumen that 535 amino acid form, be a kind of B-esterase, can catalytic hydrolysis contain aliphatics and the aromatic hydrocarbons organic compound of carboxylic acid ester groups.

3. a kind of mouse liver carboxylesterase gene clonal expression as claimed in claim 1 is characterized in that: recombinant plasmid PET-32a-Mces1f.

4. a kind of mouse liver carboxylesterase gene clonal expression as claimed in claim 1, it is characterized in that: recombinant microorganism is e. coli bl21.