CN1212336C

CN1212336C - Prepn of recombinant human parathyroid hormone PTH (1-34)

Info

Publication number: CN1212336C
Application number: CN 02153384
Authority: CN
Inventors: 胡昌华; 李明; 何建军; 徐辉
Original assignee: XINAN BIOENGINEERING INDUSTRIALIZATION INTERMEDIATE TEST BASE CO Ltd
Current assignee: XINAN BIOENGINEERING INDUSTRIALIZATION INTERMEDIATE TEST BASE CO Ltd
Priority date: 2002-11-29
Filing date: 2002-11-29
Publication date: 2005-07-27
Anticipated expiration: 2022-11-29
Also published as: CN1417231A

Abstract

The present invention relates to a preparation method for a recombination human parathyroid hormone PTH(1-34), which enables expression yield to be improved, enables purification technology to be simple and enables cost to be reduced by constructing a novel engineering bacteria strain. The present invention modifies a cDNA corresponding to a sequence by a coli degenerate codon, and a fundamental sequence is L-V-P-R-PTH(1-34), wherein the L-V-P-R is an enzyme cutting point of thrombin. The tail end of the nucleotide sequence enlarges a coli termination codon TAA and is convenient to clone, the enzyme cutting point of EcoRI is added to the sequence 5'-terminal, and the enzyme cutting point of SalI is added to the 3'-terminal. A recombinant human parathyroid hormone PTH(1-34) with high purity is obtained by the steps of engineering bacterium fermentation, inclusion body extraction, dilution refolding, thrombin enzyme cutting, anion exchange chromatography, reversed phase chromatography, cation exchange chromatography, etc.

Description

The preparation method of recombinant human parathyroid hormone PTH (1-34)

Technical field:

The present invention relates to the preparation method of human parathyroid hormone PTH (1-34), particularly use gene recombination technology to prepare the method for this product, this product can be used for treating osteoporosis.

Background technology:

Osteoporosis is a class disease relevant with the metabolism of bone, is the disease of non-specific dysostosis, mainly occurs in spongioplasm bone parts in the bone.Bone metabolism comprises that the bone split of the bone forming of scleroblast mediation and osteoclast mediation is separated and absorbs two aspects again.The metabolism of bone is that a resorbent homeostasis process is separated in the bone forming of scleroblast mediation and the bone split of osteoclast mediation.

Along with the increase at age, the activity of absorption again of bone surpasses the bone forming activity, causes bone-loss, and bone density reduces gradually, is that bone presents phenomenons such as loose, the fragile easily fracture of hollow, is called osteoporosis.The patient of osteoporosis locates to fracture at buttocks, wrist, vertebrae etc. easily.The elderly is the women after the menelipsis particularly, occurs this symptom easily.On the other hand, the elderly especially the women after the menelipsis become the group of people at high risk of osteoporosis because estrogenic minimizing and enteron aisle to the calcium absorption ability drop, cause mineral substance in the bone to reduce.The medicine of treatment osteoporosis comprises calcium preparation, V at present _D, tethelin, fluorochemical, diphosphate, thyrocalcitonin, oestrogenic hormon and derivative thereof etc., mainly be conceived to the metabolism of calcium, by suppressing the activity of osteoclast, slow down the decomposition of sclerotin and reduce the loss of sclerotin, reach the purpose of releasing osteoporosis disease.

(Parathyroid Hormone is to regulate the metabolic important factor of osteocyte PTH) to Rat parathyroid hormone 1-34, both can stimulate the propagation of osteoclast and to the decomposition of aging sclerotin, have the effect that promotes the synthetic new bone of scleroblast again.Rat parathyroid hormone 1-34 can activate osteoblastic activity, decomposes to absorb aging sclerotin, increases bone mass and bone density.In addition, Rat parathyroid hormone 1-34 can promote the again absorption of renal tubular cell to calcium phosphorus, increases the content of blood calcium, serium inorganic phosphorus; Activate kidney and produce hydroxylase, the latter makes 25-hydroxyvitamin D ₃Be converted into 1, the 25-dihydroxyvitamin D ₃Thereby, promote the absorption of intestinal epithelial cell, for the synthetic new bone of scleroblast provides material guarantee to calcium ion.Therefore, Rat parathyroid hormone 1-34 may become the ideal medicament of treatment osteoporosis.

PTH is secreted by principal cell, initial synthetic product is 115 amino acid whose Pre Pro PTHs, in secretion process, cut away 31 amino acid of N end, form ripe PTH (1-84) 1.Potts J TJr, et al Adv.Protein Chem.1982 35:323-396,2.Habener J F, et alJ.Biol.Chem.1976 2511:3893-3899,3.Kronenberg H M, et al PNAS 197976:4981-4985.After it enters blood, very fast metabolism is a N-end 1-34 amino acid fragment (PTH (1-34)), PTH (1-34) thus combine initiating signal conducting system performance biological action 6.Tregear G W with specific receptors in kidney, the osseous tissue, et al Greene E.Endocrinology 1973 93:1349,7.Potts JT Jr, et al PNAS 1971 68:63-67,8.Martz A Thomas M L.Biochem.Pharmcol.1983 32:3429-3433.。Discover that 1-34 little peptide PTH (1-34) of PTH (1-84) N-end has the physiologically active of natural Rat parathyroid hormone 1-34 completely, and under the concentration of lower or median dose, long duration of action is in bone, can stimulate the formation of bone, and effectively treat osteoporosis 4.Reeve J, et al.Br.Med.J.1980 280:1340-1344 by inducing the Synthesis in the bone.Because PTH (1-34) does not contain halfcystine, and is very unstable in vivo, and content is very low, separation and Extraction hardly may from natural tissues.Therefore, be used widely clinically, must find a kind of method that can obtain large-tonnage product reliably for making hPTH (1-34).The cost and risk of chemosynthesis is too high, is not suitable for mass production PTH (1-34).Along with the development of genetically engineered recombinant technology, people can utilize this technology to produce recombined parathyroid hormone, but 10.Nakagawa not fully up to expectations such as productive rate, S., et al 1994. Biochem.Biophys.Res.Commun.200:1735-1741.9.Applied Environ.Microbiol.1998 such as Yuji Suzuki, (2): 562-569 has reported the method for the acquisition PTH (1-34) of high yield.This report is with the escherichia coli expression fusion rotein, obtains recombinant human PTH (1-34) by cultivation, purifying.They adopt pBR332 is expression vector, and Top10 is the host bacterium; Adopt enteropeptidase to carry out enzyme and cut, obtain to have the reorganization PTH (1-34) of natural structure to remove fusion rotein.But enteropeptidase costs an arm and a leg, and makes to be unfavorable for scale operation by the with high costs of this technology.

The present invention is by making up new expression vector, problems such as expensive, the process complexity that has solved successfully not only that purifying process causes, also obtained high expression level amount, highly purified product, become possibility thereby make PTH (1-34) obtain large-scale production by genetic engineering technique.

Summary of the invention:

The purpose of this invention is to provide a kind of engineering strain, recombinant human parathyroid hormone PTH (1-34) the purity height that this bacterial strain can make expression amount improve greatly, produce, be easy to purifying, this bacterial strain has been stored in China Microbial Culture Preservation Commission common micro-organisms center, and deposit number is: CGMCC NO.0732.

Another object of the present invention provides the method for using this bacterial strain to prepare PTH (1-34).

Rat parathyroid hormone 1-34 PTH provided by the present invention (1-34) molecular weight is 4100 dalton.Its aminoacid sequence is seen 1-34 aminoacid sequence of sequence table sequence 1.

The preparation method of Rat parathyroid hormone 1-34 PTH of the present invention (1-34) may further comprise the steps:

(1) synthetic corresponding cDNA fragment

(2) described cDNA is expressed in the intestinal bacteria system

(3) PTH (1-34) in the separation and purification thalline

The present invention prepares PTH (1-34) by following steps: the gene engineering expression carrier that makes up a PTH (1-34) and fusion rotein; After the clone obtains engineering bacteria, extract inclusion body, solubilization of inclusion bodies and subsequent purification, make this product through fermentation, collection thalline, broken wall.

The present invention elder generation is with artificial-synthetic DNA's method, obtain the cDNA of PTH (1-34) and fusion rotein, the applying gene recombinant technology, this fragment is imported Bacillus coli cells, through cultivating and inducing, acquisition exists with the inclusion body form, and molecular weight is about the fusion rotein of 18 kilodaltons, and its expression amount is more than 20%; Centrifugal collection thalline, broken wall extract inclusion body, and through solubilization of inclusion bodies, renaturation after the zymoplasm enzyme is cut, is further purified, and obtains PTH (1-34), and its purity is greater than 95%.The target protein that obtains is through amino acid sequence analysis, and 15 amino acid of its N-end are: SVSEIQLMHNLGKHL, the amino acid of C-end are HNF.Through mass spectroscopy, its molecular weight is about 4100 dalton.Have identical biological activity with natural parathyroid hormone, the biological specific activity of recombinant human parathyroid hormone (1-34) is greater than 8000 units/mg.

The detailed description of invention:

1. sequences Design:

Adopt following dna sequence dna (seeing sequence table sequence 2):

catgatttac gaattc

EcoR I

ctg gtt ccg cgt tct gta tct gaa atc caa ctg atg cac aac ctg ggt aaa cac ctg aac tct

LeuValProArgSerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsnSer

atg gaa cgt gta gaa tgg ctg cgt aaa aaa ctg cag gat gta cac aac ttc taa gtcgacctgcag

MetGlu ArgValGluTrpLeuArgLysLysLeuGlnGluValHisAsnPhe Stop Sal I

This sequence has following feature: (1) adopts intestinal bacteria degenerate codon to revise the corresponding cDNA of sequence, avoids the too much repetition of base in the sequence, to guarantee the stability and the translation efficiency of sequence.(2) basic sequence is L-V-P-R-PTH (1-34), and wherein L-V-P-R is the restriction enzyme site of zymoplasm.(3) add intestinal bacteria terminator codon TAA at its nucleotide sequence end; (4) for ease of the clone, add the restriction enzyme site of EcoR I at 5 ' of sequence-end, 3 '-hold the restriction enzyme site that adds Sal I.

2. gene is synthetic

Above-mentioned nucleotide sequence adopts the chemical synthesis process preparation.This sequence clone is in carrier PUC18, and is through dna sequencing, consistent with design.

3. the structure of expression strain

1) selecting pThio-HisA for use is expression vector, it is characterized by: plasmid replicon is the coli E I of E.coli.The selection resistance marker is Amp ^r, transcripting promoter is intestinal bacteria hybrid promoter trc, transcription terminator is the terminator sequence of aspA gene, inserts site EcoR I-Sal I.

2) cut out gene fragment with EcoR I-Sal I from the PUC-18 plasmid, separate with 1%Agarose glue, purifying is standby.Enzyme tangent condition: 10*Buffer:5 μ l, DNA:30 μ l, EcoR I 1 μ l, Sal I 1 μ l, water 13 μ l, 37 ℃ of water-baths 2 hours.

3) cut pThio His A carrier with EcoR I-Sal I enzyme, handle with CAP, purifying is standby.Enzyme tangent condition: 10*Buffer:5 μ l, DNA:30 μ l, EcoR I 1 μ l, Sal I 1 μ l, water 13 μ l, 37 ℃ of water-baths 2 hours.

4) with step 2) in cloning of small fragment to the big fragment of step 3) gained, carrier segments and purpose fragment are pressed 1: 3 mixed, use T ₄Construction recombination plasmid is spent the night in 16 ℃ of connections of ligase enzyme.

5) the plasmid transformation escherichia coli BL21 competent cell after will recombinating, and be coated on the LB flat board that contains 100ug/ml Amp, picking list bacterium colony extracts plasmid and carries out enzyme and cut and identify and dna sequencing that detection of expression is screened optimum engineering bacteria.

Method is as follows: transform: (1) competent preparation: 1. the host bacterium is rule on the nutrient agar flat board, cultivated 16～20 hours for 37 ℃.2. take out single bacterium colony of 1 2-3mm size from flat board, move in the 500ml triangular flask of the substratum that contains 100mlLB, 37 ℃ of strong shaking culture of 220rpm 3 hours make the concentration of cell reach 5 * 10 ⁷Individual cell/ml, at this moment, the 0D of bacterium ₆₀₀Generally between 0.2-0.4, be logarithmic phase.3. with culture in placing 10 minutes on ice, transfer to then in two 50ml centrifuge tubes, 4000r/min, 4 ℃ are centrifugal 10 minutes.4. abandon supernatant, be inverted centrifuge tube 1 minute, flow to end remaining liq, add the ice-cold 0.1mol/L C of 10ml then _dCl ₂Sensitization liquid suspension cell placed 10 minutes on ice.5. 4000r/min, 4 ℃ were reclaimed cells in centrifugal 10 minutes, abandoned supernatant, and the stock culture of every 50ml adds the ice-cold oil of 2ml again, places-70 ℃ to freeze storage.(2) transform: 1. add in dna solution to the 200 μ l competent cell that 10 μ l contain 40ng, gentle mixing placed 30 minutes on ice.2. 42 ℃ of heat-shockeds are 90 seconds, put into ice rapidly, cell is cooled off 1-2 minute after, add 800 μ l LB substratum, 37 ℃ of 225r/min swayed culturing bacterium 45 minutes, allowed plasmid expression antibiotics resistance albumen in the bacterium.3. get 200 μ l transformation mixtures shops and be distributed on the LB agar plate of 90mm, placed 30 minutes under the room temperature, treat that solution is absorbed fully by agar after, be inverted plate in 37 ℃ of overnight incubation.

The screening of positive colony: the enzyme of (1) plasmid is cut evaluation: plasmid extracts, plasmid EcoR I and Sal I double digestion, with the 1%agarost electrophoresis characteristic strip is arranged at the 120bp place, the restriction enzyme digestion and electrophoresis collection of illustrative plates is as follows: (2) express experiment: 1. picking list bacterium colony (selecting big and full) from the flat board is inoculated in the middle pipe that the 5mlLB nutrient solution is housed.2. every middle pipe add 5 μ l50mg/ml card that, 37 ℃ of 260 rev/mins of shaking tables are cultivated and (are numbered 1.2. respectively ...).3. at OD ₆₀₀When 0.6 left and right sides, begin to induce.From every, get 100 μ l in the pipe and in another aseptic LB, manage, respectively reference numeral 1 ' 2 '. ... do original strain original 1 2. ... in by its last volume, add the IPTG inductor of 5mM, 37 ℃ of 220 rev/mins of shaking tables are cultivated.4. after inducing 4 hours, 12% separation gel runs SDS-PAGE electrophoresis detection expression, the screening engineering bacteria, it is centrifugal to get about 1.5ml, 12000 rev/mins 2 minutes.5. supernatant adds sample buffer on 50 μ l dd water+50 μ l electrophoresis to falling.6. boiling water boiled 4 minutes, last sample 20 μ l/ samples, 180 volts/hour of electrophoresis, 50 minutes.7. coomassie dyeing, shaking table 30 minutes; Decolouring, shaking table 1 hour, observations.

6) engineering strain is through fermentation culture and after adding IPTG and inducing, and SDS-PAGE detects, and the engineering bacteria inclusion body of gained is expressed the protein that a kind of molecular weight is 18 kilodaltons, and expression amount is about 20%.

4.PTH acquisition

Engineering bacteria is collected thalline through fermentation culture, and broken bacterium is extracted inclusion body.With the damping fluid of 1 times of volume 50mM Tris (pH8.0), 150mMNaCl and add slow adding Solid urea and dissolve inclusion body to 6M, centrifugal remove insolubles after, add 50mM Tris (pH8.0), 150mMNaCl and make the concentration of urea reduce to 1M, the renaturation of spending the night.The centrifugal foreigh protein removing post precipitation that removes, the zymoplasm enzyme that adds 5mg/U is cut.Reaction solution is crossed Q-sepharose FF post, and effluent liquid is collected in washing, with about acetate adjust pH to 3, removes foreigh protein removing.The centrifugal supernatant liquor that removes behind the foreigh protein removing is crossed Source 15RPC post, and behind wash-out, the target protein peak is after SP-Sepharose FF post, wash-out, and sampling is analyzed.Detect through SDS-PAGE and RP-HPLC, purity is greater than 99%.

Description of drawings:

Fig. 1 is preparation method's of the present invention process flow sheet.

Embodiment:

Further specify the present invention by the following examples

Embodiment 1: the structure of engineering bacteria

1) selecting pThio-HisA for use is expression vector,

Method is as follows: transform: (1) competent preparation: 1. the host bacterium is rule on the nutrient agar flat board, cultivated 16～20 hours for 37 ℃.2. take out single bacterium colony of 1 2-3mm size from flat board, move in the 500ml triangular flask of the substratum that contains 100mlLB, 37 ℃ of strong shaking culture of 220rpm 3 hours make the concentration of cell reach 5 * 10 ⁷Individual cell/ml, at this moment, the OD of bacterium ₆₀₀Generally between 0.2-0.4, be logarithmic phase.3. with culture in placing 10 minutes on ice, transfer to then in two 50ml centrifuge tubes, 4000r/min, 4 ℃ are centrifugal 10 minutes.4. abandon supernatant, be inverted centrifuge tube 1 minute, flow to end remaining liq, add the ice-cold 0.1mol/L C of 10ml then _aCl ₂Sensitization liquid suspension cell placed 10 minutes on ice.5. 4000r/min, 4 ℃ were reclaimed cells in centrifugal 10 minutes, abandoned supernatant, and the stock culture of every 50ml adds the ice-cold oil of 2ml again, places-70 ℃ to freeze storage.(2) transform: 1. add in dna solution to the 200 μ l competent cell that 10 μ l contain 40ng, gentle mixing placed 30 minutes on ice.2. 42 ℃ of heat-shockeds are 90 seconds, put into ice rapidly, cell is cooled off 1-2 minute after, add 800 μ l LB substratum, 37 ℃ of 225r/min swayed culturing bacterium 45 minutes, allowed plasmid expression antibiotics resistance albumen in the bacterium.3. get 200 μ l transformation mixtures shops and be distributed on the LB agar plate of 90mm, placed 30 minutes under the room temperature, treat that solution is absorbed fully by agar after, be inverted plate in 37 ℃ of overnight incubation.

The screening of positive colony: the enzyme of (1) plasmid is cut evaluation: plasmid extracts, plasmid EcoR I and Sal I double digestion, with the 1%agarost electrophoresis characteristic strip is arranged at the 120bp place, the restriction enzyme digestion and electrophoresis collection of illustrative plates is as follows: (2) express experiment: 1. picking list bacterium colony (selecting big and full) from the flat board, the sub middle pipe that the 5mlLB nutrient solution is housed of inoculation.2. every middle pipe add 5 μ l50mg/ml card that, 37 ℃ of 260 rev/mins of shaking tables are cultivated and (are numbered 1.2. respectively ...).3. at OD ₆₀₀When 0.6 left and right sides, begin to induce.From every, get 100 μ l in the pipe and in another aseptic LB, manage, respectively reference numeral 1 ' 2 '. ... do original strain original 1 2. ... in by its last volume, add the IPTG inductor of 5mM, 37 ℃ of 220 rev/mins of shaking tables are cultivated.4. after inducing 4 hours, 12% separation gel runs SDS-PAGE electrophoresis detection expression, the screening engineering bacteria, it is centrifugal to get about 1.5ml, 12000 rev/mins 2 minutes.5. abandon room temperature 00 supernatant, add sample buffer on 50 μ l dd water+50 μ l electrophoresis.6. boiling water boiled 4 minutes, last sample 20 μ l/ samples, 180 volts/hour of electrophoresis, 50 minutes.7. coomassie dyeing, shaking table 30 minutes; Decolouring, shaking table 1 hour, observations.

The purifying of embodiment 2:PTH

Make up PTH according to method of the present invention and express bacterium, through fermentation culture, centrifugal collection thalline.(50mM Tris, 150mM NaCl's thalline pH8.0) suspend, and carrying out ultrasonic bacteria breaking is diluted centrifugal collection inclusion body with lysate.And with lysate washing, and centrifugal settling, to be further purified the PTH inclusion body.

The 20g inclusion body is with the 50mM Tris of 50ml, and 150mM NaCl (pH8.0) suspends, and slowly adding Solid urea 36g under the induction stirring is 6M to final concentration, and continuation was stirred 4 hours.The centrifugal insolubles of removing, supernatant liquor 450ml are with 50mM Tris, and 150mM NaCl (pH8.0) is diluted to the concentration of urea and reduces to the 1M renaturation, spends the night.Solution centrifugal after the renaturation removes foreigh protein removing, measures the protein content in the supernatant, and the enzyme amount adding zymoplasm with 5mg albumen/U carries out enzyme and cut 4 hours.On the sample solution after enzyme is cut with the Q-Sepharose FF anion-exchange column of 250ml 50mM Tris (pH8.0) pre-equilibration.Sample peak in the collection.Last sample peak is transferred about pH to 3 with acetate, leaves standstill the centrifugal foreigh protein removing that removes in back.Supernatant liquor is crossed the Source 15RPC post with the 5% acetonitrile solution pre-equilibration of 200ml 0.1%TFA, behind same 2 column volumes of damping fluid washing, the 80% acetonitrile solution 200ml that contains 0.1%TFA is carried out linear elution, collects elution peak.Contain on the Source 15RPC post elution peak of target protein SP-Sepharose FF post with 250ml 50mMPBS pH6.0 pre-equilibration, behind same damping fluid washing 100ml, contain 50mM PBS with 200ml, 0.4M the buffer solution elution of NaCl (pH6.0), collect the target protein peak, sampling analysis, the target protein that obtains detects through SDS-PAGE and RP-HPLC, purity obtains the 30mg target protein greater than 99%.

Sequence table

＜110〉Xinan Bioengineering Industrialization Intermediate Test Base Co., Ltd.

＜120〉preparation method of recombinant human parathyroid hormone PTH (1-34)

<160>2

<210>1

<211>38

<212>PRT

＜213〉people (huamn)

<400>

Leu Val Pro Arg Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu

1 5 10 15

Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe

20 25 30

<210>2

<211>145

<212>DNA

＜213〉people (human)

<400>

catgatttac gaattcctgg ttccgcgttc tgtatctgaa atccaactga 50

tgcacaacct gggtaaacac ctgaactcta tggaacgtgt agaatggctg 100

cgtaaaaaac tgcaggatgt acacaacttc taa gtcgacc tgcag 145

<210>3

<211>145

<212>DNA

＜213〉people (human)

<400>

catgatttacgaattc ctg gtt ccg cgt tct gta tct gaa atc caa ctg atg 52

Leu Val Pro Arg Ser Val Ser Glu Ile Gln Leu Metcac aac ctg ggt aaa cac ctg aac tct atg gaa cgt gta gaa 94His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val GluTgg ctg Cgt aaa aaa ctg cag gat gta cac aac ttc taa 133Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn PheGtcgacctgcag 145

Claims

1. recombinant human parathyroid hormone analogue, its aminoacid sequence is seen sequence 1 in the sequence table.

2. the dna sequence dna of the parathyroid hormone analogs of coding claim 1 is seen sequence 2 in the sequence table.

3. engineering strain pThi-PTH/BL21 who is used to prepare the human parathyroid hormone analogue of claim 1, preserving number is CGMCC NO.0732.

4. a method for preparing recombinant human parathyroid hormone (1-34) comprises the steps:

(1) cultivates the described bacterial strain of claim 3;

(2) the human parathyroid hormone analogue of the rough claim 1 of extraction from culture;

(3) enzyme is cut the human parathyroid hormone analogue in step and is made human parathyroid hormone (1-34);

(4) step income earner's Rat parathyroid hormone 1-34 (1-34) on the purifying.

5. the preparation method of claim 4 wherein cultivates in the substratum of bacterial strain and contains yeast extract paste, peptone, NaCl.

6. the preparation method of claim 4, wherein step 2 method of extracting rough human parathyroid hormone from culture comprises: centrifugal collection thalline, with lysate suspend thalline, carrying out ultrasonic bacteria breaking, dilution centrifugal, collect inclusion body products.

7. the preparation method of claim 4, wherein step 3 and 4 comprises: dilution refolding, zymoplasm enzyme are cut, anion-exchange chromatography, reversed phase chromatography, cation-exchange chromatography.

8. the construction process of the engineering strain of claim 3 comprises the steps: 1. to adopt the cDNA of claim 2; 2. be expression vector with pThioHis; 3. construction recombination plasmid; 4. transformed into escherichia coli.

9. method according to claim 8, wherein LVPR is the zymoplasm restriction enzyme site in the aminoacid sequence of cDNA sequence encoding, do not hold at its nucleotide sequence to add intestinal bacteria terminator codon TAA, add the restriction enzyme site of EcoR I at sequence 5` one end, the 3` end adds Sal I restriction enzyme site.

10. method according to claim 8, wherein the feature of step expression vector 2. is: promotor is that intestinal bacteria hybrid promoter trc, transcription terminator are the terminator sequence of asp gene, inserting the site is EcoRI-Sal I, step construction of recombinant plasmid method 3. comprises: with cDNA sequence EcoR I and SalI double digestion, in the PUC-18 plasmid of insertion after two kinds of identical enzyme enzymes are cut, again with this fragment subclone to expression vector pThioHis, the coli strain that 4. step is transformed is BL21.