CN102191264A

CN102191264A - Production method of recombined hirudin

Info

Publication number: CN102191264A
Application number: CN 201110083156
Authority: CN
Inventors: 曹鹏; 卢悟广; 蔡雪婷
Original assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Current assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Priority date: 2011-04-02
Filing date: 2011-04-02
Publication date: 2011-09-21

Abstract

The invention discloses a production method of a recombined hirudin, which comprises the following steps of: constructing an SUMO-HV fusion gene by using an overlapped PCR (Polymerase Chain Reaction) technology; connecting the SUMO-HV fusion gene containing double endonuclease sites and a plasmid processed through the double-enzyme digestion to construct a recombined hirudin expression vector; transferring the recombined hirudin expression vector to an escherichia coli expression SUMO-HV fusion gene; splitting a recombination escherichia coli cell by using a physical method or a chemical method so as to release an SUMO-HV fusion protein, and using nickel ions for affinity, chromatography and purification so as to obtain the purified SUMO-HV fusion protein; carrying out an enzyme digestion reaction on the purified SUMO-HV fusion protein so as to release HV, and obtaining an SUMO protein containing a His6 label and an HV protein without the His6. By using the production method to prepare the recombined hirudin protein, the efficiency is greatly increased, the cost is saved and the operation is simple and convenient.

Description

A kind of production method of lepirudin 023 ludon

Technical field

The invention belongs to the medical biotechnology field, be specifically related to especially in intestinal bacteria, prepare, and obtain the method for the lepirudin 023 ludon albumen rHV of high biological activity at prokaryotic cell prokaryocyte.

Background technology

In China, leech is a kind of traditional Chinese medicine, and the traditional Chinese medical science thinks that it has broken blood, stimulates the menstrual flow, and by curative effects such as silts, is mainly used in treatment lump in the abdomen disease, blood stasis, amenorrhoea and wound.From leech with and sialisterium extracted plurality of active ingredients, r-hirudin is the maximum a kind of of wherein active the most remarkable research.The micromolecule polypeptide that r-hirudin (HV) is made up of 65-66 amino acid is the natural special inhibitor of finding up to now of the strongest zymoplasm.R-hirudin can form non-covalent stable compound with the alpha-zymoplasm, thereby the fibrinogenic ability of completely destroy zymoplasm cracking, thus the cohesion of antagonism blood.Natural hirudin has a series of variants, comprises r-hirudin variant 1 (HV1) and variant 2 (HV2), variant 3 (HV3).Experimentation on animals and clinical study show that r-hirudin is anticoagulation efficiently, and antithrombotic forms, and further blood stasis phenomenons such as the thrombin activation of prevention catalyzed by thrombin and platelet response.In addition it can also Trombin inhibiting inductive fibroblast proliferation and zymoplasm to the stimulation of endotheliocyte.Therefore, r-hirudin is a kind of up-and-coming anti-freezing stagnation resolvation medicine, can be used for various thrombus diseases, phlebothrombosis especially, the treatment of disseminated intravascular coagulation and cerebral thrombosis, the formation of the artery thrombus that also can be used to perform the operation.R-hirudin also can play a role in oncotherapy, and it can stop the transfer of tumour cell, as fibrosarcoma, and osteosarcoma, angiosarcoma, melanoma, lymphoma and leukemia cell's knurl, r-hirudin cooperates radiation and chemotherapy, can significantly heighten the effect of a treatment.

Experimentation on animals and clinical study prove that subcutaneous and intravenous injection r-hirudin does not all have obvious toxic-side effects, no matter is acute, or subacute toxicity test, blood pressure, and heart rate, blood phase bleeding time and hematochemistry composition are all unaffected.Respiratory system is not influence also, and no anaphylaxis does not generally have specific antibody and produces.And r-hirudin can be oral, and protein is more stable, certain temperature, and under the acid-base condition, and Chymotrypsin trypsinase does not exert an influence to its activity under the certain condition.

R-hirudin has important development and application values, but because leech is limited, it is extremely low that natural hirudin extracts productive rate, can't satisfy clinical and the research needs at all.Many countries and regions are all valued by genetic engineering technique production lepirudin 023 ludon.But, the research of r-hirudin and clinical application are restricted because of the leech source is deficient.Along with the development of molecular biology and genetic engineering technique, make its synthetic become possibility.Since 1986, existing abroad several laboratories are structure and the pharmacologically active and the essentially identical lepirudin 023 ludon of natural hirudin of using gene engineering method acquisition a great deal of successfully.Lepirudin 023 ludon is made up of 63-66 amino acid, and three pairs of disulfide linkage are arranged.Compare with natural hirudin, lepirudin 023 ludon not sulfation on the 63rd amino acids tyrosine, active lower slightly, character is basic identical.

Escherichia expression system since its growth rapidly, the expression amount height, with low cost, simple to operate is the most widely used operating system at present, since 1988, has had many research and utilization intestinal bacteria successful expression to go out highly active lepirudin 023 ludon.But because r-hirudin has three pairs of disulfide linkage, extremely low in the expression in escherichia coli amount, be difficult to be folded into correct conformation, the recombinant protein biological activity of acquisition is significantly less than natural hirudin.So many measures are applied to increasing the solubility expression of r-hirudin.The for example use of secreting signal peptide, the selection of amalgamation and expression technology and some high copy expression carriers and use of strong promoter or the like.

Amalgamation and expression is meant and utilizes the DNA extracorporeal recombination, with the gene of two or more different sourcess or gene fragment gram gallery together, is built into fusion gene, expresses in host cell then.Fusion gene product behind the transcription and translation in host cell should be single peptide sequence, i.e. fusion rotein.The purpose of carrying out gene fusion expression have usually following some: the expression regulation of (1) prokaryotic hosts is easy to start, and fusion rotein can correctly fold mostly, reduces the formation of inclusion body, increases the expression amount of target protein; (2) can reduce the effect of lytic enzyme in the thalline, make target protein particularly the small molecules target protein be protected; (3) purifying and the testing process of simplification expression product; (4) make up some novel activated proteins, it normally with the gene fusion expression of two or more functional proteins, produces the protein with multi-functional or new function.The systematic comparison of SUMO amalgamation and expression has two remarkable advantages in traditional expressing fusion protein system: (1) SUNO (small ubiquitin-related modifier, the little ubiquitin relevant modifications factor) not only can improve protein at colibacillary expression amount, especially some toxicity or little peptide, and can increase proteic solubility greatly.The SUMO system has been successfully applied to SARS CoV 3Cl proteolytic enzyme, GFP, the solubility expression of metalloprotease (MMP13).(2) SUMO proteolytic enzyme has high specific acitivity and high degree of specificity, and the SUMO proteolytic enzyme of 1U can cut 100 μ gSUMO fusion roteins.Different with other enzyme incision principles is the space structure of SUMO proteolytic enzyme identification SUMO, rather than primary structure, and this has just been avoided the non-special cutting of target protein.Because the N-of SUMO proteolytic enzyme end has added the His label, so simplify the removal of this enzyme and being further purified of target product greatly.And the target product N-end of process cutting can not carry unnecessary amino acid.

Summary of the invention

Technical problem to be solved by this invention provides a kind of high-efficiency method for producing of high reactivity lepirudin 023 ludon.

For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:

A kind of production method of lepirudin 023 ludon, this method comprises the steps:

(1) use overlapping pcr to make up the SUMO-HV fusion gene;

(2) the SUMO-HV fusion gene that will contain double enzyme site is connected with the plasmid of handling through double digestion, structure recombinant hirudin expression carrier;

(3) the recombinant hirudin expression carrier is changed over to escherichia coli expression SUMO-HV fusion rotein;

(4) recombinant Bacillus coli cells that obtains with physical method or chemical process cleavage step (3) discharges the SUMO-HV fusion rotein, uses the nickel ion affinity chromatograph purifying, obtains the SUMO-HV fusion rotein behind the purifying;

(5) with the SUMO-HV fusion rotein behind the purifying through endonuclease reaction, discharge HV, separate the HV albumen obtain containing the SUMO albumen of His6 label and do not contain His6 through nickel ion affinity chromatograph.

Wherein, described HV gene is the gene of coding r-hirudin variant, comprise the gene of the gene of the HV1 that encode or coding HV2 or encode HV3 gene or coding and HV1, HV2 and HV3 in any one has 95% above amino acid sequence homology and has identical active proteic gene.Be among HV1, HV2 and the HV3 any one; its amino acid residue sequence through replacement, the disappearance of one or several amino-acid residue or add make with HV1, HV2 and HV3 in any one 95% above amino acid sequence homology is arranged and has identical active albumen, encode this proteic gene also in protection scope of the present invention.

The gene order of coding SUMO is Genebank sequence number NM_001180818.

The aminoacid sequence of HV1 is Genebank sequence number P01050, and the gene order of the above-mentioned HV1 that encodes can be Genebank sequence number A19993, but is not limited to this gene order.

The aminoacid sequence of HV2 is Genebank sequence number P09945, and the gene order of the above-mentioned HV2 that encodes can be for shown in SEQ ID No.4, but is not limited to this gene order.

The aminoacid sequence of HV3 is Genebank sequence number P09944, and the gene order of the above-mentioned HV3 that encodes can be for shown in SEQ ID No.5, but is not limited to this gene order.

In the step (1), the N of described SUMO end contains the His6 label, and HV is positioned at the C end of SUMO.

For example,

When HV was HV1, if the gene order of coding HV1 is Genebank sequence number A19993, constructed SUMO-HV1 fusion gene sequence was shown in SEQ ID No.1.

When HV was HV2, if the gene order of coding HV2 is SEQ ID No.4, constructed SUMO-HV2 fusion gene sequence was shown in SEQ ID No.2.

When HV was HV3, if the gene order of coding HV3 is SEQ ID No.5, constructed SUMO-HV3 fusion gene sequence was shown in SEQ ID No.3.

In the step (2), the restriction enzyme enzyme recognition site that is added in fusion gene head, last two ends can be an II class restriction enzyme enzyme recognition site arbitrarily.

In the step (2), described recombinant hirudin expression carrier is pET28a/SUMO-HV.

In the step (4), during with the chemical process lysing cell, the lysate of employing is 0.01M PBS, pH 7.2,5～10mM imidazoles.

In the step (4), in the nickel ion affinity chromatograph purification process, Binding Buffer is 0.01M PBS, and pH 7.2, the 5-10mM imidazoles; Wash Buffer is 0.01M PBS, and pH 7.2, the 20-100mM imidazoles; Elute Buffer is 0.01M PBS pH 7.2, the 500mM imidazoles.

In the step (5), it is Ulp1 that enzyme is cut the employed enzyme of SUMO-HV fusion rotein.

In the step (5), in the nickel ion affinity chromatograph purification process, Binding Buffer is 0.01M PBS, and pH 7.2, the 5-10mM imidazoles; Wash Buffer is 0.01M PBS, and pH 7.2, the 20-100mM imidazoles; Elute Buffer is 0.01M PBS pH 7.2, the 500mM imidazoles.

In the step (5), separate the HV albumen that obtains,, obtain lepirudin 023 ludon albumen HV again through the anion-exchange chromatography purifying through nickel ion affinity chromatograph.Described anionite-exchange resin be Hi-TrapTM Q XL (GE Healthcare, 1ml).

Beneficial effect: the inventive method has made up SUMO-HV expressing fusion protein system first, originally r-hirudin solubility expression in SUMO amalgamation and expression system that output is extremely low in the pET system is greatly enhanced, the 1L culture can be gathered in the crops 80mg SUMO-HV fusion rotein, this has brought convenience for the separation and purification in downstream, and the proteic space structure of r-hirudin that obtains more approaches native state.Adopt the inventive method, calculate with the 1L bacterial cultures, the r-hirudin that finally can obtain solubility is 20mg, be higher than the albumen that other escherichia expression systems obtain far away.Use the inventive method and prepare lepirudin 023 ludon albumen, improved productive rate greatly, save cost, easy and simple to handle.

Description of drawings

Fig. 1 is a SUMO-HV1 fusion gene design of graphics.

Fig. 2 is a pET28a/SUMO-HV1 recombinant expression vector design of graphics.

Fig. 3 is the SDS-PAGE analysis chart of SUMO-HV1 fusion rotein abduction delivering and purifying, wherein, and 1: do not induce full bacterium, 2: induce full bacterium, 3-8: albumen behind the purifying, M: molecular weight of albumen standard.

Fig. 4 is that the SUMO-HV1 fusion rotein is through nickel ion affinity chromatograph His-Trap HP (GE Healthcare 5ml) ultraviolet oscillogram.

Fig. 5 cut for the SUMO-HV1 fusion protease and the HV1 protein purification of recombinating after the SDS-PAGE analysis chart, wherein, the 1:SUMO-HV1 fusion rotein, 2: enzyme is cut mixture, 3-4: reorganization HV1 albumen behind the purifying, 5: do not cut SUMO-HV1 fusion rotein and SUMO egg white mixture, M: the molecular weight of albumen standard.

Fig. 6 is that reorganization HV1 albumen is through nickel ion affinity chromatograph His-Trap HP (GE Healthcare 5ml) ultraviolet oscillogram.

Fig. 7 is the SDS-PAGE analysis chart behind the Q anionresin purification of Recombinant HV1 albumen, wherein, and 1-2: reorganization HV1 albumen, reorganization HV1 albumen behind the 3:Q anionresin purifying, M: molecular weight of albumen standard.

Fig. 8 is that reorganization HV1 albumen is through anionresin Hi-TrapTM Q XL (GE Healthcare, 1ml) ultraviolet oscillogram.

Embodiment

According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.

Embodiment 1: the preparation of lepirudin 023 ludon HV1.

1) leech albumen HV1, its aminoacid sequence are Genebank sequence number P01050; The nucleotide sequence of coding HV1 adopts Genebank sequence number A19993;

For the coding proteic nucleotide sequence of HV1 (Genebank sequence number A19993), in the end add the recognition site of terminator codon TAA and restriction enzyme after amino acid code;

2) SUMO albumen, its aminoacid sequence are Genebank sequence number NP_010798.1; The proteic nucleotide sequence of coding SUMO, its Genebank sequence number NM_001180818;

For the coding proteic nucleotide sequence of SUMO (Genebank sequence number NM_001180818), before first amino acid code, added the recognition site of restriction enzyme;

It is as follows to design primer respectively according to above-mentioned sequence:

P1：5’ATC?ACT?CAT?ATG?GGG?TCG?GAC?TCA?GAA?G-3’

P2：5’GT?ACA?ATC?AGT?GTA?AAC?AAC?ACC?TCC?AAT?CTG?TTC?GCG?GTG?3’

P3：5’GGA?GGT?GTT?GTT?TAC?ACT?GAT?TGT?AC 3’

P4：5’CG?GGA?TCC?TTA?TTG?CAA?GTA?CTC?CTC 3’

Wherein P1 contains the NdeI restriction enzyme site, initiation codon, His6 label and SUMO upstream complementary sequence, P2 contains downstream SUMO complementary sequence and HV1 upstream complementary sequence, P3 contains and HV1 upstream complementary sequence, P2 and P3 contain complementary region, and P4 contains HV1 downstream complementary sequence and BamH I restriction enzyme site and terminator codon.

First round PCR, respectively with P1, P2 amplification SUMO gene, reaction system and program are as follows:

Mend H ₂O to 50 μ l reaction conditions is as follows:

After reaction finishes, carry out evaluation of 1.0%Agarose electrophoresis and rubber tapping and reclaim the purpose band.

With P3, P4 amplification HV1 gene.Reaction system and program are as follows:

Mend H ₂O to 50 μ l reaction conditions is as follows:

As template, with P1, P4 is the upstream and downstream primer with two kinds of products of first round amplification, and reaction system and program are as follows:

Mend H ₂O to 50 μ l reaction conditions is as follows:

After reaction finishes, carry out evaluation of 1.0%Agarose electrophoresis and rubber tapping and reclaim the purpose band, obtain the SUMO-HV1 gene, its base sequence is shown in SEQ ID No.1.

3) structure contains the SUMO-HV expression carrier;

Use expression vector pET28a, with synthetic SUMO-HV1 gene, (initiating terminal contains the NdeI restriction enzyme site, and clearing end contains the BamHI restriction enzyme site), and to handle through NdeI and BamHI double digestion respectively with pET28a, the enzyme tangent condition is 37 ℃ of water-baths 6 hours.Then enzyme is cut product with 1～2% agarose gel electrophoresis, electrophoresis 15 minutes, 120 volts of voltages.Reclaim test kit with gel and reclaim above-mentioned two dna fragmentations.Its process is: a. downcuts the sepharose that contains target DNA under ultraviolet lamp, exhausts the liquid and the chopping of gel surface with paper handkerchief.Calculated for gel weight (writing down 1.5ml centrifuge tube weight in advance), this weight is as a gel volume (as 100mg=100 μ l volume).B. the Buffer DE-A that adds 3 gel volumes mixes the back in 70 ℃ of heating, be interrupted to mix (per 2～3min), melt (about 6～8min) fully until gel piece.C. add the Buffer DE-B of 0.5 Buffer DE-A volume, mix; When separated DNA fragment during, add the Virahol of 1 gel volume less than 400bp.D. draw the mixed solution among the step c, transfer to DNA and prepare in the pipe, the centrifugal 1min of 12,000 * g.Abandon filtrate.E. will prepare pipe and put back in the 2ml centrifuge tube, and add 500 μ l Buffer W1, the centrifugal 30s of 12,000 * g abandons filtrate.F. will prepare pipe and put back in the 2ml centrifuge tube, and add 700 μ l Buffer W2, the centrifugal 30s of 12,000 * g abandons filtrate.Wash the centrifugal 1min of 12,000 * g with 700 μ l Buffer W2 again with same method.G. will prepare pipe and put back in the 2ml centrifuge tube, the centrifugal 1min of 12,000 * g.H. will prepare pipe and put back in the 1.5ml centrifuge tube, central authorities add 25～30 μ l Eluent or deionized waters at the preparation film, and room temperature leaves standstill 1min.The centrifugal 1min eluted dna of 12,000 * g.It is mixing after to add T4 ligase enzyme at 3: 1 by volume that enzyme is cut back gene fragment and carrier, and 16 ℃ of connections are spent the night.With 3) connection liquid 20 μ l in the step all add in the 200 μ l competence bacteriums, put 1h on ice; 42 ℃ of heat-shocked 90sec put 5min in the ice rapidly; The LB nutrient solution that adds 37 ℃ of preheatings of 800 μ l; 37 ℃, 220rpm jolting 1h all coats the LB flat board that contains 50 μ g/ml Kan after centrifugal, is inverted overnight incubation for 37 ℃.The picking mono-clonal is to the test tube of the 3ml LB nutrient solution that contains 30 μ g/ml Kan at random, 37 ℃ of 220rpm jolting 12h, the extracting plasmid (by the Axygen plasmid in a small amount extraction agent box specification sheets carry out), 1.0%Agarose electrophoresis detection purity is also roughly quantitative; Its step is briefly as follows: a. gets the bacterium liquid of 1～4ml overnight incubation in the LB substratum, and the centrifugal 1min of 12,000 * g abandons most supernatant.B. add 250 μ l Buffer S1 suspension bacterial precipitations, suspending needs evenly should not leave little bacterium piece.C. add 250 μ l Buffer S2, gentleness also spins upside down fully to mix for 4～6 times and makes the abundant cracking of thalline, until forming bright solution.This step should not surpass 5min.D. add 350 μ l Buffer S3, gentle also spinning upside down fully mixed the centrifugal 10min of 12,000 * g 6～8 times.E. draw the centrifugal supernatant in the steps d and transfer to the preparation pipe in, the centrifugal 1min of 12,000 * g abandons filtrate.F. will prepare pipe and put back in the centrifuge tube, and add 500 μ l Buffer W1, the centrifugal 1min of 12,000 * g abandons filtrate.G. will prepare pipe and put back in the centrifuge tube, and add 700 μ l Buffer W2, the centrifugal 1min of 12,000 * g abandons filtrate; In kind more once with 700 μ l Buffer W2 washing.Abandon filtrate.H. will prepare pipe and put back in the 2ml centrifuge tube, the centrifugal 1min of 12,000 * g.I. will prepare pipe and move in the new 1.5ml centrifuge tube, central authorities add 60～80 μ l Eluent or deionized waters at the preparation periosteum, and room temperature leaves standstill 1min.The centrifugal 1min of 12,000 * g.Above-mentioned extractive plasmid is carried out enzyme cut evaluation.

4) be fit to cultivate 4 under the condition of express recombinant SUMO-HV1) in the transformant cell.

The mono-clonal that picking transforms on the flat board is inoculated in the test tube of the 3ml LB nutrient solution that contains 50 μ g/ml Kan, and 37 ℃ of 220rpm joltings are spent the night; Next day is by in the 50ml LB nutrient solution that is inoculated in 50 μ g/ml Kan at 1: 100,37 ℃ of 220rpm joltings to thalline OD600 be 0.6 (about 2h); Take out the 1ml culture, the centrifugal 2min of 12000g room temperature abandons supernatant, with the resuspended bacterial sediment of 100 μ l1 * sample-loading buffers; Adding IPTG in remaining culture is 0.2mmol/l to final concentration, and 37 ℃ of 220rpm jolting 12h induce SUMO-HV to express; Take out the 1ml culture, the centrifugal 2min of 12000g room temperature abandons supernatant, and with the resuspended bacterial sediment of 100 μ l1 * sample-loading buffers, 4 ℃ of centrifugal 12min of 4000g of residue culture abandon supernatant, precipitate put-20 ℃ frozen; Polyacrylamide gel electrophoresis detects fusion rotein and accounts for 30% of bacterial protein.

5) with physical method or chemical reagent lysing cell, extracting, purification of Recombinant fusion rotein.

With the cultivation bacterial sediment of 1L abduction delivering with 50ml Binding-Buffer resuspended after, ultrasonication (power 200W, work 9sec, intermittently 5sec, 30min altogether), 4 ℃ of centrifugal 15min of 12000rpm get supernatant, precipitate frozen in-20 ℃; Utilize the AKTA chromatographic system, supernatant liquor is with the His-Trap HP (GE Healthcare 5ml) of sample on the 5ml/min flow velocity to the Binding-Buffer pre-equilibration; Wash with the 5ml/min flow velocity with Binding-Buffer, to effluent liquid OD280 value arrival baseline; Wash with the 5ml/min flow velocity with Washing-Buffer, to effluent liquid OD280 value arrival baseline; Use Elution-Buffer with 3ml/min flow velocity wash-out then, collect elution peak, use the SDS-PAGE electrophoresis detection.

6) enzyme is cut the SUMO-HV fusion rotein.

With 5) in the SUMO-HV fusion rotein of purifying to 2L 0.01M PBS pH 7.2 dialysis 24 hours, changed one time dialyzate in per 6 hours.To removing imidazoles fully.Dialysis back albumen and SUMO proteolytic enzyme Ulp1 are according to 1: the volume ratio mixing of 20-50,20 ℃ water-bath 2-4 hour.

7) purifying leech fibroin HV.

With 6) in enzyme cut the mixture of back in the dialysis tubing and carry out purifying, purifying is as 5) described in adopt the His affinity chromatography.At first use 0.01M PBS pH 7.210mM imidazoles balance chromatography column, then enzyme is cut sample on the mixture, collect and flow out part, the SDS-PAGE electrophoresis detects.

8) Q anion-exchange chromatography purifying r-hirudin

At first, use 0.01M PBS pH 7.2 then, 0.1M NaCl wash-out foreign protein with at first using 0.01M PBS pH 7.2 balance pillars.Target protein 0.01M PBS pH 7.2,0.4M NaCl wash-out.

9) Chromozym TH colorimetric method for determining hirudin activity

(1) with the Tris damping fluid zymoplasm national standard product being diluted to final concentration is 10IU/ml, and r-hirudin to be measured and lepirudin 023 ludon standard substance are dissolved as 1mg/ml with the Tris damping fluid respectively.

(2) r-hirudin to be measured and lepirudin 023 ludon standard substance are done dilution in 1: 500,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000 respectively

(3) get 96 hole enzyme plates, add the r-hirudin solution after diluting, every hole 25 μ l respectively A～F row; The Tris damping fluid that adds 25 μ lP holes G row; Each extent of dilution is done 2 multiple holes, the Tris damping fluid in A row adding 25 μ lP holes, and B～G row adds the 10IUPml thrombin solution, every hole 25 μ l, 37 ℃ of insulation 5min then

(4) in institute is porose, add Chromozym TH solution, every hole 25 μ l; 37 ℃ of reaction 2min; (4) add the acetate stop buffer, every hole 125 μ l detect the A405 value.

(5) calculation result: with r-hirudin concentration (ng/ml) is X-coordinate, and the A405 value is an ordinate zou, with statistics software or graphing method calculation result, carries out straight-line regression with equation y=A+Bx.The activity of A405 value reflection zymoplasm, the A405 value is high more, and thrombin activity is also high more, shows that hirudin activity is lower, on the contrary the A405 value is low more, and hirudin activity is higher; When the A405 value near 0 the time, show that thrombin activity is suppressed fully, this moment, hirudin activity was suitable with thrombin activity, antithrombin activity sees Table 1 as a result.

Table 1

Embodiment 2: the preparation of lepirudin 023 ludon HV2.

Leech albumen HV2, its aminoacid sequence are P09945; The nucleotide sequence of coding HV2 adopts nucleotide sequence shown in SEQ ID No.4;

For coding HV2 proteic nucleotide sequence, in the end add the recognition site of terminator codon TAA and restriction enzyme after amino acid code;

SUMO albumen, its aminoacid sequence are Genebank sequence number NP_010798.1; The proteic nucleotide sequence of coding SUMO, its Genebank sequence number NM_001180818;

P1：5’ATC?ACT?CAT?ATG?GGG?TCG?GAC?TCA?GAA?G-3’

P2：5’GTGCAGTCGGTGTAGGTGAT?ACC?TCC?AAT?CTG?TTC?GCG?GTG?3’

P3：5’GGA?GGT?ATCACCTACACCGACTGCAC 3’

P4：5’CG?GGA?TCC?TTA?CTGCAGGTATTCTTC 3’

Wherein P1 contains the NdeI restriction enzyme site, initiation codon, His6 label and SUMO upstream complementary sequence, P2 contains downstream SUMO complementary sequence and HV2 upstream complementary sequence, P3 contains and HV2 upstream complementary sequence, P2 and P3 contain complementary region, and P4 contains HV2 downstream complementary sequence and BamH I restriction enzyme site and terminator codon.

PCR condition and method obtain the SUMO-HV2 gene with embodiment 1, and its base sequence is shown in SEQ ID No.2.

The structure of recombinant hirudin expression carrier pET28a/SUMO-HV2, change expression in escherichia coli over to, proteic enzyme cut and process such as purifying all with embodiment 1, the HV2 protein-active that obtains sees Table 1.Embodiment 3: the preparation of lepirudin 023 ludon HV3.

Leech albumen HV3, its aminoacid sequence are P09944; The nucleotide sequence of coding HV3 adopts nucleotide sequence shown in SEQ ID No.5;

For the coding proteic nucleotide sequence of HV3 (SEQ ID No.5), in the end add the recognition site of terminator codon TAA and restriction enzyme after amino acid code;

P1：5’ATC?ACT?CAT?ATG?GGG?TCG?GAC?TCA?GAA?G-3’

P2：5’GTGCAGTCGGTGTAGGTGAT?ACC?TCC?AAT?CTG?TTC?GCG?GTG?3’

P3：5’GGA?GGT?ATCACCTACACCGACTGCAC?3’

P4：5’CG?GGA?TCC?TTATTCGTCGTACGCGTC?3’

Wherein P1 contains the NdeI restriction enzyme site, initiation codon, His6 label and SUMO upstream complementary sequence, P2 contains downstream SUMO complementary sequence and HV3 upstream complementary sequence, P3 contains and HV1 upstream complementary sequence, P2 and P3 contain complementary region, and P4 contains HV3 downstream complementary sequence and BamH I restriction enzyme site and terminator codon.

PCR condition and method obtain the SUMO-HV3 gene with embodiment 1, and its base sequence is shown in SEQ ID No.3.

The structure of recombinant hirudin expression carrier pET28a/SUMO-HV3, change expression in escherichia coli over to, proteic enzyme cut and process such as purifying all with embodiment 1, the HV3 protein-active that obtains sees Table 1.

Claims

1. the production method of a lepirudin 023 ludon is characterized in that this method comprises the steps:

(1) use overlapping pcr to make up the SUMO-HV fusion gene;

2. the production method of lepirudin 023 ludon according to claim 1, it is characterized in that the gene of described HV gene, comprise that any one has 95% above amino acid sequence homology and has identical active proteic gene among the gene of the gene of the HV1 that encode or the gene of coding HV2 or the HV3 that encodes or coding and HV1, HV2 and the HV3 for coding r-hirudin variant.

3. the production method of lepirudin 023 ludon according to claim 1 and 2 is characterized in that in the step (1), and the N of described SUMO end contains the His6 label, and HV is positioned at the C end of SUMO.

4. the production method of lepirudin 023 ludon according to claim 1 is characterized in that in the step (2), described recombinant hirudin expression carrier is pET28a/SUMO-HV.

5. the production method of lepirudin 023 ludon according to claim 1 is characterized in that in the step (5), separates the HV albumen that obtains through nickel ion affinity chromatograph, again through Q anionresin purifying, obtains lepirudin 023 ludon albumen HV.