NZ753297B2 - Method for preparing heterodimeric snake venom protein - Google Patents

Abstract

Provided are recombinant plasmids containing the heterodimeric snake venom protein Agkisacutacin A chain gene and Agkisacutacin B chain gene, respectively, cell strains containing the recombinant plasmids, and a method for expressing the heterodimeric snake venom protein Agkisacutacin. The expression level of Agkisacutacin in the present method exceeds 10mg/L, and the purity level can reach more than 95% by means of two steps of purification. n level of Agkisacutacin in the present method exceeds 10mg/L, and the purity level can reach more than 95% by means of two steps of purification.

Description

Method for preparing heterodimeric snake venom protein Technical field The present disclosure relates to a preparation method of recombinant protein, in particular to a preparation method of a recombinant protein drug heterodimer snake venom.

Background Cardio-cerebrovascular diseases, especially thrombus-induced cerebral and dial infarction seriously threat human health and affect the quality of life of ts. These diseases are now the third g cause of death in the world, not only bringing great threat to the patient's life during acute attack, but also leading to huge expenditure of material resources and financial resources because of the easily occurring bad recovery. Therefore, it is urgent to find new safe and ive drugs for these es. The effect of snake venom on blood coagulation system has been paid more and more attention, and some ingredients of snake venom are effective drugs to treat these diseases.

Antiplatelet thrombolysin from Agkistrodon acutus venom is a C-type lectin like protein, which was found on the basis of separation, cation and activity study on snake venom protein of the life science school, Chinese University of e and Technology (CUST) for many years, which is named Agkisacutacin, and Chinese name antiplatelet thrombolysin. Agkisacutacin is a dimer, and the nt molecular weights of the two subunits are very close, both around 14-15kDa. The molecular weight of Agkisacucetin was accurately determined by TOF MS to be 30 kDa. The sequence and crystal ure of Agkisacucetin were finally obtained by N-terminal sequence determination of amino acid, LC-MS, crystal structure of Agkisacucetin protein, etc. Agkisacutacin has the activity of inhibiting platelet aggregation, and previous studies have laid an important foundation for the pment of this protein into a new class of antithrombotic drugs.

At t, the main source of Agkisacutacin is the extraction of natural snake venom. The source of natural snake venom is limited, the purification process is complex and the yield is low, which limits the scientific research and clinical application of Agkisacutacin as an antithrombotic drug.

Summary of the Invention One of the purposes of the present disclosure is to provide a recombinant plasmid pPIC9K-A, as shown in figure 1, the sequence of Agkisacutacin A chain gene was shown in SEQ ID NO.1; a plasmid pUCZ-B, as shown in figure 2, the sequence of Agkisacutacin B chain gene is shown in SEQ ID NO.2.

The present disclosure also provides a transformant, comprising one of the recombinant plasmid pPIC9K-A, plasmid pUCZ-B or their combination.

The present disclosure also provides a Pichia is (ie transformant), which comprises and expresses recombinant heterodimer snake venom protein, wherein the heterodimer snake venom n ts of two peptide chains of A chain and B chain, the A chain amino acid sequence is shown in SEQ ID NO.3, and the B chain amino acid ce is shown in SEQ ID NO.4; n the Pichia pastoris comprises the ce of Agkisacutacin A chain gene shown in SEQ ID NO. 1 and the ce of Agkisacutacin B chain gene shown in SEQ ID NO.2; and wherein the Pichia pastoris has the accession number CCTCC No. M2016358 and is classified as Pichia pastoris GS115.

Pichia pastoris GS115 expresses recombinant heterodimer snake venom protein, consisting of two peptide chains named A chain and B chain, wherein the A chain amino acid sequence is shown as SEQ ID NO.3 and the B chain amino acid sequence is shown as SEQ ID NO.4.

The present disclosure also provides use of the plasmids and the transformants in the manufacture of a medicament for the treatment of thrombosis.

The present disclosure also provides a method of mass expression of a recombinant heterodimer snake venom protein, comprising the steps of: Batch phase: introducing the Pichia pastoris as described in this sure or the progeny thereof into a basic salt medium with pH 4.0 to 6.0, adding glycerin to 4% to amplify bacteria and when the dissolved oxygen (DO) content rises sharply, the batch phase ends; Conversion phase: continuously supplying 50% glycerol solution containing l/L PTM1, with a flow rate of 10-15 ml /h/L, which process lasting until reaching a wet weight of 190-200 g/L; ol induction phase: stopping adding glycerin when the wet weight reaches 190-200g/L, adding methanol on containing 12 ml/L PTM1 at a constant restrictive methanol flow rate, with a stirring speed of 800-1200rpm, and a total fermentation time of 70-90 hours, and wet weight reaching 400-600 / L; Protein purification: after the fermentation, adjusting a fermentation broth pH to 8.0, conducting a high speed centrifugation, and cation by passing through a column to obtain the recombinant heterodimer snake venom protein.

Preferably, the whole process has (ie method is conducted at) a temperature of 28-34 °C.

The present disclosure also provides a recombinant protein prepared by the described method.

The present disclosure adopts the whole gene synthesis method, obtains its coding gene from the known protein sequence and structure form of Agkisacutacin. It was found that the methanol- utilizing Pichia pastoris was an ideal cutacin expression system, and a large amount Agkisacutacin of high purity was ed by inant expression. The experiment verified the recombinant Agkisacutacin expressed by yeast is a heterodimer protein and has biological activities.

At the first time the present sure adopts Pasteurus pastoris to express Agkisacutacin, and pilot scale study on fermentation process was performed. Under the condition of high density fermentation, the amount of expression was more than 10 mg/L; after three steps of purification, the purity was over 95%. In addition, the present disclosure also provides a simple but robust fermentation process for the production of Agkisacutacin.

Biomaterial deposition The cell strain with the accession number CCTCC No. M2016358 was preserved at the Chinese Typical Culture Conservation Center on June 30, 2016 at Wuhan University, Wuhan City, China, which taxonomic name is Pichia pastoris GS115.

Description of the drawings Figure 1 shows the structure of Agkisacutacin A chain expression vector.

Figure 2 shows the structure of Agkisacutacin B chain expression vector.

Figure 3 shows the results of Western Blot detection of Agkisacutacin sion strain.

Figure 4 shows the results of Western Blot detection of high expression strain of Agkisacutacin.

Figure 5 shows the fermentation expression time curve of the pilot scale of recombinant Agkisacutacin.

Figure 6 shows the expression of double strand, A chain and B chain in the pilot scale of recombinant Agkisacutacin.

Figure 7 shows the identification of n Agkisacutacin; Figure 8 shows the purification of n Agkisacutacin; Figure 9 shows the results of GP1b g ty test of recombinant Agkisacutacin. ic mode of implementation The present disclosure discloses a preparation method of a heterodimer snake venom protein.

Those skilled in the art can practise it by reference to this disclosure with proper improvement to the s parameters. In particular, it should be stated that all similar replacements and changes are obvious to those skilled in the art and are considered to be included in the present ion.

The product and method of the present sure has been described through embodiments. It is apparent that those skilled in the art can modify or appropriately modify and combine the methods and applications described herein within the contents, spirit and scope of the present disclosure, so as to realize and apply the technology of the present disclosure.

The t disclosure will be further elaborated in combination with examples below: 1. Experimental materials 1.1 Strain and plasmid P. pastoris strain GS115, pPIC9K vector purchased from Invitrogen company, PUCZ vector(Zeocin resistant) was ucted in our laboratory. 1.2 Reagents Yeast nitrogen base (YNB) purchased from BD Company; RPMI-1640 medium purchased from Hyclone Company; Fetal bovine serum purchased from Gibco Company; Yeast t and Trypton purchased from OXOID Company; BCA protein detection kit purchased from Pierce Company; d-Sorbitolam d-biotin, plasmid extraction kit, PCR product recovery kit, gel recovery kit, DNA polymerase, T4 DNA g enzyme, restriction endonuclease, protein marker and PAGE related reagents purchased from Shanghai Bioengineering; PHA and Endoglycosidase H (Endo H) sed from Sigma-Aldrich Company; EZ-ECL chemiluminescence test kit purchased from Biological Industries Company; other chemical reagents purchased from the Chinese Medicines Group. Si-containing defoamer purchased from Jiangsu Saiouxinyue Defoamer Agent Co., Ltd.; and Anti-Agkisacutacin antibody 1B9 derived from Zhaoke Pharmaceutical Company Ltd. 1.3 Instruments Electronic analytical e and pH meter purchased from Mettler Toledo Co., Ltd; vortex oscillometer oscillator sed from Scientific Industries Company; electrophoresis instrument, vertical electrophoretic tank, horizontal electrophoretic tank, gel imaging system purchased from Tianeng y; ry film conversion instrument, electric rotary instrument purchased from Bio-Rad Company; chemiluminescence detector purchased from UVITEC Company; high pressure sterilizer purchased from Hirayama Company; table refrigeration centrifuge (Centrifuge 5810R, Centrifuge 5415R) purchased from Eppendorf Co., Ltd; super clean workbench, rocker, constant temperature tor purchased from Shanghai Zhicheng Co., Ltd.; PCR instrument purchased from Germany Biometra Company; enzyme labeling ment (BLX-800) sed from Bio-Tek Company; micro liquid remover purchased from Gilson Company of France; cell tor purchased from Themo Company. AKTA explorer Labscale TFF System and Pellicon XL (10kD) purchased from Millipore company; BioFlo®115 op Fermentor purchased from New Brunswick Scientific company; large izer, Flex Stand system and 0.45μm microfiltration cartridge purchased from GE healthcare; LTQ linear IT MS purchased from ; and EttanTM MDLC HPLC system purchased from GE healthcare. 1.4. Primer 1. AgkiA-Fw-Xho1: GAGAAAAGAGATGTCGATTGTCTCCCTGGTTGGTC 2. AgkiA-Rv-Not1-ng: ATATGCGGCCGCTTATGGCGGGGACTTGCAGACGAAAG 3. AgkiB-Fw-EcoR1: CTGAATTCGGTTTCTGTTGTCCCTTGCGTTGTTCG 4. Rv-Not1-ng: ATATGCGGCCGCTTATAGCTTGAACTTGCAGACGAAATAG 2. Experimental method 2.1 Construction of cutacin A chain expression vector I. Amplification of A chain target gene with EcoR1 and Not1 restriction enzyme g sites The whole gene synthesis A chain DNA ce was amplified by PCR under the guidance of primer AgkiA-Fw-Xho1 and AgkiA-Rv-Not1-ng. The PCR reaction conditions were as follows: First 94 °C for 2 minutes, then 94 °C for 30 seconds, 55 °C for 40 seconds, 72 °C for 30 s, a total of 32 cycles; finally at 72 °C for 10 minutes. After the reaction was finished, the PCR product was detected by 1% e gel electrophoresis, and a band of about 396bp was obtained, which was consistent with the expected results. PCR t recovery kit was used to recover the target band.

II. Construction of Pichia pastoris expression vector with recombinant Agkisacutacin A chain The PCR products obtained from the above step were subjected to double enzyme digestion with restriction endonuclease EcoR1 and Not1, then the restriction fragment was linked to the plasmid pPIC9K subjected to the same double enzyme digestion by T4 DNA ligase. The linking product was transformed into Escherichia coli DH5α competent cells, and ve ination was screened. Plasmids were extracted and identified by enzyme digestion, the identified positive clones were named pPIC9K-A, and sent to ai Bioengineering for sequencing. The sequencing results showed that the DNA sequence between recognition sites EcoR1 and Not1 inserted into pPIC9K was in line with the expected results. 2.2 uction of Agkisacutacin B chain expression vector I. Amplification of B chain target gene with EcoR1 and Not1 restriction enzyme cutting sites The whole gene synthesis B chain DNA sequence was amplified by PCR under the guidance of primer AgkiB-Fw-EcoR1 and AgkiB-Rv-Not1-ng. The PCR reaction conditions were as follows: First 94 °C for 2 minutes, then 94 °C for 30 seconds, 55 °C for 40 seconds, 72 °C for 30 seconds, a total of 32 cycles, finally at 72°C for 10 minutes. After the reaction was finished, the PCR product was detected by 1% agarose gel electrophoresis, and a band of about 381bp was obtained, which was consistent with the expected results. PCR product recovery kit was used to recover the target band.

II. Construction of Pichia pastoris expression Vector with recombinant Agkisacutacin B chain The PCR products obtained from the above step were ted to double enzyme digestion with restriction endonuclease EcoR1 and Not1, then the restriction nt was linked to the plasmid pPIC9K subjected to the same double enzyme digestion by T4 DNA ligase. The linking product was transformed into Escherichia coli DH5α competent cells, and positive recombination was screened. Plasmids were extracted and identified by enzyme digestion. The identified ve clones were named pPIC9K-B, and sent to Shanghai Bioengineering for sequencing. The sequencing results was completely in line with the expected results. The cloned plasmid was subjected to double enzyme digestion by BamH1 and Not1, and the small fragment was recovered. The recovered small fragment was linked to the plasmid pUCZ subjected to the same double enzyme digestion with T4 DNA ligase. The linking product was transformed into ichia coli DH5α competent cells, and positive recombination was screened. Plasmids were extracted and fied by enzyme digestion. The identified positive clones were named pUCZB , and sent to Shanghai ineering for sequencing. The sequencing results was completely in line with the expected results. 2.3 Screening of recombinant Agkisacutacin expression clones The Pichia pastoris strain GS115 was streaked on YPD plates. After two days, the growing monoclone was picked out and cultured in YPD liquid medium. Two days later, the yeast grew to milky white, and this first s was introduced into YPD liquid , which was a secondary s. This secondary species was used to prepare yeast competent cells, which was electrotransformed into pPIC9K-A plasmid which had been linearized with Sal1 enzyme. MD plate was coated. After 48 to 72 hours, the monoclone grown on the plate were picked out and cultured in MGY liquid . After two days, the culture was centrifuged at 1500g, the supernatant was discarded, and BMMY medium with pH 5.0 was added thereto and induced for 72 hours. During the process, 100% ol was replenished every 24 hours to maintain methanol concentration in BMMY medium at 0.5%. After the end of induction, centrifuged at 12000g, the supernatant was collected, and the expression of A chain protein was detected by SDS-PAGE, and the one expression of A chain was screened out.

The GS115 monoclone expressing the A chain protein was ed on the MD plate. After two days, the grown monoclone was picked out and cultured in MGY liquid medium. Two days later, the yeast grew to milky white, and this first species was introduced into YPD liquid medium, which was a secondary species. This secondary s was used to prepare yeast competent cells, which was electrotransformed into pUCZ-B plasmid which had been linearized with Spe1 enzyme. MD plate was coated with antibiotic Zeocin. After 48 to 72 hours, the monoclone grown on the plate were was picked out and cultured in MGY liquid medium supplemented with antibiotic Zeocin. After two days, the culture was centrifuged at 1500g, the supernatant was discarded, and the BMMY medium with pH 5.0 was added thereto and induced for 72 hours.

During the process, 100% methanol was ished every 24 hours to maintain methanol concentration in BMMY medium at 0.5%. After the end of induction, centrifuged at 12000g. the supernatant was collected. The expression of double chain protein was ed by Western Blot using one antibody against Agkisacutacin. Monoclone Pichia pastoris GS115 expressing double strand was screened out, which was deposited on June 30, 2016 at the Chinese Typical Culture Conservation Center, with the access number CCTCC No. M2016358, and was named as Pichia pastoris GS115. 2.4 Fermentation expression of inant Agkisacutacin (1) First species: Inoculated from frozen working strain bank to 4ml MGY medium (MGY+Z) added with Zeocin, cultured at 30 °C, 250rpm for 36-48h to OD600=2-6, which was the fermentative first species; (2) Secondary species: 1ml of first species was inoculated into 200ml BMGY with pH .0,cultured at 30℃ and 250rpm for 12-18h to OD600=2-6, which was a fermentative secondary species. (3) Fermentation preparation: fermentation tank was assembled and related reagents were prepared, sterilized and so on; (4) Batch phase: The secondary species were inoculated into a 14L fermentor containing 4L basic salt medium of pH5.0. Basic salt medium containing 4% glycerol (pH 5.0) was used to y the bacteria in the batch phase wherein the culture temperature was 30 °C. When DO rised sharply, the batch phase ended. (5) Conversion period: When DO began to rise rapidly, it meant that glycerol in the original medium depleted, requiring nt supplementation of 50% glycerol solution containing 12ml/L PTM1, the flow rate was 12 . This process lasted 4 hours to wet weight near 190g/L; (6) Methanol induction phase: When the wet weight reached near 190g/L, the glycerine flow was stopped, and the ved oxygen rapidly increased, then a ol solution containing 12 ml/L PTM1 was added at restricted flow of methanol at constant flow rate. The temperature was controlled at 30℃, the stirring rate was about 1000rpm, the total fermentation time was about 80 hours, and the wet weight was about 500g/L. 2.5 Purification of recombinant Agkisacutacin (1) Solid-liquid separation: after fermentation, the fermentation broth pH was slowly adjusted to 8.0 with NaOH, centrifugation at 10000g was performed for 20min, and supernatuant was collected; (2) Sample clarification: Flex Stand system 0.22μm filter was used to clarify; (3) Protein capture: inant cutacin in the fermentation broth was captured by Ion exchange column. The fermentation broth was dialysis and desalted by Flexstand ultrafiltration / filtration system. After desalting, the samples passed through the CM FF column, and 30% buffer B was used to elute the samples; (4) Fine purification: some samples of the 30% elution were further purified by Q HP, eluted by gradient. The samples of different peaks were successively ted and the purity of the recombinant Agkisacutacin was detected by SDS-PAGE. (5) Storage: stored with PBS frozen n. 2.6 Activity detection of recombinant Agkisacutacin (1) Naturally extracted Agkisacutacin can bind to the GP1b site, and the binding ability of recombinant Agkisacutacin and recombinant GPIb was tested. (2) Naturally extracted cutacin can inhibit et agglutination, and the inhibition of platelet agglutination by recombinant Agkisacutacin was studied. 3 Experimental results 3.1 The construction of cutacin expression vector is shown in figure 1 and figure 2.

The A chain of Agkisacutacin has a full length of 132 amino acids. The coding sequence of A chain was linked to the 3' terminal of α-Factor signal peptide of Pichia pastoris, and cloned into pPIC9K yeast expression vector. In this way, AOX I er was induced by methanol, so as to drive its downstream protein expression.

The B chain of Agkisacutacin has a full length of 127 amino acids. The coding sequence of B chain was linked to the 3' terminal of α-factor signal peptide of Pichia pastoris, and cloned into pUCZ yeast expression vector. In this way, AOX I promoter was induced by methanol, so as to drive its downstream protein sion. 3.2 Screening of cutacin expression strains After multiple screenings, the clone of sing cutacin was obtained. The Western Blot s are shown in figure 3.

No. 1 clones was screened again, and seven expression clones were obtained. Western Blotting was used to detect the expression of Agkisacutacin in these clones, showing that all effectively expressed recombinant Agkisacutacin, as shown in Fig.4. 3.3 Fermentation expression of recombinant Agkisacutacin in pilot scale Methanol was added at restricted constant flow rate to perform fed-batch cultivation of the expression bacteria. At first, basic salt medium containing 4% glycerol (pH 5.0) was used to amplify the bacteria at 30 °C in the batch stage. When DO rose y, the batch phase ended, and the wet weight of the ia reached 134.7g/L. Then was the conversion period, that is, the glycerol flow period, after about 4 hours of growth, the wet weight of the bacteria increased further to 193.6 g/L. At this time, the flow of glycerol was stopped, and the respiration activity of bacteria decreased and DO increased rapidly. Then methanol induction was med, the ation activity of the bacteria recovered, and the DO began to decrease gradually. In the ol induction stage, because the bacteria have a phase of adaptation to methanol (2-4 hours), it is necessary to gradually increase the concentration of methanol during this period to avoid the toxicity of excessive accumulation of methanol to the bacteria. The whole fermentation s was very stable and the parameters were stable within the set ranges. At the end of fermentation, OD600 exceeded 300, and the wet weight of bacteria d 511.5 g/L. The results showed that the death rate of bacteria remained at a low level during the whole fermentation process, and methanol concentration was also stabilized at very low levels, maintaining the restricted growth of the bacteria, as seen in Figure 5 and Figure 6. Although the wet weight of the bacteria continuously increased during the induction, the expression of protein substantively d balance after 80 hours of culture (or 60 hours of ion), and no longer accumulated.

Therefore, it was more suitable to end fermentation at this time. 3.4 Purification and identification of Agkisacutacin After fermentation, pH of tation broth was adjusted to 8.0, after high-speed centrifugation and filtration and clarification via Flexstand 0.22um, BCA was used to determine the total n content in fermentation broth which was 1.6 g/L. The concentration of rAgkisacutacin was estimated by GE to be more than 100 mg/L. Because there was no ELISA kit for rAgkisacutacin detection in our ering center, so accurate quantification was not available.

Because rAgkisacutacin does not carry any tag, and we do not have a rAgkisacutacin antibody affinity column in our Engineering center, so we tried to use an ion exchange column to capture the rAgkisacutacin in the fermentation broth. By using Flexstand ultrafiltration/filtration system, the fermentation broth was dialyzed and desalted, and the ed samples flowed through the CM FF column, and the samples were eluted with 10% and 30% buffer B (the purification conditions had been preliminarily explored by using a small ion exchange column of 1ml in the early stage). It can be known from the test results that rAgkisacutacin was mainly present in the % elution (The presence of rAgkisacutacin in the sample can be determined by whether there are two closely connected bands of 15kDa in the SDS-PAGE of DTT under the condition of reduction, and the protein purity can be analyzed by SDS-PAGE under non-reductive conditions). % n also contained a small amount of rAgkisacutacin, and the t of A chain was higher than that of B chain, therefore, there should be a small amount of AA homologue dimer in % elution (Fig.7A). Some samples of 30% elution were further purified by Q HP, and there were five peaks in gradient elution, wherein two peaks were uously collected with 2-3 tubes (P1 and P2). By ing the two bands at the 15kDa position in SDS-PAGE under the condition of reduction, it was found that rAgkisacutacin was mainly present in P2 peak, especially in P2-2 sample with the least impurity. The content of A chain in P3 peak was higher than that in B chain, therefore, there would be AA homologous dimer pollution. P1, P4, and P5 did not contain A or B chain (Fig.7B).

Since the P2-2 sample mainly contained two bands, and the locations thereof were quite different, so molecular sieve was used to purify. As expected, these two bands were well separated. It was confirmed via detection that the P2 in molecular sieve was the target protein rAgkisacutacin.

Under the condition of non-reduction, there was a uniform band, and under the condition of reduction, there were two bands with basically the same content (see Fig.8, P1' and P2' denote the peaks of P1 and P2 respectively). At t, the purity of P2 and P2' in these molecular sieves has not been identified, visual measurement shows that is should achieve a purity of 90%.

By preliminary estimation, about 10 mg of purified samples can be obtained from 1L fermentation broth. 3.5 Determination of Agkisacutacin activity GPIb binding activity and antiplatelet ination activity of purified product were tested. The results of GPIb binding activity showed that, recombinant Agkisacutacin can combine well with recombinant GPIb, and the relative affinity constant was estimated to be 10-7M (Fig.9). The antiplatelet agglutination activity was detected by the Zhaoke Pharmaceutical company, and the experimental s showed that when the amount of protein d 2ug, platelet aggregation could be inhibited by 100% in the detection system; when the amount of n was 1ug, there was no inhibitory activity. All these experiments show atively that, inant Agkisacutacin from yeast has biological activity and physiological function.

The above is only preferred embodiments of the present sure, it should be noted that a number of improvements and modifications may also be made by those skilled in the art t departing from the principle of the present disclosure, which should also be regarded fallen within the scope of protection of the present invention.

SEQUENCE LISTING <110> ZHAOKE PHARMACEUTICAL (HEFEI) COMPANY LIMITED <120> Method for preparing heterodimer snake venom protein <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 508 <212> DNA <213> artificial sequence <400> 1 cmtcdsatgg ggcgattcat cttcgtgagc ttcggcttgc tggtcgtgtt cctctccctg 60 agtggaactg gagctgatgt cgattgtctc cctggttggt ctgcctatga tcagtcttgc 120 taccgggtct tcaaactcct caagacctgg gcag agaagttctg cacggagcgg 180 ccgaaaggcg ggcatctggt ctctatcgaa agcgcgggag agagagactt tgtggcccag 240 ctggtctctg agaacaaaca gacggacaat gtctggcttg gactgaagat caaa 300 ggacagcaat gcagcacaga gtggaccgat ggctccagcg tcagttatga gaatttttct 360 caat ccaaaaagtg ttttgtgctg gaaaaaaaca cagggtttcg cacatggctc 420 aatt gtggaagtga atatgctttc gtctgcaagt ccccgccata agat 480 gcagctgagt gaagtctgga gaagcaag 508 <210> 2 <211> 487 <212> DNA <213> artificial sequence <400> 2 cgat tcatcttcgt gagcttcggc ttgctggttg tgttcctttc cctaagggga 60 actggagctg gtttctgttg tcccttgcgt tggtcctcct ggca ttgctacctg 120 gtcgtcaaag aaaagaagac ctgggatgat gcagagaaat tctgcacaga acagcgcaaa 180 ggcggccatc tggtctccgt tcacagcagg gcag tggt acacctcgcc 240 tacccaattt tggatctcag tttgatctgg atgggactga gcaacatgtg gaacgactgc 300 gagt ggagtgatgg caccaagctt gacttcaaat cctgggcaaa aacatctgat 360 atcg gcaagacaga tggtgataac caatggttaa atatggactg cagcaagaaa 420 cactatttcg tctgcaagtt caagctatag tctgaagatg cagctgagtg aagtctggag 480 aagcaag 487 <210> 3 <211> 130 <212> PRT <213> artificial sequence <400> 3 Asp Val Asp Cys Leu Pro Gly Trp Ser Ala Tyr Asp Gln Ser Cys Tyr 1 5 10 15 Arg Val Phe Lys Leu Leu Lys Thr Trp Asp Asp Ala Glu Lys Phe Cys 25 30 Thr Glu Arg Pro Lys Gly Gly His Leu Val Ser Ile Glu Ser Ala Gly 40 45 Glu Arg Asp Phe Val Ala Gln Leu Val Ser Glu Asn Lys Gln Thr Asp 50 55 60 Asn Val Trp Leu Gly Leu Lys Ile Gln Ser Lys Gly Gln Gln Cys Ser 65 70 75 80 Thr Glu Trp Thr Asp Gly Ser Ser Val Ser Tyr Glu Asn Phe Ser Glu 85 90 95 Tyr Gln Ser Lys Lys Cys Phe Val Glu Lys Asn Thr Gly Phe Arg Thr 100 105 110 Trp Leu Asn Leu Asn Cys Gly Ser Glu Tyr Ala Phe Val Cys Lys Ser 115 120 125 Pro Pro <210> 4 <211> 125 <212> PRT <213> cial sequence <400> 4 Gly Phe Cys Cys Pro Leu Arg Trp Ser Ser Tyr Glu Gly His Cys Tyr 1 5 10 15 Leu Val Val Lys Glu Lys Lys Thr Trp Asp Asp Ala Glu Lys Phe Cys 25 30 Thr Glu Gln Arg Lys Gly Gly His Leu Val Ser Val His Ser Arg Glu 40 45 Glu Ala Asp Phe Leu Val His Leu Ala Tyr Pro Ile Leu Asp Leu Ser 50 55 60 Leu Ile Trp Met Gly Leu Ser Asn Met Trp Asn Asp Cys Lys Arg Glu 65 70 75 80 Trp Ser Asp Gly Thr Lys Leu Asp Phe Lys Ala Trp Ala Lys Thr Ser 85 90 95 Asp Cys Leu Ile Gly Lys Thr Asp Asp Asn Gln Trp Leu Asn Met Asp 100 105 110 Cys Ser Lys Lys His Tyr Phe Val Cys Lys Phe Lys Leu 115 120 125

Claims (4)

Claims

1. A Pichia pastoris comprising and expressing recombinant heterodimer snake venom protein, wherein the heterodimer snake venom protein consists of two peptide chains of A chain and B chain, the A chain amino acid sequence is shown in SEQ ID NO.3, and the B chain amino acid sequence is shown in SEQ ID NO.4; wherein the Pichia pastoris comprises the sequence of cutacin A chain gene shown in SEQ ID NO. 1 and the sequence of Agkisacutacin B chain gene shown in SEQ ID NO. 2; and wherein the Pichia pastoris has the accession number CCTCC No. M2016358 and is classified as Pichia pastoris GS115.

2. Use of the Pichia pastoris according to claim 1 in the manufacture of a ment for the treatment of thrombosis.

3. A method of mass sion of a recombinant heterodimer snake venom n, sing the steps of: Batch phase: introducing the Pichia pastoris according to claim 1 or the progeny thereof into a basic salt medium with pH 4.0 to 6.0, adding glycerin to 4% to amplify the Pichia pastoris and when the dissolved oxygen (DO) t rises sharply, the batch phase ends; Conversion phase: continuously supplying 50% glycerol on containing 10-15ml/L PTM1, with a flow rate of 10-15 ml /h/L, which process lasting until ng a wet weight of 190-200 g/L; ol induction phase: stopping adding glycerin when the wet weight reaches 190-200g/L, adding methanol solution containing 12 ml/L PTM1 at a constant restrictive methanol flow rate, with a stirring speed of 800-1200rpm, and a total fermentation time of 70-90 hours, and wet weight reaching 400-600 / L; and Protein purification: after the fermentation, adjusting a fermentation broth pH to 8.0, conducting a high speed centrifugation, and purification by passing through a column to obtain the recombinant heterodimer snake venom protein.

4. The method according to claim 3, wherein the method is conducted at a temperature of 28- 34 °C. 5'AOXll 3‘AOX1 A chain genei A TT / ■D-jOC bit ■ Kani HIS4