CN1986813A - Batroxobin and its preparing process and specific coding gene - Google Patents

Abstract

The present invention discloses batroxobin and its preparation process and specific coding gene. The batroxobin coding gene possesses one of the following nucleotide sequence: 1. the sequence 2 in the sequence list; 2. the sequence 2 in the sequence list with 1-3 mutant nucleotides; and 3. the nucleotide sequence obtained through connecting IL-2 signal peptide gene sequence to the 5' end of the said sequence. During preparation process of batroxobin, any one coding gene of the nucleotide sequence 1-3 is introduced through eukaryon expression vector into yeast or mammal cell line for expression to obtain batroxobin protein. The process of the present invention expresses batroxobin protein in high bioactivity successfully, and obtains purified yeast yield of 10 mcg/ml or 14 BU/ml and in specific activity of 1400 BU/mg. The present invention has high yield and is suitable for industrial production.

Description

A kind of batroxobin and preparation method thereof and own coding gene

Technical field

The present invention relates to a kind of batroxobin and preparation method thereof and own coding gene.

Background technology

1963, Klobusitzi separated from America spearhead pallas pit viper (Bothrops atrox) venom with Konig and obtains a kind of serine proteinase enzyme, i.e. batroxobin.Up to now, obtained the whole and partial amino-acid series of more than 30 kind of Thrombin-like enzyme, their primary structure is closely similar, and these zymoplasms can both be cut mammiferous plasma fibrinogen by enzyme, make it to change into scleroproein, thereby influence hemorrhage-coagulation process of animal.In these snake venom class blood coagulating protein enzymes, especially to batroxobin and the peace clo (Ancrod) physics, chemical property and clinical application, research comparatively clear (Stocker K andBariow G.H.Methods Enzymol.1976,45:214-223).

The specific effect substrate of batroxobin is a Fibrinogen, and different with zymoplasm is, the A chain of its cutting fibre proteinogen, and do not act on the B chain.Arg in its hydrolysis plasma fibrinogen A chain ¹⁶-GLy ¹⁷The position is during key, can discharge fibrinopeptide A, thereby apace the Fibrinogen in the blood is transformed into scleroproein, then these scleroproeins just can be gathered into loose being easy to and come wound closure by the thrombus of scleroproein enzymic hydrolysis, the effect of realization quick-acting haemostatic powder.Using heavy dose of time marquis, batroxobin has fibrinogen concentration in the blood of reduction, improve the hydrodynamic characteristic of blood viscosity and blood, thereby play effect (Pirkle H and Stocker K.Thromb Haemost.1991, the 65:444-450 of fiber eliminating enzyme; MarshN.A.Blood Coagul Fibrinolysis.1994,5:339-410).Based on more such biochemical characteristics, batroxobin successfully has been developed to hemostatic drug and has been fallen fine medicine.The preparation (external trade(brand)name Reptilase, Chinese translation " reptilase " also contains the factor X activator in addition) that efficient hemostatic function is arranged; The preparation (external trade(brand)name Defibrase, Chinese translation " fiber eliminating enzyme ") that falls fine effect is arranged.In Europe, batroxobin is substituting human thrombin as haemostatic medicament.

The Thrombin-like enzyme preparation of present separation and purification in 7 kinds of snake venom venom of China's listing.Clinical use Thrombin-like enzyme with the longest history is the peace clo of red mouthful of pallas pit viper (A.rhodostoma) snake venom of batroxobin and Malaysia.Domestic have dispel fine enzyme, northeast agkistrodon halys ussuriensis embolism-resisting enzyme (Ahylysantinfarctase), Jiangsu and Zhejiang Provinces Ahylysantinfarctase and ' reptilase ' imitation ' Ba Quting ' (this medicine (protection period is 2001.8.21-2007.8.21〉also be to extract to be prepared from from snake venom) etc. of agkistrodon acutus venom to be used for clinical.But the production cost that extracts batroxobin from the venom of snake is too high.

Though batroxobin protein has only a peptide chain, 12 hemiamic acids are arranged in the batroxobin molecule, form six kinds of intramolecular disulfide bonds; Glycosylation modified in addition, its intramolecularly has two N-glycosylation site: Asn ¹⁴⁶-Asn ¹⁴⁷-Thr ¹⁴⁸And Asn ²²⁵-Lys ²²⁶-Thr ²²⁷The single chain protein that sophisticated batroxobin molecule is made up of 231 amino-acid residues, it is 25.5kD that Theoretical Calculation goes out its molecular weight, iso-electric point is 7.39, abroad the actual molecular weight of the batroxobin of biological extraction is 42kD from the B.atrx.moojuni venom, and the deviation of this molecular weight is because glycosylation modified cause.In addition, also can extract in other subspecies of B.atrox and the venom of other kind poisonous snake and have the active albumen of batroxobin, but its molecular weight does not wait from 29kD to 42kD, and this also is owing to last difference of amino acid composition or glycosylated degree difference between the different subspecies are caused.Though the investigator has had many understandings to the character of batroxobin,, the molecular biology research of this snake venom composition is made slow progress always.1991, Japanese scientist Maeda etc. utilized recombinant DNA technology to obtain the gene of B.atrox moojenii Thrombin-like enzyme first, and was cloned into and obtains amalgamation and expression (MAEDA M in the intestinal bacteria, SATOH S, SUZUKI S, et al.J Biochem.1991,109:632-637).2002, long eyebrow Pallas pit viper Thrombin-like enzyme Gussruobin in vain of report such as Yang Qing and Dalian snake obtain expression to Pallas pit viper Thrombin-like enzyme Gloshedobin in pichia yeast (Yang Qing learns military affairs recklessly, is permitted Xiao Ming, Deng. Acta Biochimica et Biophysica Sinica .2002,34 (1): 6-10).2004, K.H.Chung etc. adopt pichia yeast to express the batroxobin of B.atrox.moojuni, have the biologic activity identical with natural batroxobin, output reaches 6.95 μ g/ml (Weon-Kyoo You behind the purifying, Kwang-Hoe Chung, et al.FEBS.2004,571:67-73).2004, (the Chinese patent notification number are CN1534093) such as Huang Xiudong of China adopted pichia yeast to express the batroxobin of B.atrox.moojuni, and output reaches 20KU/ml behind the purifying.

Adopt engineered means production to be rich in disulfide linkage and glycosylation modified albumen always is a technical barrier, especially producing has many serine proteinase enzyme molecules to disulfide linkage.This is that what obtain in protokaryon almost is inclusion body entirely because two thin key pairing error rates are very high.Can guarantee higher disulfide linkage pairing accuracy in the eukaryotic cell expression system (yeast, CHO and insect cell etc.).Folding and the assembling of secretory protein is that (endoplasmicreticulum finishes on ER), and many molecular chaperoneses (molecular chaperone) are arranged on endoplasmic reticulum in endoplasmic reticulum in eukaryote.The notion of molecular chaperones is: play synergism in biomacromolecule folding (folding), processes such as assembling (assembly), transhipment and degraded, participate in assisting antigenic presenting and the duplicating, transcribe and the establishment of conformation of genetic material; Participate in cell cycle regulating, anti-ageing, apoptosis regulation etc., but the big class that any variation self do not take place extensively is present in biological intravital protein molecule.(Bip), it can discern the proteic signal sequence of endoplasmic reticulum (signal sequence) for Kar2p, binding protein as conjugated protein; Bip albumen also has atpase activity, can provide it to act on the required energy of protein folding by hydrolysising ATP; (protein disulfide isomerase PDI) can assist the correct pairing of disulfide linkage to protein disulfide bond isomerase.

Summary of the invention

The purpose of this invention is to provide a kind of batroxobin and preparation method thereof and own coding gene.

The special-purpose gene of preparation batroxobin provided by the present invention has one of following nucleotide sequence:

1) sequence 2 in the sequence table;

2) in the sequence table sequence 2 through replacing 1-3 base, the proteic nucleotide sequence of identical function of encoding;

3) 1) 5 ' end of described sequence connects the nucleotide sequence that IL-2 signal peptide gene sequence obtains.

4) can be with 1 under the rigorous condition of height), 2) or 3) nucleotide sequence of the dna sequence dna hybridization of described sequence;

The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.

The nucleotides sequence of described IL-2 signal peptide gene is classified as from GENBANK number and is 5 of NM_000586 ' end 295-354 position Nucleotide.

Described encoding gene has one of following nucleotide sequence:

1) sequence 2 in the sequence table;

2) sequence 4 in the sequence table;

3) sequence 6 in the sequence table;

4) sequence 8 in the sequence table.

The method for preparing batroxobin provided by the present invention is above-mentioned encoding gene to be imported in yeast or the mammal cell line by carrier for expression of eukaryon express, and obtains batroxobin protein.

In the described method, described yeast can be pichia spp, is preferably pichia spp GS115/His-.

Described mammal cell line is a Chinese hamster ovary celI system.

When described encoding gene imported in the yeast, described carrier for expression of eukaryon was pPIC9; When described encoding gene imported in the mammal cell line, described carrier for expression of eukaryon was pcDNA3.1.

Described encoding gene importing saccharomycetes to make fermentation is expressed used fermention medium and is contained sorbyl alcohol and soy peptone.The method that described encoding gene imports the saccharomycetes to make fermentation expression is preferably at 30 ℃, pH5.0, after basic medium fermented 20 hours eventually, add 5% glycerine 100ml by 0.8ml/min, hungry 30 minutes, add the 3g soy peptone again, transfer pH to 6.9, add the methanol induction that contains 0.2% volume sorbyl alcohol by 0.2ml/min and cultivated 90 hours; Described basic medium is a 40g/L glycerine, 18g/L K ₂SO ₄, 15g/L MgSO ₄.7H ₂O, 4.13g/L KOH, 0.9g/L CaSO ₄.2H ₂O, 13.3mL/L H ₃PO ₄

Described method also comprises carries out purifying with expressing the batroxobin protein that obtains.Wherein, the purification process of yeast expression batroxobin earlier through hydrophobic chromatoghaphy medium (STREAMLINETMPhenyl), passes through cation exchange medium (SP Sepharose for after fermented supernatant fluid being added pre-treatment such as 2M ammonium sulfate again ^TMFF) and heparin sepharose FF medium (HeparinSepharose FF), carry out again at last can preparing the pure product of batroxobin once step gel permeation chromatography (Sephacryl  S-100).

The batroxobin protein of above-mentioned method preparation also is protection scope of the present invention.

The nucleotide sequence of described batroxobin protein is:

1) sequence 1 in the sequence table;

2) the 59th Ala of the aminoterminal of sequence 1 sports the aminoacid sequence (sequence 5 in the sequence table) of Arg;

3) the 128th Thr of the aminoterminal of sequence 1 sports the aminoacid sequence (sequence 7 in the sequence table) of Arg;

4) aminoterminal of sequence 1 connects the aminoacid sequence (sequence 3 in the sequence table) of IL-2 signal peptide;

The aminoacid sequence of described IL-2 signal peptide is to be the aminoterminal 1-20 amino acids sequence of NP_000577 from GENBANK number.

The genetic codon that the present invention selects yeast and Chinese hamster ovary celI to like, synthetic the batroxobin full-length gene order, this gene is inserted into respectively among the expression vector pcDNA3.1 of the secreted expression carrier pPIC9 class carrier of Pichia pastoris and Chinese hamster ovary celI and carries out secreting, expressing, successfully given expression to the high batroxobin protein of biologic activity.By optimizing a series of conditions in the fermenting process, as (helping yeast produces more basic albumen and comprises molecular chaperones to add a certain amount of sorbyl alcohol, glycosylase etc.) and soy peptone (can delay the degraded of secretory protein), output reaches every milliliter of fermented liquid 10 μ g/ml or 14 batroxobin units (14BU/ml) behind the purifying, and specific activity is 1400BU/mg.This output has surpassed other expression level of having delivered and having reported, is fit to large-scale production.

Method of the present invention is utilized the special-purpose gene of above-mentioned preparation batroxobin, and by yeast, the glycosylated batroxobin of Chinese hamster ovary celI generation, the batroxobin of sudden change, they have makes the animal plasma agglomerative characteristic that contains antithrombotics; Said antithrombotics comprises heparin, EDTA, Citrate trianion, oxalate, HIRULOG and r-hirudin etc.; Identical or the several amino acid that suddenlyd change of batroxobin (Batroxobin) aminoacid sequence in the aminoacid sequence (SEQ-1) of the batroxobin that method of the present invention is produced and the Bothrops atrox moojeni venom, but the glycosylation modified characteristics with yeast or mammalian cell of its molecule, and this class modification is that its biological activity is necessary; And in yeast, express this batroxobin with method of the present invention, can utilize zymic α-signal peptide, wherein inserted the KEX2 protease recognition sequence between signal peptide sequence and the batroxobin protein, can make expressed proteins behind the KEX2 proteolytic cleavage, have the N end same with the batroxobin of nature.

The batroxobin of method preparation of the present invention and the batroxobin of sudden change can be used as a kind of main component of hemostatic drug, also can be directly as the usefulness of falling the fiber medicine.

Description of drawings

Fig. 1 is the structure of batroxobin gene expression vector pPIC9-BG0

Fig. 2 is the structure schema of batroxobin mutant gene (all representing BGm1, BGm2, BGm3, BGm4 with BGm among the figure) expression vector pPIC9-BGm1, pPIC9-BGm2, pPIC9-BGm3, pPIC9-BGm4 (all representing with pPIC9-BGm among the figure)

Fig. 3 transforms bacterium colony PCR qualification result behind the GS115 for pPIC9-BG0

Fig. 4 is the SDS-PAGE electrophoretogram of the different fermentations time (0-90 hour) of the batroxobin protein of BG0 expression

Fig. 5 analyzes collection of illustrative plates (STREAMLINE for the hydrophobic chromatoghaphy medium of the batroxobin protein that BG0 expresses ^TMPhenyl)

Fig. 6 analyzes collection of illustrative plates (SP Sepharose for the cation exchange medium of the batroxobin protein that BG0 expresses ^TMFast Flow)

Fig. 7 analyzes collection of illustrative plates for the heparin sepharose FF medium (Heparin Sepharose FF) of the batroxobin protein that BG0 expresses

Fig. 8 is the gel permeation chromatography collection of illustrative plates (Sephacryl  S-100) of the batroxobin protein of BG0 expression

The SDS-PAGE electrophoresis of the batroxobin protein that Fig. 9 expresses in yeast for BG0

Figure 10 is the molecular weight mass spectrum of the batroxobin protein of BG0 expression

Figure 11 is the SDS-PAGE electrophoresis of the batroxobin protein of CH0 expression

Figure 12 is the influence of batroxobin (2 NIH Units/kg) to the mouse bleeding time

Embodiment

Method among the following embodiment if no special instructions, is ordinary method.

The effect detection of the synthetic and expression batroxobin of embodiment 1, recombinant batroxobin gene BG0

1, the structure of the acquisition of recombinant batroxobin gene BG0 and expression vector

1) acquisition of recombinant batroxobin gene BG0

Aminoacid sequence data according to disclosed batroxobin among the Genebank (GENBANK Accession Number is X12747), select the genetic codon of hobby of yeast and Chinese hamster ovary celI, synthetic recombinant batroxobin full-length gene order (nucleotide sequence) with sequence 2 in the sequence table.For making goal gene can insert the pPIC9 carrier, at batroxobin gene (Batroxobin Gene, BG) 5 of (GENBANK Accession Number is X12747 from 5 ' end 1-693 position Nucleotide) '-end adds the point of contact of restriction enzyme XhoI, 3 '-end interpolation TAA termination codon and EcoRI point of contact, hold the corresponding codon AAA AGA of adding KEX2 protease recognition sequence Lys-Arg between first amino acid Val codon at the XhoI point of contact with batroxobin protein N-in addition, this just guarantees can successfully excise the a-signal peptide sequence when target protein is in being secreted into fermented supernatant fluid, and the batroxobin of engineering bacterium expression is had with the same n terminal amino acid sequence of this albumen that extracts in the snake venom.The nucleotide sequences and the Genebank Accession Number of design recombinant batroxobin full-length gene are that synthetic batroxobin gene nucleotide sequences (the Chinese patent notification number is CN1534093) such as X12747 and Huang Xiudong have 18.9% and 15.9% difference respectively.

The splicing of gene adopts recursion PCR (Recursive PCR) method, and concrete grammar is: whole gene is divided into faces 12 fragments of eclipsed mutually and synthesize, specific as follows:

T1：5′-CTCGAGAAAAGAGTCATTGGAGGTGATGAATGTGACATCAACGAACACCCTTTCCTTGCCTTCATGTACTACTCTCCACG-3′；

T2：5′-CAGTGTGCAGCGGTCAGGACCCATTCCTGGTTGATCAAAGTCATACCACAGAAGTAACGTGGAGAGTAGTACATGAAGGC-3′；

T3：5′-CCTGACCGCTGCACACTGTAACAGAAGATTTATGCGTATCCACCTTGGTAAGCACGCCGGATCTGTCG-3′；

T4：5′-CTTATTAGGACAAATGAACTTCTCCTTTGGGTATCTAACGACCTCATCGTAGTTGGCGACAGATCCGGCGTGC-3′；

T5：5′-GGAGAAGTTCATTTGTCCTAATAAGAAGAAAAACGTCATTACCGACAAGGACATTATGTTGATCAGACTGGACAGACCTG-3′；

T6：5′-CAACACTTGGAGGGTTGGAAGGCAAAGAGAGAGGAGCGATGTGTTCGGAGTTTTTGACAGGTCTGTCCAGTCTGATCAAC-3′；

T7：5′-CCAACCCTCCAAGTGTTGGTTCCGTTTGCCGTATTATGGGATGGGGAGCAATCACAACTTCTGAAGACACTTACCCAG-3′；

T8：5′-GTAAGCCTCACGACAGACAGTATTGTTGAACAGGTTAATGTTAGCACAGTGAGGGACATCTGGGTAAGTGTCTTCAGAAG-3′；

T9：5′-GTCTGTCGTGAGGCTTACAACGGTTTGCCAGCTAAGACCTTGTGTGCAGGTGTCCTGCAAGGAGGTATCGATACATGTGG-3′；

T10：5′-CCCCAAGACAAGATTCCCTGGAATTGTCCATTACAGATCAGTGGTCCACCAGAGTCACCACCACATGTATCGATACCTCC-3′；

T11：5′-GGGAATCTTGTCTTGGGGAAGTGATCCATGTGCCGAACCACGTAAGCCTGCCTTCTACACCAAGGTCTTTGATTACCTTC-3′；

T12：5′-GAATTCTTATGGACAAGTAGCAGTCTTGTTTCCAGCAATGATAGACTGAATCCATGGAAGGTAATCAAAGACCTTGG-3′。

12 fragment T1-T12 are template with above-mentioned synthetic, with 5 ' end primer P1:5 '-CAGCTCGAGAAAAGAGTCATTGGAG-3 '; 3 ' end primer P2:5 '-CAGGAATTCTTATGGACAAGTAGCAGTCTTG-3 ' carries out pcr amplification for primer.PCR reaction system: each 0.1 μ L of 12 sections templates (7.5uM), each 1 μ L of P1 (15uM) and P2 (15uM), dNTP (each 2.5mM) 5 μ L, KOD DNA Polymerase (5U/ μ L) 0.5 μ L, 10 * KOD buffer, 5 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L.PCR response procedures: 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 1min, 25 circulations.The final PCR product that obtains shows that through order-checking this fragment has the nucleotide sequence of sequence 2 in the sequence nucleotide sequence table, with its called after BG0.The 13-705 position Nucleotide of sequence 2 is the batroxobin encoding sequence in sequence table, the amino acid residue sequence that coding has sequence 1 in the sequence table; The 1-12 position Nucleotide of sequence 2 is the encoding sequence of KEX2 protease recognition sequence in sequence table.

2) structure of the recombinant vectors pPIC9-BG0 of expression BG0 (making up schema as shown in Figure 1) in yeast

Final PCR product B G0 is reclaimed, through XhoI and EcoRI double digestion, 1% sepharose reclaims the BG0 fragment again, be inserted between the XhoI and EcoRI enzyme recognition site of pPIC9 carrier (available from Invetrogen company), the recombinant plasmid that obtains is cultivated with LA (LB+100 μ g/ml Amp+) is dull and stereotyped, select the mono-clonal on LA (the LB+100 μ g/ml Amp+) flat board, the LA medium liquid is cultivated, extract plasmid, XhoI and the screening of EcoRI double digestion contain the clone of BG0, with α-factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' sequencing primer from 5 of BG0 ' end and 3 ' hold the detection of checking order, shows detection the pPIC9 recombinant vectors called after pPIC9-BG0 that contains BG0 respectively.Contain expression vector pPIC9 among the pPIC9-BG0 and can be used for transforming pichia spp host bacterium GS115/His-.With the above-mentioned expression plasmid carrier pPIC9-BG0 of a large amount of preparations of plasmid extraction kit, for the need of transformed yeast.

2, to utilize host bacterium GS115/His-to produce batroxobin be its active detection to BG0

1) contains the acquisition of pPIC9-BG0 reorganization bacterium

With pPIC9-BG0 Bgl II linearizing, transform Pichia pastoris host bacterium GS115/His-, coating histidine auxotroph substratum MD (1.34%YNB, 4 * 10-5 vitamin H, 1% glucose, 2g/100mL agar) obtain the GS115 that recombinates on the flat board.With a little yeast colony that on the MD flat board, grows of rifle point picking, carry out bacterium colony PCR checking, with 5 ' AOX1 primer: 5 '-GACTGGTTCCAATTGACAAGC-3 '; 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' is a primer.The PCR reaction system: reorganization GS115 bacterium colony is a template, each 0.5 μ L of 5 ' AOX1 (15uM) and 3 ' AOX1 (15uM), dNTP (each 2.5mM) 2 μ L, rTaq DNA Polymerase (5U/ μ L) 0.1 μ L, 10*rTaq buffer 2 μ L, ddH2O 15 μ L.The PCR response procedures: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ 1 minute, 25 the circulation.In regrouping process,, can form Mut if pPIC9-BG0 is inserted among wild-type GS115 self methanol oxidase gene 5 ' AOX1 or the 3 ' AOX1 ⁺(methyl alcohol utilizes normal type) if pPIC9-BG0 substitutes wild-type GS115 self methanol oxidase gene, can form Mut ^s(methyl alcohol utilizes slow type), so the 2200bp band is the PCR product of wild-type GS115 self methanol oxidase gene, the 750bp band is the PCR product of BG0, i.e. Mut ⁺Two band 2200 and 750bp are arranged, Mut ^sOne band 750bp is arranged, Mut ⁺And Mut ^sAll are the positive colonies that contain pPIC9-BG0.50 positive colonies that contain pPIC9-BG0 have been screened altogether.The part qualification result as shown in Figure 3, swimming lane 1,3 and 4 is Mut among Fig. 3 ⁺The bacterium colony PCR of (methyl alcohol utilizes normal type) swimming lane 2 and 5 as a result is Mut ^sThe bacterium colony PCR result of (methyl alcohol utilizes slow type), swimming lane GS115 are the GS115/His-bacterium colony PCR result of unconverted, and swimming lane BG0 is the PCR result of pPIC9-BG0, and swimming lane marker is a molecular weight standard.

2) fermentative production of batroxobin

The determination of activity of batroxobin is to utilize batroxobin can enzyme cut Fibrinogen, and the human plasma that contains Trisodium Citrate condensed, and this method carries out.Concrete grammar is: the positive colony bacterium colony that above-mentioned PCR checking is contained pPIC9-BG0 inserts respectively and 3mL BMG is housed (contains 100mM potassiumphosphate pH 6.0 among the 1L, 1.34%YNB, 4 * 10 ^-5Vitamin H, 1% glycerine) in the test tube of nutrient solution, cultivated 24 hours for 30 ℃,, ferment to OD with 0.5% methanol induction 48 hours ₆₀₀=25, with reference to the measuring method for activity of the batroxobin that from snake venom, extracts, the enzymic activity (expression amount) that gives expression in the fermented supernatant fluid is measured with the people's standard blood plasma that has added Trisodium Citrate.Batroxobin activity (expression amount) is, under 37 ℃, 100 μ l fermented to OD ₆₀₀=25 zymic fermented supernatant fluids join 300 μ l and contain in people's standard blood plasma of Trisodium Citrate, behind the mixing, condense the required time.50 clones that contain pPIC9-BG0, batroxobin activity (expression amount) is 10mins-30mins.

Pichia spp can be in simple culture media well-grown and expression bioactive recombinant batroxobin is arranged.But the excretory recombinant protein is easily degraded in simple culture media, and the yield of product is reduced.In the experiment, on the basis of yeast basic medium, choose soy peptone respectively and sorbyl alcohol has carried out comparative study.Concrete grammar is: be respectively that (phenotype is Mut to the 10mins bacterial classification that contains pPIC9-BG0 with the above-mentioned batroxobin expression amount that sifts out ^s) go up a jar fermentation, be inoculated into 3 1L that the 200mLBMG substratum is housed respectively and shake that propagation is forwarded to 3L basic medium (40g/L glycerine, 18g/LK are housed as seed liquor in the bottle ₂SO ₄, 15g/L MgSO ₄.7H ₂O, 4.13g/L KOH, 0.9g/L CaSO ₄.2H ₂O, 13.3mL/L H ₃PO ₄) the 5L fermentor tank in, at 30 ℃, pH 5.0 fermentations, ferment after 20 hours, add 5% glycerine 100ml by 0.8ml/min, hungry 30 minutes, in two fermentor tanks, add the 3g soy peptone more therein, wherein one jar with the soybean protein peptone, transfer pH to 6.9, a fermentor tank that adds soy peptone adds methyl alcohol (containing 0.2% volume sorbyl alcohol) by 0.2ml/min therein again, and other two fermentor tanks are pressed 0.2ml/min and added methyl alcohol, the inducing culture fermentation, fermented supernatant fluid was got in fermentation every 12 hours, with the people's standard blood plasma that has added Trisodium Citrate the enzymic activity (expression amount) that gives expression in the fermented supernatant fluid is measured.Batroxobin activity (expression amount) is, under 37 ℃, 100 μ l fermented supernatant fluids joined 300 μ l contain in people's standard blood plasma of Trisodium Citrate, behind the mixing, condenses the required time.Calculate human plasma rate of setting (1/ condense required time), result of experiment is shown in A among Fig. 4, the result shows, the albumen human plasma rate of setting that adds sorbyl alcohol and soy peptone fermentation in the basic medium is the highest, show, add sorbyl alcohol and help yeast and produce more basic albumen and comprise molecular chaperones, glycosylase etc., promoted the assembling and correct the folding of batroxobin, soy peptone has delayed the degraded of batroxobin simultaneously.1 be basic medium among the A among Fig. 4,2 for adding soy peptone in basic medium, and 3 be adding sorbyl alcohol and soy peptone in basic medium.

With the above-mentioned batroxobin expression amount that sifts out is that (phenotype is Mut to the 10mins bacterial classification that contains pPIC9-BG0 ^s) go up a jar fermentation, be inoculated into the 1L that 200mL BMG substratum is housed and shake that propagation is forwarded to 3L basic medium (40g/L glycerine, 18g/L K are housed as seed liquor in the bottle ₂SO ₄, 15g/L MgSO ₄.7H ₂O, 4.13g/L KOH, 0.9g/L CaSO ₄.2H ₂O, 13.3mL/LH ₃PO ₄) the 5L fermentor tank in, at 30 ℃, pH 5.0 fermentations, ferment after 20 hours, add 5% glycerine 100ml, hungry 30 minutes by 0.8ml/min, add the 3g soy peptone again, transfer pH to 6.9, added methyl alcohol (containing 0.2% volume sorbyl alcohol) inducing culture 90 hours by 0.2ml/min, this moment, the concentration of fermented liquid was OD ₆₀₀=360, the expression amount (enzymic activity) that records batroxobin according to the method described above is 30secs (B among Fig. 4, arrow shows band).

3) fermentation obtains the purifying and the activity identification thereof of batroxobin

With above-mentioned ferment to concentration be OD ₆₀₀After the fermented supernatant fluid of=360 fermented liquids adds the pre-treatment of 2M ammonium sulfate, through what use in advance that 10mM PBS (pH6.0) and 2M ammonium sulfate balance cross hydrophobic chromatoghaphy medium (STREAMLINE is housed earlier ^TMPhenyl, Amersham Biosciences, article No.: chromatography column 17-5121-02), with 50mM PBS (pH 6.0) gradient elution that contains 2M ammonium sulfate, collect active peak, analyze collection of illustrative plates as shown in Figure 5, "-" is labeled as active peak among Fig. 5; To collect liquid and carry out the SDS-PAGE electrophoresis, the result is shown in 1 of swimming lane among Fig. 9;

With above-mentioned collection peak, through what cross cation exchange medium (SPSepharose is housed again with 10mM PBS (pH6.0) balance ^TMFast Flow, Amersham Pharmacia Biotech AB, article No.: chromatography column 17-0729-01), with the 10mM PBS that contains 0-0.5M sodium-chlor (pH 6.0) gradient elution, collect active peak, analyze collection of illustrative plates as shown in Figure 6, "-" is labeled as active peak among Fig. 6; To collect liquid and carry out the SDS-PAGE electrophoresis, the result is shown in swimming lane among Fig. 92;

Above-mentioned cation exchange medium is collected the peak, through what cross heparin sepharose FF (Heparin Sepharose FF is housed again with 10mM Tris-HCl (pH7.4) balance, Beijing Zhuo Guan Science and Technology Ltd., article No.: chromatography column CS-A13-01), with the 10mM Tris-HCl that contains 0-0.5M sodium-chlor (pH 6.0) gradient elution, collect active peak, analyze collection of illustrative plates as shown in Figure 7, "-" is labeled as active peak among Fig. 7; To collect liquid and carry out the SDS-PAGE electrophoresis, the result is shown in swimming lane among Fig. 93;

Above-mentioned heparin sepharose FF is collected the peak, carry out a step again and use gel permeation chromatography (Sephacryl  S-100, Pharmacia LKB Biotech AB, article No.: 17-0612-01), collect the batroxobin solution that active peak obtains purifying with 10mMTris-HCl (pH7.4) wash-out that contains 0.15M NaCl then, analyze collection of illustrative plates as shown in Figure 8, "-" is labeled as active peak among Fig. 8, and the result shows that this output of utilizing BG0 to produce batroxobin is 10mg/L fermented liquid (OD ₆₀₀=360), i.e. 10mg/10 ¹³Cfu.

People's standard blood plasma with Trisodium Citrate carries out the active detection of batroxobin, concrete grammar is: under 37 ℃, the fermented supernatant fluid of 100 μ l is joined 300 μ l contain in people's standard blood plasma of Trisodium Citrate, behind the mixing, observe the required time of condensing and compare with human thrombin standard substance under the same terms.A batroxobin unit (BU) is equivalent to 0.17 NIH zymoplasm unit (NIHUnit, 1 NIH zymoplasm unit definition is under 28 ± 1.0 ℃ of conditions, the zymoplasm amount of condensing 1ml people's standard fibers proteinogen solution in 15 ± 0.5 seconds).

The result shows, is substrate with people's standard blood plasma, and the batroxobin activity is every milliliter of fermented liquid 14 batroxobin units (14BU/ml), and the specific activity of the batroxobin behind the above-mentioned purifying is 1400BU/mg.

Carry out the active detection of batroxobin with bovine fibrinogen, concrete grammar is: under 37 ℃, the fermented supernatant fluid of 100 μ l is joined 300 μ l, 0.4% bovine fibrinogen (Tris-HCl pH7.4, contain 0.15M NaCl) in, behind the mixing, observe under condense required time and the same terms the human thrombin standard substance and compare time of coagulation.A batroxobin unit is equivalent to 0.17 NIH zymoplasm unit (NIH Unit, 1 NIH zymoplasm unit definition is under 28 ± 1.0 ℃ of conditions, the zymoplasm amount of condensing 1ml people's standard fibers proteinogen solution in 15 ± 0.5 seconds).The result shows that the specific activity that with the bovine fibrinogen is the substrate batroxobin is 1800BU/mg (table 1)

The batroxobin solution of purifying is carried out the SDS-PAGE electrophoresis, and the result is shown in swimming lane among Fig. 94.

The recombinant batroxobin of purifying after deglycosylating enzyme PNGase F (New England BioLabs) hydrolysis, is carried out the SDS-PAGE electrophoresis, and the result is shown in swimming lane among Fig. 95.

Swimming lane M is an albumen lower molecular weight standard among above-mentioned Fig. 9; Swimming lane 1 is STREAMLINE ^TMThe active peak of Phenyl; Swimming lane 2 is SP Sepharose ^TMThe active peak of FF; Swimming lane 3 is the active peak of Heparin Sepharose FF; Swimming lane 4 is the active peak (batroxobin protein of purifying) of Sephacryl  S-100; Swimming lane 5 is deglycosylated batroxobin protein.

The methanol yeast expression system as long as the glycosylated possibility of N-site is arranged in the protein molecular primary structure, generally all can have N-type glycosylation phenomenon when the foreign protein of expressing.It is 25.5KD that batroxobin aminoacid sequence data calculates this proteic molecular weight, the above-mentioned batroxobin that is purified into, the apparent molecular weight that SDS-PAGE electrophoresis (Fig. 9) draws is between 30-33KD, the molecular weight mass spectrum of batroxobin protein shows that the molecular-weight average of BG0 production batroxobin is 30.55KD as shown in figure 10.As shown in Figure 9, batroxobin is the about 25.5KD of proteic molecular weight after deglycosylating enzyme PNGase F hydrolysis, illustrates that the batroxobin that BG0 of the present invention produces has the glycosylation phenomenon.

BG0 is produced the batroxobin protein order-checking, and the first five amino acid whose sequence is VIDGG, shows that the batroxobin protein of secreting, expressing is consistent with expected result.

3, the mouse bleeding time of batroxobin protein is detected

The test mouse is male mice (20-25g, each handles 10), batroxobin protein (the 2 NIH Units/kg of the Pichia anomala expression behind the above-mentioned purifying of difference tail vein injection, 8.4 μ g albumen/kg body weight) or natural batroxobin protein (2 NIHUnits/kg, 8.8 μ g albumen/kg body weight) or according to the dosage of 5ml/kg body weight inject 10mmol/L PBS (pH 7.4) (contrast), after 60 minutes, apart from tail point 2-3mm place's crosscut, 1.5cm in 37 ℃ of physiological saline is immersed in fixing back, the record bleeding time.The result shows batroxobin protein (the 2 NIH Units/kg of the Pichia anomala expression behind the above-mentioned purifying of injection, 8.4 μ g albumen/kg body weight) or natural batroxobin protein (2 NIH Units/kg, 8.8 μ g albumen/kg body weight) or the control treatment mouse bleeding time is about 55 seconds respectively, 60 seconds, 90 seconds; The result is shown in 1 among Figure 13,2,3, and the result shows that batroxobin protein has shortened the bleeding time of mouse, promotes hemostasis, and its haemostatic effect and natural batroxobin protein are suitable.Among Figure 13,1: 5ml/kg body weight 10mmol/L PBS (pH 7.4) (contrast); 2: natural batroxobin protein (2 NIH Units/kg, 8.8 μ g albumen/kg body weight); 3: the batroxobin protein of yeast expression (2NIH Units/kg, 8.4 μ g albumen/kg body weight).

Embodiment 2, contain batroxobin gene (IBG0) synthetic of IL-2 signal peptide gene and express the effect detection of batroxobin.

1, contains synthetic, the order-checking of batroxobin gene (IBG0) of IL-2 signal peptide gene and the structure of expression vector

Utilizing the overlapping extension PCR of montage (SOE-PCR) is that template (sequence 2 in the sequence table) is connected with IL-2 signal peptide gene (being 5 of NM_000586 ' end 295-354 position nucleotide sequence from GENBANK number) with batroxobin gene BG0, and concrete grammar is:

With IL-2 signal peptide gene (is 5 of NM_000586 ' end 295-354 position nucleotide sequence from GENBANK number) is template, use the IL25 primer: 5 '-CTGGATCCACGATGTATAGGATGCAACTGCTG-3 ' and IL23 primer: 5 '-AGCGCTGTTGGTGACCAG-3 ' carries out PCR for primer, PCR reaction system: IL-2 signal peptide gene (7.5uM) 0.1 μ L, each 0.5 μ L of IL25 primer (15uM) and IL23 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10*KOD buffer 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L; PCR response procedures: 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 70bp; With pPIC9-BG0 is template, with the ILB primer: 5 '-CTGGTCACCAACAGCGCTGTCATTGGAGGTGATGAATG-3 ' and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' is that primer carries out PCR, PCR reaction system and PCR response procedures are as mentioned above; The result obtains the fragment of 800bp.Two fragments that obtain with above-mentioned IL25 primer and IL23 primer and ILB primer and 3 ' AOX1 primer amplification are template, with IL25 primer and 3 ' AOX1 primer is that primer carries out PCR, the IL-2 signal peptide gene is connected with the recombinant batroxobin gene, obtain having the recombinant batroxobin gene (IBGO) of IL-2 signal peptide, show through order-checking, this fragment has the nucleotide sequence of sequence 4 in the sequence table, the amino acid residue sequence of sequence 3 in the code sequence tabulation.The 1-60 position nucleotide coding IL-2 signal peptide sequence of sequence 4 in the sequence table, it is the 1-20 amino acids residue sequence of sequence 3 in the sequence table, with IBGO through BamHI and EcoRI double digestion, 1% sepharose reclaims the dna fragmentation that meets 800bp again, be inserted between the multiple clone site BamHI and EcoRI restriction enzyme site of pcDNA3.1 carrier, then, use the T7 primer:

5 '-TAATACGACTCACTATAGGG-3 ' and BGH primer: 5 '-TAGAAGGCACAGTCGAGG-3 ' sequencing primer respectively from 5 of BG gene '-end and 3 '-hold the evaluation of checking order, will show the recombinant vectors called after pcDNA3.1-IBGO that contains IBGO through order-checking.

2, contain the expression of batroxobin gene (IBG0) in Chinese hamster ovary celI of IL-2 signal peptide

Use Liofectamine ^TM2000 method transforms Chinese hamster ovary celI, with plasmid pcDNA3.1-IBG0 and Liofectamine ^TM2000 mix in serum-free DMEM substratum [every liter: 1 bag of DMEM cultivates powder (4500mg glucose, 4.0mM L-glutaminate, 110mg Sodium.alpha.-ketopropionate), 2.0g sodium bicarbonate, each 100mg of penicillin and Streptomycin sulphate], and hatch at room temperature 20 minutes; Mixture is joined in 24 orifice plates of completing cell, soft mixing, and place 37 ℃, 5%CO ₂Cultivate in the cell culture incubator after 24 hours, the substratum of cell is replaced with the DMEM substratum that contains 10%DF (Defined FBS, superfine foetal calf serum) continues to cultivate 48 hours; Use the DMEM substratum that contains 10%DF that the contains 750mg/L G418 screening of pressurizeing instead, changed liquid once in per 2 days, pressurization screening 14 days.Limiting dilution assay picking mono-clonal.When the cell of normal growth is paved with 24 orifice plates after waiting to pressurize, its density about 10 ⁶/ hole is diluted to 10,20 cell/ml respectively with growth medium respectively after it is digested suspension, and 200 μ l/ holes are inoculated in 96 well culture plates, put 37 ℃ of cultivations.After 10 days, promptly visible single cell clone growth is by carrying out the detection of target protein in the hole.Screened 96 clones that can express batroxobin altogether, expression amount does not wait from 90mins to 180mins.

Be that [every liter: 1 bag of DMEM cultivates powder (4500mg glucose at no albumen, serum-free DMEM substratum for the single cell clone of 90mins with above-mentioned expression amount, 4.0mM L-glutaminate, the 110mg Sodium.alpha.-ketopropionate), 2.0g sodium bicarbonate, each 100mg of penicillin and Streptomycin sulphate] middle enlarged culturing to 10 ¹⁰Cell/L substratum, with 1 times of culture supernatant dilution, heparin sepharose FF (the HeparinSepharose FF of method according to embodiment 1 through crossing with 10mM Tris-HCl (pH8.4) balance, Beijing Zhuo Guan Science and Technology Ltd., article No.: chromatography column CS-A13-01), with 10mM Tris-HCl (pH8.4) gradient elution that contains 0-0.5M sodium-chlor, collect active peak and carry out the SDS-PAGE electrophoresis, the result is shown in swimming lane among Figure 12 1;

Above-mentioned heparin sepharose FF is collected the peak, through what cross anionic exchange medium (Q Sepharose is housed again with 10mM Tris-HCl (pH8.4) balance ^TMFast Flow, Pharmacia LKB Biotech AB, article No.: chromatography column 17-0510-01), with 10mM Tris-HCl (pH8.4) gradient elution that contains 0-0.5M sodium-chlor, collect active peak and carry out the SDS-PAGE electrophoresis, the result is shown in swimming lane among Figure 12 2;

Above-mentioned anionic exchange medium is collected the peak, carry out a step again and use gel permeation chromatography (Sephacryl  S-100, Pharmacia LKB Biotech AB, article No.: 17-0612-01), then with 10mMTris-HCl (pH7.4) wash-out that contains 0.15M NaCl, collect active peak and carry out SDS-PAGE electrophoresis (being the batroxobin solution of purifying), the result is shown in swimming lane among Figure 12 3.Carry out with people's standard blood plasma of Trisodium Citrate that batroxobin is active to be detected, concrete grammar is: under 37 ℃, the nutrient solution supernatant liquor of 100 μ l is joined 300 μ l contain in people's standard blood plasma of Trisodium Citrate, behind the mixing, observe and condense the required time.Carry out the active detection of batroxobin with bovine fibrinogen, concrete grammar is: under 37 ℃, the nutrient solution supernatant liquor of 100 μ l is joined 300 μ l, 0.4% bovine fibrinogen (Tris-HCl pH7.4, contain 0.15M NaCl) in, behind the mixing, observe the required time of condensing and compare with the human thrombin standard substance 0 knot time of coagulating under the same terms.A batroxobin unit is equivalent to 0.17 NIH zymoplasm unit (NIH Unit, 1 NIH zymoplasm unit definition is under 28 ± 1.0 ℃ of conditions, the zymoplasm amount of condensing 1ml standard fibers proteinogen solution in 15 ± 0.5 seconds).The result shows that this output of utilizing CHO to produce batroxobin is the 0.1mg/L nutrient solution, promptly 10 ¹⁰Cell/L nutrient solution, it is more as shown in table 1 than vigor detected result, shows that CHO produces batroxobin and still has very high ratio vigor.

The recombinant batroxobin that the CHO of purifying is expressed carries out the SDS-PAGE electrophoresis after deglycosylating enzyme PNGase F (New England BioLabs) hydrolysis, the result is shown in swimming lane among Figure 12 4.

Swimming lane M is an albumen lower molecular weight standard among above-mentioned Figure 12; Swimming lane 1 is the active peak of heparin sepharose FF; Swimming lane 2 is Q Sepharose ^TMThe active peak of Fast Flow; Swimming lane 3 is the active peak (being the batroxobin protein of purifying) of Sephacryl  S-100; Swimming lane 4 is deglycosylated batroxobin protein.

As long as the glycosylated possibility of N-site is arranged in the protein molecular primary structure, generally all can there be N-type glycosylation phenomenon in the expressing cho cell system when the foreign protein of expressing.It is 25.5KD that batroxobin aminoacid sequence data calculates this proteic molecular weight, the above-mentioned batroxobin that is purified into, and the apparent molecular weight that SDS-PAGE electrophoresis (Figure 12) draws is between 32-35KD.As shown in figure 12, batroxobin is the about 25.5KD of proteic molecular weight after deglycosylating enzyme PNGase F hydrolysis, illustrates that the batroxobin of Chinese hamster ovary celI production of the present invention has the glycosylation phenomenon.

3, the mouse bleeding time of the batroxobin protein of CHO (pcDNA3.1-IBG0) expression is detected

The test mouse is male mice (body weight 20-25g, 10 of each experiment treatment group), batroxobin protein (the 2 NIH Units/kg that CHO behind the above-mentioned purifying of difference tail vein injection expresses, 8.4 μ g albumen/kg body weight) or 5ml/kg body weight 10mmol/LPBS (pH 7.4) (contrast), after 60 minutes, apart from tail point 2-3mm place's crosscut, 1.5cm in 37 ℃ of physiological saline is immersed in fixing back, the record bleeding time.The result shows the batroxobin protein (2 NIH Units/kg, 8.4 μ g albumen/kg body weight) that the CHO of injection behind the above-mentioned purifying expresses or 5ml/kg body weight 10mmol/L PBS (pH 7.4) (contrast) the mouse bleeding time is about 51 seconds respectively, 90 seconds; The result is shown among Figure 13 1,4, and the result shows that the batroxobin protein that CHO expresses has shortened the bleeding time of mouse, promotes hemostasis, and the batroxobin protein of its haemostatic effect and yeast expression and natural batroxobin protein are suitable.Among Figure 13,1: injection 5ml/kg body weight 10mmol/L PBS (pH 7.4) (contrast), 4: the batroxobin protein of injecting the CHO expression behind the above-mentioned purifying.

Embodiment 3, to the glycosylation research of the batroxobin protein of BG0 genetic expression

1, batroxobin glycosylation site mutator gene (BGm1, acquisition BGm2) and the structure of expression vector (Fig. 2)

Adopt site-directed mutagenesis technique that the BG0 gene is suddenlyd change respectively Asn (from the 146th of the aminoterminal of sequence 1) is sported Gln (glycosylated motif is Asn-X-Thr), be about to that 5 ' end 448-450 position Nucleotide (AAC) of sequence 2 sports CAA in sequence table; Concrete grammar is:

With pPIC9-BG0 is template, uses the a-Factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and M13 primer: 5 '- TTGGAACAGGTTAATGTTAGCACAG-3 ' ( The sudden change codon) carry out pcr amplification for primer, PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of a-Factor primer (each 2.5mM) and M13 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10*KODbuffer 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L.PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 470bp.Use the M15 primer: 5 '-CTGTGCTAACCTGTTC CAAAATACTGTCTGTCGTGAGG-3 ' ( The sudden change codon) and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' carries out PCR for primer, PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of M15 primer (15uM) and 3 ' AOX1 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O35 μ L.PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 340bp.After above-mentioned two fragments that obtain are reclaimed, as template, with the a-Factor primer:

5 '-TACTATTGCCAGCATTGCTGC-3 ' and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' carries out PCR for primer, the program of PCR is: the PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of a-Factor primer (15uM) and 3 ' AOX1 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L.PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 800bp, shows through order-checking, and this fragment has the nucleotide sequence (being sequence 9) that 5 of sequence in the sequence table 2 ' end 448-450 position Nucleotide (AAC) is sported CAA, with its called after BGm1.

The BGml fragment with XhoI and EcoRI double digestion, is inserted between the XhoI and EcoRI restriction enzyme site of pPIC9 carrier, is built into the pPIC9 recombinant vectors that contains BGml, this recombinant vectors called after pPIC9-BGml.

Asn (from the 225th of the aminoterminal of sequence 1) is sported Gln, be about to that 5 ' end 685-687 position Nucleotide (AAC) of sequence 2 sports CAA in sequence table; Concrete grammar is: with pPIC9-BG0 is template, uses the a-Factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and M23 primer: 5 '-GGAATTCTTATGGACAAGTAGCAGTC TTGTTTCCAGCAATG-3 ' (sudden change codon)For primer carries out PCR, the program of PCR is: the PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of a-Factor primer (15uM) and M23 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L.PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 730bp, shows through order-checking, and this fragment has the nucleotide sequence (being sequence 10) that 5 of sequence in the sequence table 2 ' end 685-687 position Nucleotide (AAC) is sported CAA, with its called after BGm2.

The BGm2 fragment that above-mentioned PCR is obtained is inserted between the XhoI and EcoRI restriction enzyme site of pPIC9 carrier through XhoI and EcoRI double digestion, is built into the pPIC9 recombinant vectors that contains BGm2, will identify correct recombinant vectors called after pPIC9-BGm2.

With pPIC9-BGm1 and pPIC9-BGm2 Bgl II linearizing, according to the described method of embodiment 1 step 2, transform pichia spp GS115/His-host bacterium, obtain containing pPIC9-BGm1 or pPIC9-BGm2 reorganization bacterium respectively, at 30 ℃, MD is dull and stereotyped to be cultivated 2-3 days, PCR identifies and (carries out pcr amplification with PCR 5 ' AOX1 primer and 3 ' AOX1 primer, reaction system: the 25ul system, the GS115 bacterium colony of recombinating on a small quantity is a template, each 0.5 μ L of 5 ' AOX1 primer (15uM) and 3 ' AOX1 primer (15uM), dNTP (each 2.5mM) 2 μ L, rTaq DNA Polymerase (5U/ μ L) 0.1 μ L, 10 * rTaq buffer, 2 μ L, ddH ₂O 15 μ L.PCR response procedures: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, 25 circulations.); Identify positive colony, PCR identified show that the reorganization bacterium that contains pPIC9-BGm1 or pPIC9-BGm2 cultivates and methanol induction with 1BMG according to the method for embodiment that the batroxobin that a glycosylation site has been removed in the fermentation expression sudden change fermented OD 96 hours ₆₀₀=360, with the fermented supernatant fluid purifying, to carry out activity and detect, detection method is with the step 2 of embodiment 1.

The result shows: the expression amount that contains the batroxobin that pPIC9-BGm1 or pPIC9-BGm2 reorganization bacterium fermentation obtains reaches 10mg/L fermented liquid (OD respectively ₆₀₀=360), i.e. 10mg/10 ¹³Cfu, specific activity is respectively 500BU/mg, 170BU/mg (table 1);

BG0 is 30.55KD at the batroxobin molecular weight that yeast GS115 expresses, and total sugar content 16.5% is about 1400BU/mg than living; The molecular weight of the natural batroxobin that extracts from the abdomen snake is born in the year of snake venom is 34.9 KD, and total sugar content 27% is about 1350BU/mg than living, and this shows that different biological glycosylation batroxobins are more little than the influence of living to it.

Illustrating that sudden change removes glycosylation site in the batroxobin protein that any one BG0 expresses, the enzyme activity of batroxobin reduces greatly; Existence and glycosylation thereof that these two glycosylation sites in the batroxobin protein that BG0 expresses are described are extremely important to the vigor of keeping enzyme.

Embodiment 4, amino acid mutation are to active influence

1, Ala (from the 59th of the aminoterminal of sequence 1) sports the acquisition of batroxobin and the encoding gene thereof of Arg

Ala (from the 59th of the aminoterminal of sequence 1) is sported Arg, be about to that 5 ' end 187-189 position Nucleotide (GCC) of sequence 2 sports CGA in sequence table; Concrete grammar is: with pPIC9-BG0 is template, uses the a-Factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and M33 primer: 5 '- TCGGACAGATCCGGCGTGCTTAC-3 ' ( The sudden change password Son) for primer carries out PCR, the program of PCR is: the PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L, each 0.5 μ L of a-Factor primer and M33 primer, dNTP 2 μ L, KOD DNA Polymerase 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄2 μ L, ddH ₂O 35 μ L; PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 210bp; Use the M35 primer: 5 '-GCACGCCGGATCTGTC CGAAACTACGATGAGGTCGTTAG-3 ' ( The sudden change codon) and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' carries out PCR for primer, the program of PCR is: the PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of a-Factor primer (15uM) and 3 ' AOX1 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase 0.1 μ L (5U/ μ L), 10 * KOD buffer, 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L; PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 690bp.Two fragments that above-mentioned PCR is obtained reclaim the back as template, with the a-Factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' is that primer carries out PCR, PCR reaction system and PCR response procedures are as mentioned above; The result obtains the fragment of 800bp, detects through order-checking, detects to show that this fragment has the nucleotide sequence (being sequence 6 in the sequence table) that 5 of sequence in sequence table 2 ' end 187-189 position Nucleotide (GCC) is sported CGA, with fragment called after BGm3.The amino acid residue sequence that the BGm3 coding has sequence 5 in the sequence table.

BGm3 through XhoI and EcoRI double digestion, is inserted among the XhoI and EcoRI enzyme recognition site of pPIC9 carrier, is built into the pPIC9 recombinant vectors that contains BGm3, will identify correct recombinant vectors called after pPIC9-BGm3.

2, Thr (from the 128th of the aminoterminal of sequence 1) sports the acquisition of batroxobin and the encoding gene thereof of Arg

Thr (from the 128th of the aminoterminal of sequence 1) is sported Arg, soon 5 of sequence 2 ' end 394-396 position Nucleotide (ACT) sports CGA in sequence table, concrete grammar is: with pPIC9-BG0 is template, uses the a-Factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and M43 primer: 5 '- TCGTGTGATTGCTCCCCATCC-3 ' ( The sudden change codon) for primer carries out PCR, PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of a-Factor primer (15uM) and M43 (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L; PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 420bp, uses the M45 primer: 5 '-ATGGGGAGCAATCACA CGATCTGAAGACACTTACCCAG-3 ' ( The sudden change codon) and 3 ' AOX, 1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' carries out PCR for primer, the PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L, each 0.5 μ L of M45 and 3 ' AOX1 primer, dNTP 2 μ L, KOD DNA Polymerase 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄2 μ L, ddH ₂O 35 μ L; PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 380bp.Two fragments that above-mentioned PCR is obtained reclaim the back as template, with the a-Factor primer: 5 '-TACTATTGCCAGCATTGCTGC-3 ' and 3 ' AOX1 primer: 5 '-GCAAATGGCATTCTGACATCC-3 ' is that primer carries out PCR, PCR reaction system: pPIC9-BG0 plasmid 0.1 μ L (0.04ug), each 0.5 μ L of a-Factor primer (15uM) and 3 ' AOX1 primer (15uM), dNTP (each 2.5mM) 2 μ L, KOD DNA Polymerase (5U/ μ L) 0.1 μ L, 10 * KOD buffer, 2 μ L, MgSO ₄(25mM) 2 μ L, ddH ₂O 35 μ L; PCR response procedures: 94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, 25 circulations; The result obtains the fragment of 800bp, detects through order-checking, detects to show that this fragment has the nucleotide sequence (being sequence 8 in the sequence table) that 5 of sequence in sequence table 2 ' end 394-396 position Nucleotide (ACT) is sported CGA, with fragment called after BGm4.The amino acid residue sequence that the BGm4 coding has sequence 7 in the sequence table.

BGm4 through XhoI and EcoRI double digestion, is inserted among the XhoI and EcoRI enzyme recognition site of pPIC9 carrier, is built into the pPIC9 recombinant vectors that contains BGm4, will identify correct recombinant vectors called after pPIC9-BGm4.

3, BGm3 and the BGm4 expression in pichia spp

According to embodiment 1 described method, pPIC9-BGm3 or pPIC9-BGm4 are transformed GS115/His-, carry out PCR then and identify, will identify that correct positive colony ferments to OD ₆₀₀=360, and according to embodiment 1 described method detection of active and purifying.People's standard blood plasma with Trisodium Citrate carries out the active detection of batroxobin, concrete grammar is: under 37 ℃, the fermented supernatant fluid of 100 μ l is joined 300 μ l to be contained in people's standard blood plasma of Trisodium Citrate, behind the mixing, observe under condense required time and the same terms the human thrombin standard substance and compare time of coagulation.A batroxobin unit is equivalent to 0.17 NIH zymoplasm unit (NIH Unit, 1 NIH zymoplasm unit definition is under 28 ± 1.0 ℃ of conditions, the zymoplasm amount of condensing 1ml standard fibers proteinogen solution in 15 ± 0.5 seconds).Carry out the active detection of batroxobin with bovine fibrinogen, concrete grammar is: under 37 ℃, the fermented supernatant fluid of 100 μ l is joined 300 μ l, 0.4% bovine fibrinogen (Tris-HCl pH7.4, contain 0.15M NaCl) in, behind the mixing, observe under condense required time and the same terms the human thrombin standard substance and compare time of coagulation.A batroxobin unit is equivalent to 0.17 NIH zymoplasm unit (NIH Unit, 1 NIH zymoplasm unit definition is under 28 ± 1.0 ℃ of conditions, the zymoplasm amount of condensing 1ml people's standard fibers proteinogen solution in 15 ± 0.5 seconds).The result shows: BGm4 genetic expression wherein will be mutated into the expression amount of R batroxobin from the 128th T of aminoterminal of sequence 1 for reaching 10mg/L fermented liquid (OD ₆₀₀=360), i.e. 10mg/10 ¹³Cfu, specific activity are 1050BU/mg; The expression amount that will be mutated into the R batroxobin from the 59th A of aminoterminal of sequence 1 that the BGm3 of another strain expresses is 10mg/L fermented liquid (OD ₆₀₀=360), specific activity is 1330BU/mg.With bovine fibrinogen is that substrate detects its activity and expression amount: under 37 ℃, 100 μ l are joined 300 μ l, 0.4% bovine fibrinogen (pH7.4, contain 0.9%NaCl) in, behind the mixing, write down time of coagulation, the result shows that the batroxobin that will be mutated into R from the 59th A of aminoterminal of sequence 1 that BGm3 expresses has changed the substrate specificity of batroxobin protein, and the activity of bovine fibrinogen is higher than the batroxobin that BG0 expresses, and the activity of human plasma is lower than the batroxobin (table 1) that BG0 expresses.

Table 1. batroxobin mutant contrasts than vigor with the batroxobin that BG0 expresses

	BG0 is at the batroxobin of yeast expression	The batroxobin that IBG0 expresses in Chinese hamster ovary celI	BGm1 is at the batroxobin of yeast expression	BGm2 is at the batroxobin of yeast expression	BGm3 is at the batroxobin of yeast expression	BGm4 is at the batroxobin of yeast expression
							Than vigor (BU/mg, human plasma are substrate)	1400	1360	500	170	1050	1330
Than vigor (BU/mg, bovine fibrinogen is a substrate)	1800	1730	540	216	2500	1750

4, the mouse bleeding time of the batroxobin protein of BGm3 and BGm4 expression is detected

The test mouse is male mice (20-25g, n=10), BGm3 batroxobin protein (the 2NIH Units/kg of the Pichia anomala expression behind the above-mentioned purifying of difference tail vein injection, 8.8 μ g albumen/kg body weight) or BGm4 batroxobin protein (2NIH Units/kg, 11.2 μ g albumen/kg body weight) or 5ml/kg body weight 10mmol/L PBS (pH 7.4) (contrast), after 60 minutes, apart from tail point 2-3mm place's crosscut, 1.5cm in 37 ℃ of physiological saline is immersed in fixing back, the record bleeding time.The result shows BGm3 batroxobin protein (the 2NIH Units/kg of the Pichia anomala expression behind the above-mentioned purifying of injection, 8.8 μ g albumen/kg body weight) or BGm4 batroxobin protein (2NIH Units/kg, 11.2 μ g albumen/kg body weight) or 5ml/kg body weight 10mmol/L PBS (pH 7.4) (contrast) the mouse bleeding time is about 53 seconds respectively, 57 seconds, 90 seconds; Concrete outcome is shown among Figure 13 1,5,6.Among Figure 13,1: injection 5ml/kg body weight 10mmol/L PBS (pH 7.4) (contrast); 5. the BG3 batroxobin protein of yeast expression (2 NIH Units/kg, 11.2 μ g albumen/kg body weight); 6. the BG4 batroxobin protein of yeast expression (2 NIH Units/kg, 8.8 μ g albumen/kg body weight).

Sequence table

<160>10

<210>1

<211>231

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>1

Val Ile Gly Gly Asp Glu Cys Asp Ile Asn Glu His Pro Phe Leu Ala

1 5 10 15

Phe Met Tyr Tyr Ser Pro Arg Tyr Phe Cys Gly Met Thr Leu Ile Asn

20 25 30

Gln Glu Trp Val Leu Thr Ala Ala His Cys Asn Arg Arg Phe Met Arg

35 40 45

Ile His Leu Gly Lys His Ala Gly Ser Val Ala Asn Tyr Asp Glu Val

50 55 60

Val Arg Tyr Pro Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Lys Asn

65 70 75 80

Val Ile Thr Asp Lys Asp Ile Met Leu Ile Arg Leu Asp Arg Pro Val

85 90 95

Lys Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Asn Pro Pro

100 105 110

Ser Val Gly Ser Val Cys Arg Ile Met Gly Trp Gly Ala Ile Thr Thr

115 120 125

Ser Glu Asp Thr Tyr Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu

130 135 140

Phe Asn Asn Thr Val Cys Arg Glu Ala Tyr Asn Gly Leu Pro Ala Lys

145 150 155 160

Thr Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp Thr Cys Gly Gly

165 170 175

Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Gln Phe Gln Gly Ile Leu

180 185 190

Ser Trp Gly Ser Asp Pro Cys Ala Glu Pro Arg Lys Pro Ala Phe Tyr

195 200 205

Thr Lys Val Phe Asp Tyr Leu Pro Trp Ile Gln Ser Ile Ile Ala Gly

210 215 220

Asn Lys Thr Ala Thr Cys Pro

225 230

<210>2

<211>714

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

ctcgagaaaa gagtcattgg aggtgatgaa tgtgacatca acgaacaccc tttccttgcc 60

ttcatgtact actctccacg ttacttctgt ggtatgactt tgatcaacca ggaatgggtc 120

ctgaccgctg cacactgtaa cagaagattt atgcgtatcc accttggtaa gcacgccgga 180

tctgtcgcca actacgatga ggtcgttaga tacccaaagg agaagttcat ttgtcctaat 240

aagaagaaaa acgtcattac cgacaaggac attatgttga tcagactgga cagacctgtc 300

aaaaactccg aacacatcgc tcctctctct ttgccttcca accctccaag tgttggttcc 360

gtttgccgta ttatgggatg gggagcaatc acaacttctg aagacactta cccagatgtc 420

cctcactgtg ctaacattaa cctgttcaac aatactgtct gtcgtgaggc ttacaacggt 480

ttgccagcta agaccttgtg tgcaggtgtc ctgcaaggag gtatcgatac atgtggtggt 540

gactctggtg gaccactgat ctgtaatgga caattccagg gaatcttgtc ttggggaagt 600

gatccatgtg ccgaaccacg taagcctgcc ttctacacca aggtctttga ttaccttcca 660

tggattcagt ctatcattgc tggaaacaag actgctactt gtccataaga attc 714

<210>3

<211>251

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>3

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Val Ile Gly Gly Asp Glu Cys Asp Ile Asn Glu His

20 25 30

Pro Phe Leu Ala Phe Met Tyr Tyr Ser Pro Arg Tyr Phe Cys Gly Met

35 40 45

Thr Leu Ile Asn Gln Glu Trp Val Leu Thr Ala Ala His Cys Asn Arg

50 55 60

Arg Phe Met Arg Ile His Leu Gly Lys His Ala Gly Ser Val Ala Asn

65 70 75 80

Tyr Asp Glu Val Val Arg Tyr Pro Lys Glu Lys Phe Ile Cys Pro Asn

85 90 95

Lys Lys Lys Asn Val Ile Thr Asp Lys Asp Ile Met Leu Ile Arg Leu

100 105 110

Asp Arg Pro Val Lys Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro

115 120 125

Ser Asn Pro Pro Ser Val Gly Ser Val Cys Arg Ile Met Gly Trp Gly

130 135 140

Ala Ile Thr Thr Ser Glu Asp Thr Tyr Pro Asp Val Pro His Cys Ala

145 150 155 160

Asn Ile Asn Leu Phe Asn Asn Thr Val Cys Arg Glu Ala Tyr Asn Gly

165 170 175

Leu Pro Ala Lys Thr Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp

180 185 190

Thr Cys Gly Gly Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Gln Phe

195 200 205

Gln Gly Ile Leu Ser Trp Gly Ser Asp Pro Cys Ala Glu Pro Arg Lys

210 215 220

Pro Ala Phe Tyr Thr Lys Val Phe Asp Tyr Leu Pro Trp Ile Gln Ser

225 230 235 240

Ile Ile Ala Gly Asn Lys Thr Ala Thr Cys Pro

245 250

<210>4

<211>755

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

atgtatagga tgcaactgct gtcttgcatt gctctgtctc tggcctggtc accaactccg 60

tcattggagg tgatgaatgt gacatcaacg aacacccttt ccttgccttc atgtactact 120

ctccacgtta cttctgtggt atgactttga tcaaccagga atgggtcctg accgctgcac 180

actgtaacag aagatttatg cgtatccacc ttggtaagca cgccggatct gtcgccaact 240

acgatgaggt cgttagatac ccaaaggaga agttcatttg tcctaataag aagaaaaacg 300

tcattaccga caaggacatt atgttgatca gactggacag acctgtcaaa aactccgaac 360

acatcgctcc tctctctttg ccttccaacc ctccaagtgt tggttccgtt tgccgtatta 420

tgggatgggg agcaatcaca acttctgaag acacttaccc agatgtccct cactgtgcta 480

acattaacct gttcaacaat actgtctgtc gtgaggctta caacggtttg ccagctaaga 540

ccttgtgtgc aggtgtcctg caaggaggta tcgatacatg tggtggtgac tctggtggac 600

cactgatctg taatggacaa ttccagggaa tcttgtcttg gggaagtgat ccatgtgccg 660

aaccacgtaa gcctgccttc tacaccaagg tctttgatta ccttccatgg attcagtcta 720

tcattgctgg aaacaagact gctacttgtc cataa 755

<210>5

<211>231

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>5

Val Ile Gly Gly Asp Glu Cys Asp Ile Asn Glu His Pro Phe Leu Ala

1 5 10 15

Phe Met Tyr Tyr Ser Pro Arg Tyr Phe Cys Gly Met Thr Leu Ile Asn

20 25 30

Gln Glu Trp Val Leu Thr Ala Ala His Cys Asn Arg Arg Phe Met Arg

35 40 45

Ile His Leu Gly Lys His Ala Gly Ser Val Arg Asn Tyr Asp Glu Val

50 55 60

Val Arg Tyr Pro Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Lys Asn

65 70 75 80

Val Ile Thr Asp Lys Asp Ile Met Leu Ile Arg Leu Asp Arg Pro Val

85 90 95

Lys Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Asn Pro Pro

100 105 110

Ser Val Gly Ser Val Cys Arg Ile Met Gly Trp Gly Ala Ile Thr Thr

115 120 125

Ser Glu Asp Thr Tyr Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu

130 135 140

Phe Asn Asn Thr Val Cys Arg Glu Ala Tyr Asn Gly Leu Pro Ala Lys

145 150 155 160

Thr Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp Thr Cys Gly Gly

165 170 175

Asp Ser Gly Gly Pro LeuIle Cys Asn Gly Gln Phe Gln Gly Ile Leu

180 185 190

Ser Trp Gly Ser Asp Pro Cys Ala Glu Pro Arg Lys Pro Ala Phe Tyr

195 200 205

Thr Lys Val Phe Asp Tyr Leu Pro Trp Ile Gln Ser Ile Ile Ala Gly

210 215 220

Asn Lys Thr Ala Thr Cys Pro

225 230

<210>6

<211>714

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

ctcgagaaaa gagtcattgg aggtgatgaa tgtgacatca acgaacaccc tttccttgcc 60

ttcatgtact actctccacg ttacttctgt ggtatgactt tgatcaacca ggaatgggtc 120

ctgaccgctg cacactgtaa cagaagattt atgcgtatcc accttggtaa gcacgccgga 180

tctgtccgaa actacgatga ggtcgttaga tacccaaagg agaagttcat ttgtcctaat 240

aagaagaaaa acgtcattac cgacaaggac attatgttga tcagactgga cagacctgtc 300

aaaaactccg aacacatcgc tcctctctct ttgccttcca accctccaag tgttggttcc 360

gtttgccgta ttatgggatg gggagcaatc acaacttctg aagacactta cccagatgtc 420

cctcactgtg ctaacattaa cctgttcaac aatactgtct gtcgtgaggc ttacaacggt 480

ttgccagcta agaccttgtg tgcaggtgtc ctgcaaggag gtatcgatac atgtggtggt 540

gactctggtg gaccactgat ctgtaatgga caattccagg gaatcttgtc ttggggaagt 600

gatccatgtg ccgaaccacg taagcctgcc ttctacacca aggtctttga ttaccttcca 660

tggattcagt ctatcattgc tggaaacaag actgctactt gtccataaga attc 714

<210>7

<211>231

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>7

Val Ile Gly Gly Asp Glu Cys Asp Ile Asn Glu His Pro Phe Leu Ala

1 5 10 15

Phe Met Tyr Tyr Ser Pro Arg Tyr Phe Cys Gly Met Thr Leu Ile Asn

20 25 30

Gln Glu Trp Val Leu Thr Ala Ala His Cys Asn Arg Arg Phe Met Arg

35 40 45

Ile His Leu Gly Lys His Ala Gly Ser Val Ala Asn Tyr Asp Glu Val

50 55 60

Val Arg Tyr Pro Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Lys Asn

65 70 75 80

Val Ile Thr Asp Lys Asp Ile Met Leu Ile Arg Leu Asp Arg Pro Val

85 90 95

Lys Asn Ser Glu His Ile Ala Pro Leu Ser Leu Pro Ser Asn Pro Pro

100 105 110

Ser Val Gly Ser Val Cys Arg Ile Met Gly Trp Gly Ala Ile Thr Arg

115 120 125

Ser Glu Asp Thr Tyr Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu

130 135 140

Phe Asn Asn Thr Val Cys Arg Glu Ala Tyr Asn Gly Leu Pro Ala Lys

145 150 155 160

Thr Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp Thr Cys Gly Gly

165 170 175

Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Gln Phe Gln Gly Ile Leu

180 185 190

Ser Trp Gly Ser Asp Pro Cys Ala Glu Pro Arg Lys Pro Ala Phe Tyr

195 200 205

Thr Lys Val Phe Asp Tyr Leu Pro Trp Ile Gln Ser Ile Ile Ala Gly

210 215 220

Asn Lys Thr Ala Thr Cys Pro

225 230

<210>8

<211>714

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>8

ctcgagaaaa gagtcattgg aggtgatgaa tgtgacatca acgaacaccc tttccttgcc 60

ttcatgtact actctccacg ttacttctgt ggtatgactt tgatcaacca ggaatgggtc 120

ctgaccgctg cacactgtaa cagaagattt atgcgtatcc accttggtaa gcacgccgga 180

tctgtcgcca actacgatga ggtcgttaga tacccaaagg agaagttcat ttgtcctaat 240

aagaagaaaa acgtcattac cgacaaggac attatgttga tcagactgga cagacctgtc 300

aaaaactccg aacacatcgc tcctctctct ttgccttcca accctccaag tgttggttcc 360

gtttgccgta ttatgggatg gggagcaatc acacgatctg aagacactta cccagatgtc 420

cctcactgtg ctaacattaa cctgttcaac aatactgtct gtcgtgaggc ttacaacggt 480

ttgccagcta agaccttgtg tgcaggtgtc ctgcaaggag gtatcgatac atgtggtggt 540

gactctggtg gaccactgat ctgtaatgga caattccagg gaatcttgtc ttggggaagt 600

gatccatgtg ccgaaccacg taagcctgcc ttctacacca aggtctttga ttaccttcca 660

tggattcagt ctatcattgc tggaaacaag actgctactt gtccataaga attc 714

<210>9

<211>714

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>9

ctcgagaaaa gagtcattgg aggtgatgaa tgtgacatca acgaacaccc tttccttgcc 60

ttcatgtact actctccacg ttacttctgt ggtatgactt tgatcaacca ggaatgggtc 120

ctgaccgctg cacactgtaa cagaagattt atgcgtatcc accttggtaa gcacgccgga 180

tctgtcgcca actacgatga ggtcgttaga tacccaaagg agaagttcat ttgtcctaat 240

aagaagaaaa acgtcattac cgacaaggac attatgttga tcagactgga cagacctgtc 300

aaaaactccg aacacatcgc tcctctctct ttgccttcca accctccaag tgttggttcc 360

gtttgccgta ttatgggatg gggagcaatc acaacttctg aagacactta cccagatgtc 420

cctcactgtg ctaacattaa cctgttcaac aatactgtct gtcgtgaggc ttacaacggt 480

ttgccagcta agaccttgtg tgcaggtgtc ctgcaaggag gtatcgatac atgtggtggt 540

gactctggtg gaccactgat ctgtaatgga caattccagg gaatcttgtc ttggggaagt 600

gatccatgtg ccgaaccacg taagcctgcc ttctacacca aggtctttga ttaccttcca 660

tggattcagt ctatcattgc tggaaacaag actgctactt gtccataaga attc 714

<210>10

<211>714

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>10

ctcgagaaaa gagtcattgg aggtgatgaa tgtgacatca acgaacaccc tttccttgcc 60

ttcatgtact actctccacg ttacttctgt ggtatgactt tgatcaacca ggaatgggtc 120

ctgaccgctg cacactgtaa cagaagattt atgcgtatcc accttggtaa gcacgccgga 180

tctgtcgcca actacgatga ggtcgttaga tacccaaagg agaagttcat ttgtcctaat 240

aagaagaaaa acgtcattac cgacaaggac attatgttga tcagactgga cagacctgtc 300

aaaaactccg aacacatcgc tcctctctct ttgccttcca accctccaag tgttggttcc 360

gtttgccgta ttatgggatg gggagcaatc acaacttctg aagacactta cccagatgtc 420

cctcactgtg ctaacattaa cctgttcaac aatactgtct gtcgtgaggc ttacaacggt 480

ttgccagcta agaccttgtg tgcaggtgtc ctgcaaggag gtatcgatac atgtggtggt 540

gactctggtg gaccactgat ctgtaatgga caattccagg gaatcttgtc ttggggaagt 600

gatccatgtg ccgaaccacg taagcctgcc ttctacacca aggtctttga ttaccttcca 660

tggattcagt ctatcattgc tggacaaaag actgctactt gtccataaga attc 714

Claims (10)

1, a kind of special-purpose gene for preparing batroxobin has one of following nucleotide sequence:

1) sequence 2 in the sequence table;

2) in the sequence table sequence 2 through replacing 1-3 base, the proteic nucleotide sequence of identical function of encoding;

3) 1) 5 ' end of described sequence connects the nucleotide sequence that IL-2 signal peptide gene sequence obtains.

4) can be with 1 under the rigorous condition of height), 2) or 3) nucleotide sequence of the dna sequence dna hybridization of described sequence;

2, gene according to claim 1 is characterized in that: the nucleotides sequence of described IL-2 signal peptide gene is classified as from GENBANK number and is 5 of NM_000586 ' end 295-354 position Nucleotide.

3, gene according to claim 2 is characterized in that: described encoding gene has one of following nucleotide sequence:

1) sequence 2 in the sequence table;

2) sequence 4 in the sequence table

3) sequence 6 in the sequence table;

4) sequence 8 in the sequence table.

4, a kind of method for preparing batroxobin is the arbitrary described encoding gene of claim 1-3 to be imported in yeast or the mammal cell line by carrier for expression of eukaryon express, and obtains batroxobin protein.

5, method according to claim 5 is characterized in that: described yeast is a pichia spp, is preferably pichia spp GS115/His-.

6, method according to claim 5 is characterized in that: described mammal cell line is a Chinese hamster ovary celI system.

7, method according to claim 6 is characterized in that: when described encoding gene imported in the yeast, described carrier for expression of eukaryon was pPIC9; When described encoding gene imported in the mammal cell line, described carrier for expression of eukaryon was pcDNA3.1.

8, according to the arbitrary described method of claim 4-7, it is characterized in that: described method also comprises carries out purifying with expressing the batroxobin protein that obtains.

9, the batroxobin protein of the arbitrary described method preparation of claim 4-8.

10, batroxobin protein according to claim 9 is characterized in that: the amino acid residue sequence of described batroxobin protein is:

1) sequence 1 in the sequence table;

2) the 59th Ala of the aminoterminal of sequence 1 sports the aminoacid sequence of Arg;

3) the 128th Thr of the aminoterminal of sequence 1 sports the aminoacid sequence of Arg;

4) aminoterminal of sequence 1 connects the aminoacid sequence of IL-2 signal peptide;

The aminoacid sequence of described IL-2 signal peptide is to be the aminoterminal 1-20 amino acids sequence of NP_000577 from GENBANK number.

Images