CN103374588A - Production method of recombinant human blood coagulation factor XIIa - Google Patents
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Abstract
The invention belongs to the field of genetic engineering and in particular relates to a production method of a recombinant human blood coagulation factor XIIa. The production method of the recombinant human blood coagulation factor XIIa comprises the following steps of: acquiring DNA (deoxyribonucleic acid) coding an active region of a human blood coagulation factor XIIa, amplifying by virtue of pichia pastoris, centrifuging and dialyzing a yeast culture solution and carrying out filter pressing by adopting a filter membrane on the yeast culture solution, and then carrying out column chromatographic purification by taking Ni-NTA (Ni-nitrilotriacetic acid) as medium, so that the recombinant human blood coagulation factor XIIa is obtained. By adopting the production method of the recombinant human blood coagulation factor XIIa, production cost of the human blood coagulation factor XIIa is reduced, and product activity is higher than that of blood coagulation factor XIIa extracted from blood.
Description
Technical field
The invention belongs to the genetically engineered field, particularly the production method of a kind of recombinant human blood coagulation factor XII a.
Background technology
Human blood coagulation XII be otherwise known as contact factor, surface factor or the HAGEMAN factor (hageman factor).It has participated in the intrinsic coagulation activation system in vivo.Process is mainly when blood and electronegative collagen protein (skin heart outer wall) or allosome surface (such as kaolin, glass etc.) when contacting, and factor ⅫYou Chengjiechuyinzi just becomes factor ⅫYou Chengjiechuyinzi a by activation of zymogen.Factor ⅫYou Chengjiechuyinzi a decapacitation activates outside the plasma thromboplastin antecedent, makes simultaneously again the front bradykinin of blood plasma discharge enzyme and activates.Kallikrein after the activation further makes again factor XII activate under the promotion of high molecular weight kininogen conversely.Factor ⅫYou Chengjiechuyinzi a after the activation is at Ca
2+Existence is lower and factor IX is activated.Thereby finished the blood coagulation reaction of some row.In recent years, because the expert finds its function further investigation, find that the sudden change of factor ⅫYou Chengjiechuyinzi a can cause various diseases such as coronary heart disease both at home and abroad, thrombus etc., the structure and function of therefore studying factor ⅫYou Chengjiechuyinzi a becomes a present focus.But because raw-material restriction, progress is very slow at present.
Factor ⅫYou Chengjiechuyinzi a extracts to separate from human blood and obtains at present.Two step ion exchange chromatographies have been comprised in its purge process, a step gel permeation chromatography, a step activation of zymogen process.Process is complicated, complex operation, yield low (less than 0.1mg/L blood), expense high (about 3500 yuan of 0.1mg).According to analyzing its major cause be: the one, starting material are more expensive, need a large amount of people's blood; Two: the albumen purification process is loaded down with trivial details, needs multi-section to purify; Three. purifying protein needs activation of zymogen that activity is just arranged; Four: can't carry out recombinant expressed and purifying.
Summary of the invention
The technical problem to be solved in the present invention is: the production method that a kind of convenient purification, lower-cost human blood coagulation XII a are provided.
The invention provides the production method of a kind of recombinant human blood coagulation factor XII a, step is as follows:
(1) obtains the DNA of encoding human factor ⅫYou Chengjiechuyinzi a active region (amino acid fragment 354-615) by the synthetic method of gene, the dna clone that obtains in pPICZ α B carrier, is made up pPICZ α B-factor ⅫYou Chengjiechuyinzi a carrier, transform bacillus coli DH 5 alpha;
(2) utilize plasmid extraction method extracting recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a; Recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a is carried out the linearizing of Sac I single endonuclease digestion, and stand-by after reclaiming;
(3) inoculation pichia spp X33 or GS115 is transferred to culture in the YPD liquid nutrient medium, and at 27-30 ℃, it is ℃ centrifugal to be cultured to OD600=1.3～1.5,4 under the condition of 200-250rpm, washs resuspended precipitation with the 1M Sorbitol Powder of icing sterilized water and ice successively;
(4) get above-mentioned bacterium liquid, with linearizing DNA mixing, click the Sorbitol Powder that adds the 1M of precooling after transforming, be transferred to aseptic Eppendorf tube; Get 20-200 μ L bacterium liquid after centrifugal and be applied to the YPDSZ flat board, hatch 72～96h for 30 ℃, until bacterium colony occurs, dibbling is preserved; The high copy of screening positive colony bacterial strain is cultivated centrifugal collection supernatant liquor after cultivation finishes;
(5) supernatant liquor that collection in the step (4) is obtained again by the column chromatography purification take Ni-NTA as medium, obtains recombinant human blood coagulation factor XII a through the filter membrane press filtration of dialysis, 0.45 μ m.
Wherein, the detailed operation steps of described step (5) is as follows: will express supernatant liquor and pour into dialysis tubing, 4 ℃ of lower chromatography level pad dialysis with 10 times of volumes; Behind the 48h, with the filter membrane press filtration of supernatant liquor in the bag by 0.45 μ m; With Ni-NTA medium dress post, carry out balance with level pad.The clear liquor that obtains with press filtration after the balance spends the night 4 ℃ of lower run by gravity loadings.After loading is complete, with 5～10 column volume level pad washings, use again 5～10 column volume elution buffer wash-outs; Collect the effluent of above each step; Wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.
Preferably, in the above-mentioned steps (5), can also add the activation step: the Ni-NTA medium after the complete washing of collection loading, at room temperature activate damping fluid with about 10 column volumes and softly mix 45min.Be that the EDTA of 0.1mM stops activating reaction with final concentration.Again medium is filled post, with 2～10 column volume level pad washings, use again 2～20 column volume elution buffer wash-outs; Collect the effluent of above each step, wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.
Described level pad is to be comprised of 20-100mMTris, 50-200mMNaCl and 5%～10% glycerine, and pH is the damping fluid between 7.5～8.0.
Described elution buffer is to be comprised of 20-100mMTris, 50-200mMNaCl, 5%～10% glycerine and 100-250mM imidazoles, and pH is the damping fluid between 7.5～8.0.
Described activation damping fluid is: the chromatography level pad that contains the 0.2ug/ml kallikrein.
Preferably, the molecular weight that dams of described dialysis tubing is 5-10kD; The molecular weight that dams of evaporating pipe is 10-30kD.
Beneficial effect of the present invention: compared with prior art, this be utilize first both at home and abroad vivoexpression the method success expression people source factor ⅫYou Chengjiechuyinzi a.The present invention adopts the synthetic gene technology to obtain the gene of people source factor ⅫYou Chengjiechuyinzi a, and use clone technology that the gene of factor ⅫYou Chengjiechuyinzi a is inserted in the Yeast expression carrier, the expression of recycling yeast expression system success people source recombinant protein factor ⅫYou Chengjiechuyinzi a, obtained having highly active people's recombinant blood coagulation factor XII a by affinity chromatography technology success purifying.Its yield surpasses 0.7mg/L, and activity is higher than the factor ⅫYou Chengjiechuyinzi a that extracts from blood.
The present invention has set up the complete factor ⅫYou Chengjiechuyinzi a of a cover and has expressed, synthesis technique, embody expression of recombinant proteins system and affinity chromatography and produced the superiority of factor ⅫYou Chengjiechuyinzi a, be conducive to the controlled of the simplification of technique and quality, reduced production cost, improved product quality, obtained higher yield (greater than 0.7mg/L), purity is greater than 85% recombinant protein very high with activity.The method is suitable for suitability for industrialized production simultaneously, and function, structure and the medicinal design of studying factor ⅫYou Chengjiechuyinzi a for us lay the first stone.
Detailed description of the invention
The YPD substratum has another name called yeast extract powder peptone dextrose culture-medium, is the yeast culture liquid of commonly using.
Description of drawings
Fig. 1 is the Western Blot figure of Hageman factor a recombinant protein, and 1 is blood factor XIIa recombinant protein among the figure, M﹠amp; The positive contrast of P.
Fig. 2-7 is the activity curve of the Hageman factor a recombinant protein of corresponding embodiment 1-3 respectively, and wherein Y2-14 obtains product among the embodiment 1, and Fig. 2 is the determination of activity of Y2-14 in S2302 matrix, and Fig. 3 is the determination of activity of Y2-14 in S2222 matrix; Y2-26 obtains product among the embodiment 2, and Fig. 4 is the determination of activity of Y2-26 in S2302 matrix, and Fig. 5 is the determination of activity of Y2-26 in S2222 matrix; Y3-25 obtains product among the embodiment 3, and Fig. 6 is the determination of activity of Y3-25 in S2302 matrix, and Fig. 7 is the determination of activity of Y3-25 in S2222 matrix; FXIIa β is the Hageman factor a albumen that extracts from human blood.
Embodiment
Embodiment 1
With the synthetic coding factor ⅫYou Chengjiechuyinzi a(354-615 that obtains of gene) DNA(sequence table 1) be cloned in the pPICZ α B carrier, make up pPICZ α B-factor ⅫYou Chengjiechuyinzi a carrier, transform bacillus coli DH 5 alpha.Extracting recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a, concentration is 736.28 μ g/ml.Get 20 μ g recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a and carry out the linearizing of Sac I single endonuclease digestion, be dissolved in TEBuffer after the recovery, final volume 30 μ l.Inoculate the X33 overnight culture to the 10mlYPD liquid nutrient medium, 30 ℃, 230rpm, OD600=1.366 behind about 16h.4 ℃ centrifugal, and the 1M Sorbitol Powder with ice sterilized water and ice washs resuspended precipitation successively.Making final volume is 1.5ml.Get above-mentioned bacterium liquid 60 μ l, with linearizing DNA mixing, be transferred in the 0.2cm electric shock cup of ice precooling.Ice bath 5min.Select yeast pre-set programs electric shock to transform.Add immediately the Sorbitol Powder of the 1M of 1ml precooling in the electric shock cup, be transferred to aseptic Eppendorf tube.Get 100 μ L bacterium liquid after centrifugal and be applied to YPDSZ flat board (zeocin:0.1mg/ml), hatch 96h for 30 ℃, bacterium colony occurs.Utilize universal primer and Auele Specific Primer to carry out PCR take bacterium liquid as template and filter out 27 of positive colonies, phenotype all is Mut+.The picking positive colony filters out 5 of high copy bacterial strains to the YPDS flat board that contains high density zeocin (1mg/ml).A high copy mono-clonal of picking (Y214) is seeded in the 5L shaking flask that contains 1L BMGY, and 30 ℃, 180rpm cultivates 24h to OD600=5.17.Centrifugal collection thalline is removed the BMGY substratum, uses the resuspended thalline of 1L inducing culture BMMY to OD600=1.2, and 25 ℃, 180rpm, 1% methyl alcohol final concentration continues to cultivate 4 days.Express and finish rear centrifugal collection supernatant.To express supernatant liquor and pour into dialysis tubing, 4 ℃ of lower chromatography level pad dialysis with 100L.Behind the 48h, with the filter membrane press filtration of supernatant liquor in the bag by 0.45 μ m.Ni-NTA medium dress post with 3ml carries out balance with level pad.The clear liquor that obtains with press filtration after the balance spends the night 4 ℃ of lower run by gravity loadings.After loading is complete, with the washing of 30ml level pad.Collect the Ni-NTA medium after washing, at room temperature activate damping fluid with 25ml and softly mix 45min.Be that the EDTA of 0.1mM stops activating reaction with final concentration.Again medium is filled post, with the washing of 15ml level pad, use again 30ml elution buffer wash-out.Collect the effluent of above each step.Wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.The final factor ⅫYou Chengjiechuyinzi a albumen (sequence table 2) that obtains 250ul, concentration is 3mg/ml, yield is 0.75mg/L.Detect through SDS-PAGE and Western Blot, purity surpasses 80%.Albumen sampling is carried out activity and is detected, and activity all is better than the factor ⅫYou Chengjiechuyinzi a albumen that extracts from blood in S2302 and S2222 matrix.
Embodiment 2
With the synthetic coding factor ⅫYou Chengjiechuyinzi a(354-615 that obtains of gene, C486A, the 486th amino acids that is factor ⅫYou Chengjiechuyinzi a is L-Ala by cysteine mutation) DNA(sequence table 3) be cloned in the pPICZ α B carrier, make up pPICZ α B-factor ⅫYou Chengjiechuyinzi a carrier, transform bacillus coli DH 5 alpha.Extracting recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a, concentration is 752.11 μ g/ml.Get 20 μ g recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a and carry out the linearizing of Sac I single endonuclease digestion, be dissolved in TE Buffer after the recovery, final volume 30 μ l.Inoculate the X33 overnight culture to the 10mlYPD liquid nutrient medium, 30 ℃, 230rpm, OD600=1.366 behind about 16h.4 ℃ centrifugal, and the 1M Sorbitol Powder with ice sterilized water and ice washs resuspended precipitation successively.Making final volume is 1.5ml.Get above-mentioned bacterium liquid 60 μ l, with linearizing DNA mixing, be transferred in the 0.2cm electric shock cup of ice precooling.Ice bath 5min.Select yeast pre-set programs electric shock to transform.Add immediately the Sorbitol Powder of the 1M of 1ml precooling in the electric shock cup, be transferred to aseptic Eppendorf tube.Get 100 μ L bacterium liquid after centrifugal and be applied to YPDSZ flat board (zeocin:0.1mg/ml), hatch 96h for 30 ℃, bacterium colony occurs.Utilize universal primer and Auele Specific Primer to carry out PCR take bacterium liquid as template and filter out 27 of positive colonies, phenotype all is Mut+.The picking positive colony filters out 5 of high copy bacterial strains to the YPDS flat board that contains high density zeocin (1mg/ml).A high copy mono-clonal of picking (Y226) is seeded in the 5L shaking flask that contains 1L BMGY, and 30 ℃, 180rpm cultivates 24h to OD600=3.32.Centrifugal collection thalline is removed the BMGY substratum, uses the resuspended thalline of 1L inducing culture BMMY to OD600=0.9, and 25 ℃, 180rpm, 1% methyl alcohol final concentration continues to cultivate 4 days.Express and finish rear centrifugal collection supernatant.To express supernatant liquor and pour into dialysis tubing, 4 ℃ of lower chromatography level pad dialysis with 100L.Behind the 48h, with the filter membrane press filtration of supernatant liquor in the bag by 0.45 μ m.Ni-NTA medium dress post with 3ml carries out balance with level pad.The clear liquor that obtains with press filtration after the balance spends the night 4 ℃ of lower run by gravity loadings.After loading is complete, with the washing of 30ml level pad, use again 30ml elution buffer wash-out.Collect the effluent of above each step.Wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.The final factor ⅫYou Chengjiechuyinzi a albumen (sequence table 4) that obtains 300ul, concentration is 3.5mg/ml, yield is 1.05mg/L.Detect through SDS-PAGE and Western Blot, purity surpasses 80%.Albumen sampling is carried out activity and is detected, and activity all is better than the factor ⅫYou Chengjiechuyinzi a albumen that extracts from blood in S2302 and S2222 matrix.
Embodiment 3
With synthetic coding factor ⅫYou Chengjiechuyinzi a (354-615, N433A, the C486A that obtains of gene; The 433rd amino acids that is factor ⅫYou Chengjiechuyinzi a sports L-Ala by asparagine; The 486th amino acids is L-Ala by cysteine mutation) DNA(sequence table 5) be cloned in the pPICZ α B carrier, make up pPICZ α B-factor ⅫYou Chengjiechuyinzi a carrier, transform bacillus coli DH 5 alpha.Extracting recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a, concentration is 717.52 μ g/ml.Get 20 μ g recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a and carry out the linearizing of Sac I single endonuclease digestion, be dissolved in TE Buffer after the recovery, final volume 30 μ l.Inoculate the X33 overnight culture to 10ml YPD liquid nutrient medium, 30 ℃, 230rpm, OD600=1.366 behind about 16h.4 ℃ centrifugal, and the 1M Sorbitol Powder with ice sterilized water and ice washs resuspended precipitation successively.Making final volume is 1.5ml.Get above-mentioned bacterium liquid 60 μ l, with linearizing DNA mixing, be transferred in the 0.2cm electric shock cup of ice precooling.Ice bath 5min.Select yeast pre-set programs electric shock to transform.Add immediately the Sorbitol Powder of the 1M of 1ml precooling in the electric shock cup, be transferred to aseptic Eppendorf tube.Get 100 μ L bacterium liquid after centrifugal and be applied to YPDSZ flat board (zeocin:0.1mg/ml), hatch 96h for 30 ℃, bacterium colony occurs.Utilize universal primer and Auele Specific Primer to carry out PCR take bacterium liquid as template and filter out 25 of positive colonies, phenotype all is Mut
+The picking positive colony filters out 6 of high copy bacterial strains to the YPDS flat board that contains high density zeocin (1mg/ml).A high copy mono-clonal of picking (Y325) is seeded in the 5L shaking flask that contains 1LBMGY, and 30 ℃, 180rpm cultivates 24h to OD600=3.01.Centrifugal collection thalline is removed the BMGY substratum, uses the resuspended thalline of 1L inducing culture BMMY to OD600=0.9, and 25 ℃, 180rpm, 1% methyl alcohol final concentration continues to cultivate 4 days.Express and finish rear centrifugal collection supernatant.To express supernatant liquor and pour into dialysis tubing, 4 ℃ of lower chromatography level pad dialysis with 100L.Behind the 48h, with the filter membrane press filtration of supernatant liquor in the bag by 0.45 μ m.Ni-NTA medium dress post with 3ml carries out balance with level pad.The clear liquor that obtains with press filtration after the balance spends the night 4 ℃ of lower run by gravity loadings.After loading is complete, with the washing of 30ml level pad, use again 30ml elution buffer wash-out.Collect the effluent of above each step.Wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.The final factor ⅫYou Chengjiechuyinzi a albumen (sequence table 6) that obtains 300ul, concentration is 3mg/ml, yield is 0.9mg/L.Detect through SDS-PAGE and Western Blot, purity surpasses 80%.Albumen sampling is carried out activity and is detected, and activity all is better than the factor ⅫYou Chengjiechuyinzi a albumen that extracts from blood in S2302 and S2222 matrix.
Embodiment 4
Activity determination method is according to document: M.Margarida Bernardo, Duane E.Day, Steven T.Olson, Joseph D.Shore.Surface-independent Acceleration of Factor X11Activation by Zinc Ions.THE JOURNAL OF BIOLOGICACL CHEMISTRY, 1993,268 (17): the carrying out of describing among the 12468-12476..
Concrete, the active detection of the present invention adopted direct Detection Method: in 96 microwell plates, add 160 μ l and contain the protein solution of 25nM FXIIa, then add 40 μ lS2302 or S2222 substrate solution, with the absorbance of En Vision microplate reader monitoring at 405nm.
Wherein: kallikrein chromogenic substrate solution (S2302): 2mM, dispose with reaction buffer; Kallikrein chromogenic substrate solution (S2222): 2mM disposes with reaction buffer; Reaction buffer: 20mM Hepes, 150mM sodium chloride, 0.1%PEG, pH8.0.
Determination of activity the results are shown in Figure of description 2-7, as can be seen from the figure, the recombinant blood coagulation factor XII a that embodiment 1-3 obtains, activity all is better than extracting the factor ⅫYou Chengjiechuyinzi a that obtains from human blood.
Sequence table
Claims (7)
1. the production method of a recombinant human blood coagulation factor XII a, step is as follows:
(1) obtains the DNA of encoding human factor ⅫYou Chengjiechuyinzi a active region by the synthetic method of gene, the dna clone that obtains in pPICZ α B carrier, is made up pPICZ α B-factor ⅫYou Chengjiechuyinzi a carrier, transform bacillus coli DH 5 alpha; Described active region refers to human blood coagulation XII a amino acid fragment 354-615;
(2) utilize plasmid extraction method extracting recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a; Recombinant plasmid pPICZ alpha B-factor ⅫYou Chengjiechuyinzi a is carried out the linearizing of Sac I single endonuclease digestion, and stand-by after reclaiming;
(3) inoculation pichia spp X33 or GS115 is transferred to culture in the YPD liquid nutrient medium, and at 27-30 ℃, it is ℃ centrifugal to be cultured to OD600=1.3～1.5,4 under the condition of 200-250rpm, washs resuspended precipitation with the 1M Sorbitol Powder of icing sterilized water and ice successively;
(4) get above-mentioned bacterium liquid, with linearizing DNA mixing, click the Sorbitol Powder that adds the 1M of precooling after transforming, be transferred to aseptic Eppendorf tube; Get 20-200 μ L bacterium liquid after centrifugal and be applied to the YPDSZ flat board, hatch 72～96h for 30 ℃, until bacterium colony occurs, dibbling is preserved; The high copy of screening positive colony bacterial strain is cultivated centrifugal collection supernatant liquor after cultivation finishes;
(5) supernatant liquor that collection in the step (4) is obtained again by the column chromatography purification take Ni-NTA as medium, obtains recombinant human blood coagulation factor XII a through the filter membrane press filtration of dialysis, 0.45 μ m.
2. the production method of recombinant human blood coagulation factor XII a as claimed in claim 1 is characterized in that the 486th amino acids is L-Ala by cysteine mutation in the described human blood coagulation XII a active region.
3. the production method of recombinant human blood coagulation factor XII a as claimed in claim 1 is characterized in that the 433rd amino acids sports L-Ala by asparagine in the described human blood coagulation XII a active region.
4. the production method of recombinant human blood coagulation factor XII a as claimed in claim 1 is characterized in that the 486th amino acids is L-Ala by cysteine mutation in the described human blood coagulation XII a active region, and the 433rd amino acids sports L-Ala by asparagine.
5. such as the production method of each described recombinant human blood coagulation factor XII a of claim 1-4, it is characterized in that the detailed operation steps of described step (5) is as follows:
To express supernatant liquor and pour into dialysis tubing, 4 ℃ of lower chromatography level pad dialysis with 10 times of volumes; Behind the 48h, with the filter membrane press filtration of supernatant liquor in the bag by 0.45 μ m; With Ni-NTA medium dress post, carry out balance with level pad; The clear liquor that obtains with press filtration after the balance spends the night 4 ℃ of lower run by gravity loadings; After loading is complete, with 5～10 column volume level pad washings, use again 5～10 column volume elution buffer wash-outs; Collect the effluent of above each step; Wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.
6. the production method of recombinant human blood coagulation factor XII a as claimed in claim 5 is characterized in that the molecular weight that dams of described dialysis tubing is 5-10kD; The molecular weight that dams of evaporating pipe is 10-30kD.
7. such as the production method of recombinant human blood coagulation factor XII a as claimed in claim 5, it is characterized in that also comprising in the described step (5) the activation step:
Collecting the Ni-NTA medium after the complete washing of loading, with after about 10 column volumes activate damping fluid mixings 45min, is the EDTA termination activating reaction of 0.1mM with final concentration at room temperature; Again medium is filled post, with 2～10 column volume level pad washings, use again 2～20 column volume elution buffer wash-outs; Collect the effluent of above each step, wash-out part effluent utilizes evaporating pipe to concentrate and replaces damping fluid and is level pad.
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| CN101376875A (en) * | 2007-08-27 | 2009-03-04 | 上海生物制品研究所 | Pichia yeast expressing recombinant human blood coagulation factor VII, and preparation and use thereof |
| CN103060366A (en) * | 2012-12-25 | 2013-04-24 | 武汉科技大学 | Human blood coagulation factor IX mutant pichia pastoris expression vector and construction method and application thereof |
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| CN101376875A (en) * | 2007-08-27 | 2009-03-04 | 上海生物制品研究所 | Pichia yeast expressing recombinant human blood coagulation factor VII, and preparation and use thereof |
| CN103060366A (en) * | 2012-12-25 | 2013-04-24 | 武汉科技大学 | Human blood coagulation factor IX mutant pichia pastoris expression vector and construction method and application thereof |
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| CN105985428A (en) * | 2015-02-10 | 2016-10-05 | 许健 | Procoagulant function of human recombinant blood coagulation factor XIII |
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