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Production method of recombinant human blood coagulation factor XIIa

A technology of human blood coagulation factor and production method, which is applied in the production field of recombinant human blood coagulation factor Ⅻa, can solve the problems of inability to carry out recombinant expression and purification, expensive raw materials, low yield, etc., achieve simplified quality control, and improve product quality , the effect of reducing production costs

Active Publication Date: 2013-10-30
VIVA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process is complicated, the operation is cumbersome, the yield is low (less than 0.1mg / L blood), and the cost is high (about 3500 yuan for 0.1mg)
According to the analysis, the main reasons are: first, the raw materials are more expensive and require a large amount of human blood; second, the protein purification process is cumbersome and requires multiple purifications; third, the purified protein needs to be activated by the zymogen to be active; fourth, it cannot be recombinantly expressed and purification

Method used

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  • Production method of recombinant human blood coagulation factor XIIa
  • Production method of recombinant human blood coagulation factor XIIa
  • Production method of recombinant human blood coagulation factor XIIa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The DNA (Sequence Listing 1) encoding coagulation factor XIIa (354-615) obtained by gene synthesis was cloned into the pPICZαB vector to construct the pPICZαB-coagulation factor XIIa vector, which was transformed into Escherichia coli DH5α. The recombinant plasmid pPICZαB-coagulation factor Ⅻa was extracted to a concentration of 736.28 μg / ml. Take 20 μg of the recombinant plasmid pPICZαB-coagulation factor Ⅻa for linearization by Sac Ⅰ single enzyme digestion, and dissolve it in TEBuffer after recovery, with a final volume of 30 μl. Inoculate X33 overnight culture into 10ml YPD liquid medium, 30°C, 230rpm, OD600=1.366 after about 16h. Centrifuge at 4°C, wash with ice sterile water and ice 1M sorbitol successively to resuspend the precipitate. Bring the final volume to 1.5ml. Take 60 μl of the above bacterial solution, mix it with the linearized DNA, and transfer it to an ice-cooled 0.2 cm electroshock cuvette. Ice bath for 5min. Select the yeast preset program for el...

Embodiment 2

[0026]The DNA (sequence list 3) encoding blood coagulation factor Ⅻa (354-615, C486A, that is, the 486th amino acid of blood coagulation factor Ⅻa mutated from cysteine ​​to alanine) obtained by gene synthesis was cloned into the pPICZαB vector, and constructed pPICZαB-coagulation factor Ⅻa vector, transformed into Escherichia coli DH5α. The recombinant plasmid pPICZαB-coagulation factor Ⅻa was extracted to a concentration of 752.11 μg / ml. Take 20 μg of the recombinant plasmid pPICZαB-coagulation factor Ⅻa for Sac Ⅰ single enzyme digestion and linearization, and dissolve it in TE Buffer after recovery, with a final volume of 30 μl. Inoculate X33 overnight culture into 10ml YPD liquid medium, 30°C, 230rpm, OD600=1.366 after about 16h. Centrifuge at 4°C, wash with ice sterile water and ice 1M sorbitol successively to resuspend the precipitate. Bring the final volume to 1.5ml. Take 60 μl of the above bacterial solution, mix it with the linearized DNA, and transfer it to an ice-...

Embodiment 3

[0028] The coagulation factor Ⅻa (354-615, N433A, C486A) encoded by gene synthesis; that is, the 433rd amino acid of coagulation factor Ⅻa is mutated from asparagine to alanine; the 486th amino acid is mutated from cysteine ​​to alanine Acid) DNA (sequence table 5) was cloned into the pPICZαB vector to construct the pPICZαB-coagulation factor Ⅻa vector, which was transformed into Escherichia coli DH5α. The recombinant plasmid pPICZαB-coagulation factor Ⅻa was extracted to a concentration of 717.52 μg / ml. Take 20 μg of the recombinant plasmid pPICZαB-coagulation factor Ⅻa for SacⅠ single enzyme digestion and linearization, and dissolve it in TE Buffer after recovery, with a final volume of 30 μl. Inoculate X33 overnight culture into 10ml YPD liquid medium, 30°C, 230rpm, OD600=1.366 after about 16h. Centrifuge at 4°C, wash with ice sterile water and ice 1M sorbitol successively to resuspend the precipitate. Bring the final volume to 1.5ml. Take 60 μl of the above bacterial sol...

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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 [0001] The invention belongs to the field of genetic engineering, in particular to a production method of recombinant human blood coagulation factor Ⅻa. Background technique [0002] Human coagulation factor XII is also known as contact factor, surface factor or HAGEMAN factor (hageman factor). It participates in the endogenous coagulation activation system in vivo. The main process is that when blood comes into contact with negatively charged collagen (the outer wall of skin blood vessels) or foreign body surfaces (such as kaolin, glass, etc.), coagulation factor Ⅻ is activated by zymogen to coagulation factor Ⅻa. Coagulation factor Ⅻa can not only activate coagulation factor Ⅺ, but also activate plasma pre-soothing kallikrein. The activated kallikrein in turn further activates factor XII under the promotion of high molecular weight kininogen. Activated coagulation factor Ⅻa in Ca 2+ The presence in turn activates Factor IX. Thereby completed a series o...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/745
Inventor 钱冬明胡亮李立炯
Owner VIVA BIOTECH
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