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Expression method of human anti-zymoplasm III, special-purpose expression vector and engineering bacterium thereof

An antithrombin and expression vector technology, which is applied in the field of human antithrombin III expression and its special expression vector and engineering bacteria, can solve the problems of unseen Pichia pastoris, and achieve large production scale and growth cycle. Short, easy-to-use effects

Inactive Publication Date: 2009-04-08
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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Problems solved by technology

However, there is no research on the expression of AT-III using Pichia pastoris in China

Method used

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  • Expression method of human anti-zymoplasm III, special-purpose expression vector and engineering bacterium thereof
  • Expression method of human anti-zymoplasm III, special-purpose expression vector and engineering bacterium thereof
  • Expression method of human anti-zymoplasm III, special-purpose expression vector and engineering bacterium thereof

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Embodiment 1

[0030] Embodiment 1, expression of human antithrombin III in Pichia pastoris

[0031] 1. Construction and identification of recombinant Pichia pastoris expression vector 9K-AT

[0032] (1) Restructuring design of human antithrombin III gene: According to the gene sequence of human antithrombin III (AT-III) in GenBank (GenBank number: P01008), the sequence in the sequence listing is introduced after the stop codon A nucleotide sequence shown in 1 can form a stable secondary structure to prolong the life of mRNA and prevent read-through. Recognition sites for restriction endonucleases EcoR I and Not I were introduced according to the sequence designed and restructured and the gene characteristic sequence of the restriction endonuclease. The PCR amplification primer sequence designed according to the above gene sequence is: forward primer 5'-CGGAATTCCACGGGAGCCCTGTGGACATCTGC-3' (sequence 2 in the sequence listing), reverse primer 5'-TTTATAGCGGCCGCAGGAAGAGGTGCAAAGAATAAGAAC-3' (seq...

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Abstract

The invention discloses a fermentation expression method of a human anti-thrombin III, a special expression vector and an engineered strain thereof. The Pichia pastoris expression vector for expressing the human anti-thrombin III is the recombinant Pichia pastoris expression vector containing human anti-thrombin III code gene. The Pichia pastoris engineered strain for expressing the human anti-thrombin III is obtained by introducing the recombinant Pichia pastoris expression vector containing the human anti-thrombin III into Pichia pastoris. The use of the Pichia pastoris expression vector and the engineered strain for carrying out the expression of the human anti-thrombin III can make up for the deficiencies of the traditional preparation method of the human anti-thrombin III and the expression mode of a prokaryotic expression system; in addition, if the invention is applied to the industrial production of the human anti-thrombin III, the advantages of simple operation, short growth cycle of raw material organisms, large production scale (high-density fermentation), low extraction cost and high enzyme activity are significant, thereby having important industrial application prospect and practical significance.

Description

technical field [0001] The invention belongs to the fermentation expression method of human antithrombin III and its special expression vector and engineering bacteria in the fields of genetic engineering and microbial fermentation engineering. Background technique [0002] Human antithrombin III (AT-III) is an important physiological anticoagulant factor in plasma, which plays an important role in maintaining the balance of blood coagulation and anticoagulation, and bears 70% of the physiological antithrombin in plasma Activity, under the action of heparin, it exerts an anticoagulant effect by combining with thrombin and factor Xa, and its deficiency will easily lead to thrombosis. It has been widely used clinically abroad for the treatment of various diseases, including: disseminated intravascular coagulation (DIC), congenital and acquired AT-III deficiency, sepsis, etc. (Int Angiol.2007, 26: 53- 63; Angiology. 2007, 58:85-91). Especially for the treatment of DIC, the ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/15C12N1/19C07K14/81C12P21/02C12R1/84
Inventor 于群宋丽雅李伟静贺敏谢冰洁乔志新任鹏
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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