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Synthesis of batroxobin gene and preparation method of expression product thereof

A batroxobin, gene technology, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of low activity, high disulfide bond mismatch rate, no practical production significance, etc. question

Inactive Publication Date: 2010-05-12
YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because the disulphide bond mismatch rate is high, and most of the obtained in the prokaryotic expression system are in the form of inclusion bodies, with low or almost no activity, and have no practical production significance

Method used

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  • Synthesis of batroxobin gene and preparation method of expression product thereof
  • Synthesis of batroxobin gene and preparation method of expression product thereof
  • Synthesis of batroxobin gene and preparation method of expression product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0025] 1. Construction of recombinant batroxobin expression vectors pPICZαA-bat and pPICZαA-bat-m

[0026] 1. Artificial synthesis of recombinant batroxobin gene

[0027] According to the nucleic acid sequence (X12747) and the amino acid sequence of the batroxobin in the venom of Agkistrodon brasiliensis published in Genebank, the codons preferred by Pichia pastoris were selected to artificially synthesize the full-length gene sequence of batroxobin (SEQ- 3), using the protein homology modeling online program SWISS-MODEL and the homology modeling software Modeller 9V6, using the X-ray diffraction structure of TSV-PA as the template of the three-dimensional structure model of batroxobin to predict batroxobin Three-dimensional structure of enzymes. Based on the three-dimensional structure obtained by homology modeling of batroxobin, according to the space structure of batroxobin substrate-fibrinogen, the possible active pocket for substrate binding was searched, and its docking...

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PUM

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Abstract

The invention relates to a batroxobin protein which is derived from the venom of a brothrops atrox and prepared by adopting a recombinant DNA technology. The coding gene of the batroxobin protein has one of the following nucleotide sequences of: (1) sequence 3 in a sequence table; (2) sequence 4 in the sequence table, wherein a coding batroxobin amino acid sequence is relative to a natural batroxobin amino acid sequence, and the 41 bit of His and the 178 bit of Ser are mutated to be Glu. By utilizing the fermentation and purification method optimized by the invention, the high bioactive batroxobin protein is successfully expressed in a secretion expression way. The batroxobin with recombinant expression can be used as a main ingredient for hemostatics.

Description

Technical field: [0001] The present invention relates to a Batroxobin protein derived from the venom of Agkistrodon brasiliensis produced by DNA recombination technology, the key of which is the synthesis, cloning, expression, directional transformation and fermentation expression of the batroxobin gene and separation and purification. The recombinantly expressed batroxobin can be used as the main component of the hemostatic drug. Background technique: [0002] Batroxobin was first isolated and refined from the venom of the Brazilian lancehead viper (Bothrops atrox) in 1936 by Von Klobusitzky, a scholar in Vienna, Austria. It is a single-chain glycoprotein consisting of 17 amino acids, with a total of 231 amino acids and a molecular weight of 39,000-43,000. So far, researchers have isolated and purified more than 20 serine protease components with thrombin-like activity from the venom of Agkistrodon vipers. These serine proteases specifically cleave fibrinogen in mammalia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N15/81C12N9/64A61P7/04C12R1/84
Inventor 唐先兵张同
Owner YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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