Recombinant a human peptide production method

A technology of Xili peptide and Xili, which is applied in the field of preparation of biologically active peptides, can solve the problems of low yield and complicated process steps, and achieve the effects of high expression rate, simplified production process, and improved production efficiency

Inactive Publication Date: 2006-05-10
CHENGDU XIMA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process steps are extremely complicated, and it is easy to form intermolecular polymers or isomers when the BNP disulfide bond is generated, and the yield is extremely low

Method used

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  • Recombinant a human peptide production method
  • Recombinant a human peptide production method
  • Recombinant a human peptide production method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Selection of expression vector

[0060] The pET series expression vector pET32a from Novagen was selected as the expression vector. The pET-32a is an Escherichia coli expression vector system for high-efficiency fusion expression of proteins and polypeptides. The target protein or polypeptide is thioredoxin (Trx ·Tag TM ) as a fusion partner. (See attached for map figure 1 , figure 2 ).

[0061] 2. Gene design and synthesis

[0062] According to the human nosire peptide DNA coding sequence reported in the literature (the sequence can be changed according to the principles of universality and degeneracy of the genetic code, but the amino acid composition and arrangement are not changed), the coding mature human BNP sequence is designed, such as: 5'_AGC CCT AAA ATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GAC CGTATC AGC TCT TCC AGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA_3'. At the same time, add Kpn recognition sequence GGTACC and enterokinase (Enterokin...

Embodiment 2

[0078] 1. Selection of expression vector

[0079] The pTDa of our laboratory was selected as the expression vector (see attached Figure 6 )

[0080] 2. Gene design and synthesis

[0081] According to the preference principle of Escherichia coli for the use of genetic code, the coding sequence of mature human nosire peptide was designed: 5'_AGC CCT AAA ATG GTA CAG GGT TCT GGT TGC TTC GGT CGT AAA ATG GACCGT ATC AGC TCT TCC AGC GGT CTG GGT TGC AAA GTA CTG CGT CGT CAC TAA (the sequence can be changed according to the principle of universality and degeneracy of the genetic code, but the amino acid composition and arrangement will not be changed). Add EcoRI recognition sequence GAA TTC and thrombin (Thrombin) restriction site (-LVPR-X) coding sequence CTG GTG CCT CGT at the 5'-end of the sequence at the same time; add the recognition of Sal I at the 3'-end The sequence GTC GAC thus forms the final design sequence as follows: 5'_GAA TTC CTG GTG CCT CGT AGC CCTAAA ATG GTA CAG GGT ...

Embodiment 3

[0090] According to the physicochemical property determination of the rhBNP of embodiment 1 or 2 gained

[0091] 1. Determination of the N-terminal 15 amino acid sequences

[0092] The recombinant human nosire peptide obtained by the above two methods was subjected to N-terminal determination, Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met

[0093] 2. Molecular weight mass spectrometry

[0094] The FAB method was used to determine the molecular weight of the recombinant human nosiritide obtained by the two methods was 3462, which was consistent with the theoretical prediction.

[0095] 3. Determination of biological activity

[0096] Use Phenylephrine to bind with its receptors on blood vessels to produce a vasoconstrictor effect, increase the resting tension of blood vessels and maintain it at a stable level, and then use rhBNP to combine with its natriuretic peptide receptors on blood vessels to produce a vasoconstrictive effect on rabbit chest The vasodilato...

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Abstract

The invention relates to a method of producing peptides restructured by polypeptide with bioactivity, including following steps: a. Synthesize genes with respect to coding area of peptides amino acids artificially; b Genes gained from step a fuse with 3íõside of bacteria thioredoxin genes on the proper expression carriers, among which there are enterokinase or fibrin ferment recognition site dots; c Expression carriers of the coding genes in fusion proteins gained from step b invert proper bacillus colis to get gene engineering bacterias; d said gene engineering bacteria is handled through fermentation, extraction, chromatography, purification steps to prepare peptides- thioredoxin fusion proteins; e said fusion proteins gained from step d is handled by dissection of enterokinase or fibrin ferment, separation, purification to acquire restructed peptides.

Description

Technical field: [0001] The invention relates to a preparation method of biologically active polypeptides, in particular to a method for preparing nasilitide biologically active polypeptides using gene recombination technology. Background technique: [0002] Nesiritide, also known as Brain Natreuritic Peptide (BNP), is a bioactive substance synthesized and secreted by brain tissue and heart. Sudoh et al. first isolated nosiritide from pig brain, and then found that nosiritide was also distributed in other animals and human hearts. Nosiritide synthesized in brain cells exerted physiological functions as a neurotransmitter; And secreted into the blood circulation, nosiritide acts as an endocrine of the heart, and plays an important role in regulating cardiovascular function and water-salt metabolism. Plasma BNP content increases in heart failure, and intravenous infusion of exogenous BNP can further improve heart and kidney function in heart failure. Therefore, recombinant h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12A61K38/16A61P9/04C12N1/21C12N15/63C12N15/70C12P21/02
Inventor 周裕程赵斌彭宏卫杨伟彭辉
Owner CHENGDU XIMA BIOTECH
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