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Method for preparing recombinant N-terminal brain natriuretic peptide precursor based on elastin-like label

A technology similar to elastin and brain natriuretic peptide, applied in the field of preparation of recombinant N-terminal brain natriuretic peptide precursor, can solve the problems of high cost and time-consuming, and achieve the effect of high practical value

Active Publication Date: 2016-12-14
大连东方雍和生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the NT-proBNP produced by the E. coli expression system requires separation and purification steps such as cell lysis and affinity chromatography, resulting in time-consuming and high-cost

Method used

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  • Method for preparing recombinant N-terminal brain natriuretic peptide precursor based on elastin-like label
  • Method for preparing recombinant N-terminal brain natriuretic peptide precursor based on elastin-like label
  • Method for preparing recombinant N-terminal brain natriuretic peptide precursor based on elastin-like label

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Embodiment 1

[0033] 1. Construction of NT-proBNP fusion expression vector pET28a(+) / ELP[I] 40 / NT-proBNP. Design and optimize the NT-proBNP gene based on the human BNP gene sequence (EMBL accession number AB037521.1), and entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the NT-proBNP gene according to Seq ID.NO.1. The gene structure is as follows figure 1 shown. Cloning NT-proBNP gene into pET28a(+) / ELP[I] by using EcoRI and XhoⅠ restriction sites at both ends of the gene 40On the carrier, form NT-proBNP fusion expression vector pET28a(+) / ELP[I] 40 / NT-proBNP, its nucleotide sequence is Seq ID.NO.2, and the fusion expression vector structure is as follows figure 2 shown.

[0034] 2. Transform the BL21(DE3) Escherichia coli strain (purchased from Treasure Bioengineering (Dalian) Co., Ltd.) by electroporation with the constructed expression vector, and inoculate the identified transformant into 10 ml containing kanamycin sulfate (Kan) 50 micrograms per milliliter of LB m...

Embodiment 2

[0040] 1. Construction of NT-proBNP fusion expression vector pET28a(+) / ELP[I] 40 / NT-proBNP. Design and optimize the NT-proBNP gene based on the human BNP gene sequence (EMBL accession number AB037521.1), and entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the NT-proBNP gene according to Seq ID.NO.1. The gene structure is as follows figure 1 shown. Cloning NT-proBNP gene into pET28a(+) / ELP[I] by using EcoRI and XhoⅠ restriction sites at both ends of the gene 40 On the carrier, form NT-proBNP fusion expression vector pET28a(+) / ELP[I] 40 / NT-proBNP, its nucleotide sequence is Seq ID.NO.2, and the fusion expression vector structure is as follows figure 2 shown.

[0041] 2. Transform the BL21(DE3) Escherichia coli strain (purchased from Treasure Bioengineering (Dalian) Co., Ltd.) by electroporation with the constructed expression vector, and inoculate the identified transformant into 10 ml containing kanamycin sulfate (Kan) 50 micrograms per milliliter of LB ...

Embodiment 3

[0047] 1. Construction of NT-proBNP fusion expression vector pET28a(+) / ELP[I] 40 / NT-proBNP. Design and optimize the NT-proBNP gene based on the human BNP gene sequence (EMBL accession number AB037521.1), and entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the NT-proBNP gene according to Seq ID.NO.1. The gene structure is as follows figure 1 shown. Cloning NT-proBNP gene into pET28a(+) / ELP[I] by using EcoRI and XhoⅠ restriction sites at both ends of the gene 40 On the carrier, form NT-proBNP fusion expression vector pET28a(+) / ELP[I] 40 / NT-proBNP, its nucleotide sequence is Seq ID.NO.2, and the fusion expression vector structure is as follows figure 2 shown.

[0048] 2. Transform the BL21(DE3) Escherichia coli strain (purchased from Treasure Bioengineering (Dalian) Co., Ltd.) by electroporation with the constructed expression vector, and inoculate the identified transformant into 10 ml containing kanamycin sulfate (Kan) 50 micrograms per milliliter of LB ...

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Abstract

The invention belongs to the field of biotechnology, and relates to amethod for preparing recombinant N-terminal brain natriuretic peptide precursor based on elastin-like label. The method comprises the following steps: cloning N-terminal brain natriuretic peptide gene onto a pET28a(+) / ELP[I]40 prokaryotic expression vector through genetic recombination, transferring the recombinant expression vector into Escherichia coli BL21(DE3) for expression, collecting thalli, then purifying the recombinant protein by adopting a repeated reversible transition cycling ITC method, wherein the protein is ELP[I]40-NT-proBNP recombinant protein. The method can fast, simply and efficiently purify a fusion protein with anti-NT-proBNP antibody binding activity, the recovery purity of the fusion expression protein can achieve 90% or more, thus providing a basis for preparing a standard substance with brain natriuretic peptide precursor antigenicity in high efficiency and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing recombinant N-terminal brain natriuretic peptide precursor by using an elastin-like label through a non-chromatographic method. Background technique [0002] B-type natriuretic peptide (BNP) was first isolated from pig brain by Japanese scholars in 1988, so it got its name. Later studies found that it was mainly synthesized and secreted by cardiomyocytes. The human BNP gene encodes a pro-BNP precursor composed of 134 amino acid residues, which forms a terminal brain natriuretic peptide (pro-BNP) after removing the signal peptide composed of 26 amino acids, and then is cleaved by proteolytic enzymes into a pro-BNP containing 76 amino acids. Active N-terminal brain natriuretic peptide precursor (NT-proBNP) and BNP containing 32 amino acids with physiological activity. When the myocardium is stretched, pro-BNP is released and further decomposed into NT-proBNP and B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C07K19/00
CPCC07K14/58C07K2319/00C12N15/62C12N15/70C12N2800/101
Inventor 胡学军杨春光丁宁孙慎侠韩立赤于天舒卫莹
Owner 大连东方雍和生物科技发展有限公司
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