Gene of coding recombination buman natriurea and method of producing recombination human natriurea using the gene

A technology encoding gene and natriuretic acid, applied in the field of genetic engineering, can solve problems such as difficult control, low product uniformity and activity, and undiscovered recombinant human natriuretic protein purification and activity identification, so as to avoid environmental pollution and reduce production costs low effect

Inactive Publication Date: 2009-07-15
SUZHOU LANDING BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficult control of chemical cleavage reaction conditions, chemical modification side reactions often exist, and the product has low uniformity and activity
So far, we have not found any literature related to the expression, purification and activity identification of recombinant human natriuretic hormone

Method used

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  • Gene of coding recombination buman natriurea and method of producing recombination human natriurea using the gene
  • Gene of coding recombination buman natriurea and method of producing recombination human natriurea using the gene
  • Gene of coding recombination buman natriurea and method of producing recombination human natriurea using the gene

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Experimental program
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Embodiment

[0079] 1. Construction of pET32a / hUNP expression plasmid

[0080]Construction of pET32a / hUNP expression plasmid: Using chemically synthesized hUNP gene as template, hUNP double-stranded DNA was amplified by PCR. PCR amplification conditions are: 5.0fmol hUNP template (SEQ ID NO.3), 50pmol upstream primer (SEQ ID NO.4) and downstream primer (SEQ ID NO.5), 0.25U taq enzyme, denaturation at 94°C for 2min Carry out the following 26 cycles: 94°C, 30s; 52°C, 1min; 72°C, 40s, the PCR product was purified by agarose electrophoresis, recovered with a DNA fragment recovery kit, digested with KpnI / XhoI double enzymes, and recovered a large fragment (approx. 120bp) and KpnI / XhoI digested expression plasmid pET32a were ligated with T4 DNA ligase to form pET32a / hUNP plasmid, and transformed into E.coli DH5α competent cells, positive clones were identified by KpnI / XhoI double enzyme digestion, pET32a / hUNP plasmid Perform sequence determination.

[0081] 2. Expression and purification of Th...

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Abstract

The invention discloses a gene encoding recombinant human natriuretic factor and a method for producing recombinant human urinary natriuretic factor using the gene. The nucleotide sequence of the gene is described in SEQ ID NO.1. In this method, double-stranded DNA containing SEQ ID NO.6 and SEQ ID NO.1 is connected to an expression vector containing a gene encoding a thioredoxin and a histidine tag to form a thioredoxin-urenatrien fusion protein expression vector, Transform Escherichia coli with the expression vector, induce the expression of thioredoxin-uronatriuretic fusion protein, use enterokinase to cleavage the thioredoxin-uronatriuretic fusion protein, and purify the cleavage product with Zn-Sepharose affinity column, recombine Human natriuretic sodium remains in the sample through-fluid, which can be collected, purified and identified. The sequence can be efficiently expressed in the Escherichia coli system, and the method has low cost and good effect, and is suitable for industrial application.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a gene encoding recombinant human natriuretic factor, and also relates to a method for producing recombinant human urinary natriuretic factor using the gene. Background technique [0002] Urodilatin (UNP) is a polypeptide hormone purified from human urine by German scholar Forssmann in 1988. [1] . Human natriuretic peptide (hUNP) is a polypeptide composed of 32 amino acids, and its molecular weight is 3507Da. Urin has the functions of lowering mean arterial blood pressure, diuresis and promoting urinary sodium excretion, and can inhibit the activity of renin in plasma and the secretion of aldosterone and endothelin [2] , Promote the filtration rate of glomeruli, relax vascular smooth muscle, and reduce the systemic impedance of blood vessels. [0003] Natriuretic peptide and atrial natriuretic peptide (ANP) are hormones formed from Cardiodilatin prohornone consisting of 126 am...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/62C12N15/70C12N1/21C07K14/435C07K1/14
Inventor 孙自勇刘建宁
Owner SUZHOU LANDING BIOPHARM CO LTD
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