Recombinant engineering bacteria expressing highly recombinant hgh and its construction method and application

A technology of recombinant engineering bacteria and expression vector, applied in the field of genetic engineering, can solve the problems of low activity and low purity of hGH, and achieve the effect of improving protein products, high product activity, and improving activity

Active Publication Date: 2022-02-11
SHENZHEN KEXING PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to achieve the above-mentioned purpose, the present invention provides a recombinant engineering bacterium for high-efficiency expression of recombinant hGH and its construction method and application, which overcomes the shortcomings of the existing methods of expressing recombinant hGH using Escherichia coli and the low activity and low purity of hGH obtained And other issues

Method used

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  • Recombinant engineering bacteria expressing highly recombinant hgh and its construction method and application
  • Recombinant engineering bacteria expressing highly recombinant hgh and its construction method and application
  • Recombinant engineering bacteria expressing highly recombinant hgh and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0040] 1. Optimize the acquisition of genes

[0041] According to the known natural amino acid sequence of hGH, according to the codon preference of Escherichia coli and considering the elimination of hairpin structure and other secondary structures that are not conducive to expression, the coding sequence of hGH protein is optimized, as shown in SEQ ID NO: 1 DNA sequence.

[0042] 2. Construction of expression plasmids and transformation of host bacteria

[0043] The signal peptide of the outer membrane protein of Escherichia coli is modified, and the expression of hGH is guided by the selected signal peptide of the outer membrane protein of Escherichia coli as a leader peptide, wherein the designed signal peptide is shown in SEQ ID NO:2.

[0044] Digest the synthetic ST-II-hGH gene (BGI Synthesis) with NdeI and HindIII enzymes to form sticky ends, and use 1% agarose gel electrophoresis to recover the target gene;

[0045] The PeT22b200211 expression vector was digested wit...

Embodiment 2

[0049] 1. Optimize the acquisition of genes

[0050] According to the known natural amino acid sequence of hGH, according to the codon preference of Escherichia coli and considering the elimination of hairpin structure and other secondary structures that are not conducive to expression, the coding sequence of hGH protein is optimized, as shown in SEQ ID NO: 1 DNA sequence.

[0051] 2. Construction of expression plasmids and transformation of host bacteria

[0052] The signal peptide of the outer membrane protein of Escherichia coli is modified, and the expression of hGH is guided by the selected signal peptide of the outer membrane protein of Escherichia coli as a leader peptide, wherein the designed signal peptide is shown in SEQ ID NO:3.

[0053] Digest the synthetic OmpA-hGH gene with NdeI and HindIII enzymes to form sticky ends, and use 1% agarose gel electrophoresis to recover the target gene;

[0054] The pET22b200211 expression vector was digested with NdeI and HindII...

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Abstract

The invention belongs to the technical field of biomedicine, and specifically discloses a recombinant engineering bacterium for efficiently expressing recombinant hGH and its construction method and application. The invention transforms the codon optimization of the signal peptide hGH of the outer membrane protein of Escherichia coli through design, and combines the hGH protein with STII Signal peptide or OmpA signal peptide is fused, and the fusion protein gene and signal peptide are combined with the E. coli lipoprotein LPP promoter and the E. coli LPPT7 terminator as expression control elements to enable recombinant hGH to be secreted and expressed efficiently in E. coli. The present invention overcomes the The existing methods for expressing recombinant hGH using Escherichia coli all have their own shortcomings, and the obtained hGH has low activity and low purity.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium for highly expressing recombinant hGH and its construction method and application. Background technique [0002] Human Growth Hormone (hGH) is a protein hormone with a single peptide chain secreted by the eosinophilic cells of the anterior pituitary gland. It is the most important hormone for human growth after birth. It has multiple functions such as regulating human growth and metabolism. . It contains 191 amino acid residues, the molecular weight is about 22kDa, and there is no glycosylation in the molecule. Human growth hormone has a wide range of physiological functions and can affect almost all tissue types and cells, even including immune tissue, brain tissue and hematopoietic system. [0003] Human growth hormone (hGH) plays an important role in the growth and development of the human body. It can promote the growth of bon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/18C12N15/62C12N15/70C12N1/21C12P21/02C12R1/19
CPCC07K14/61C12N15/70C07K2319/02C12N2800/22
Inventor 王忠徐钟潘志友马鸿杰柏江涛吴凤梅
Owner SHENZHEN KEXING PHARM CO LTD
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