Recombinant engineering bacteria for efficiently expressing human growth hormone, construction method and application

A technology of secretion expression and Escherichia coli, which is applied in the field of medical bioengineering, can solve the problems of low activity and low purity of hGH, and achieve the effect of simple purification process, simplified purification process and high expression level

Active Publication Date: 2014-06-25
吉林省奇健生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the shortcom

Method used

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  • Recombinant engineering bacteria for efficiently expressing human growth hormone, construction method and application
  • Recombinant engineering bacteria for efficiently expressing human growth hormone, construction method and application
  • Recombinant engineering bacteria for efficiently expressing human growth hormone, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Embodiment 1: Construction of rhGH expression engineering bacteria

[0097] 1. Optimize the acquisition of genes

[0098] According to the known natural amino acid sequence of hGH, according to the codon preference of Escherichia coli and considering the elimination of secondary structures such as hairpin structures that are not conducive to expression, the coding sequence primers of hGH were designed without changing the amino acid sequence. The full-length sequence of rhGH was obtained by PCR method. The coding sequence of the Escherichia coli heat-stable enterotoxin signal peptide was synthesized by Bao Bio.

[0099] 2. Construction of expression plasmid pET22b(+)-rhGH and transformation of host bacteria

[0100] The expression plasmid is the pET22b(+) expression vector purchased from InVitrogen (the expression vector contains an IPTG-induced promoter and peIB secretion signal peptide), and the host bacteria is Escherichia coli BL21(DE3).

[0101] Treat the synthe...

Embodiment 2

[0104] The selection of embodiment 2 suitable expression vectors

[0105] According to the method similar to Example 1, insert the hGH sequence into several different expression vectors, such as pET28a(+) (purchased from Invitorgen Company), pET29a(+) (purchased from Invitorgen Company), pET-30Ek / LIC purchased from Invitorgen Company) the same restriction site (NcoI / HindIII), to obtain plasmid pET28a (+)-rhGH, pET29a (+)-rhGH, pET-30Ek / LIC-rhhGH.

[0106] The plasmids pET28a(+)-rhGH, pET29a(+)-rhGH, and pET-30Ek / LIC-rhGH were transformed into corresponding host bacteria respectively, and resistant strains were selected. Shake flask experiments were carried out together with engineering bacteria containing plasmid pET22b(+) / rhGH. After being induced by IPTG (or arabinose) for 2 hours, the expression of the target protein expressed by the engineering bacteria containing plasmid pET22b(+)-rhGH accounted for the total protein About 20%, while the expression of the target protein ...

Embodiment 3

[0107] The selection of embodiment 3 optimum culture medium

[0108] Pick the Escherichia coli BL21 (DE3) monoclonal that transfers to pET22b (+)-rhGH prepared in Example 1, inoculate in the 250ml Erlenmeyer flask that contains 50ml LB seed liquid, cultivate 3-4hr, wait for OD 600 reach 0.8-1.0, inoculate in a 1000ml Erlenmeyer flask containing 250ml improved seed solution at a ratio of 1:10, cultivate for about 8-10h, and wait until OD 600 When it reaches 6-8, ferment in a tank according to 1:10, add carbon source (glycerol), magnesium salt, ammonia water to adjust pH6.8-7.0, temperature 37°C, DO>30%, wait for OD 600 When it reaches 15-18, add 0.5-0.8mM IPTG to start induction, add carbon source (glycerol), nitrogen source, and phosphate buffer to adjust pH to 7.2-7.4, DO>50%, temperature 30-32°C, induction 4 -5hr over. The samples were tested by SDS-PAGE to determine the protein content.

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Abstract

The invention discloses recombinant engineering bacteria for efficiently expressing human growth hormone (hGH), a construction method and an application and provides an escherichia coli operon for expressing recombinant hGH, an expression plasmid containing an operon sequence and engineering bacteria QJSW-SZ01 (CGMCC No:7258) used for secretory expression of the recombinant hGH and obtained by transformation of the expression plasmid containing the operon. The recombinant engineering bacteria are characterized in that a coding sequence of a signal peptide of an escherichia coli heat-stable enterotoxin is changed, and rare codons of escherichia coli in the coding sequence are mutated so as to avoid formation of a secondary structure; meanwhile, an amino acid is altered on the terminal of the coding sequence so that the coding sequence is more beneficial to guidance of secretory expression of hGH; the signal peptide, an escherichia coli alkaline phosphatsae promoter (phoA promoter) and an escherichia coli T7 terminator are combined to be used as an expression control element so that the recombinant hGH is efficiently secretory-expressed in the escherichia coli in a soluble form. The recombinant engineering bacteria lay the foundation of finally developing a low-cost hGH pharmaceutical product.

Description

technical field [0001] The invention relates to the field of medical bioengineering, in particular to a recombinant engineering bacterium highly expressing human growth hormone, its construction method and application. Background technique [0002] Human growth hormone (hGH), also known as somatotorpin, is a hydrophilic single chain globulin composed of 191 amino acids, its relative molecular mass is about 22kD, and its isoelectric point P1 is 4.9. There are two pairs of disulfide bonds in the molecule. hGH is an important non-glycosylated protein hormone secreted by eosinophils in the anterior pituitary gland. Under the stimulation of the hypothalamic growth hormone releasing factor, the pituitary gland can release growth hormone, while somatostatin can inhibit its release. Growth hormone is transported to the target tissue through the blood, affecting almost all tissue types and cells, even including immune tissue, brain tissue and hematopoietic system. Its main functio...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N1/21C07K14/61C07K1/36C07K1/34C07K1/14C07K1/18C07K1/22C12R1/19
Inventor 席玥
Owner 吉林省奇健生物技术有限公司
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