Application of lactose to expression of hIGF-1 (Human Insulin-like Growth Factor-1) by high density fermentation of recombinant colon bacillus

A technology of recombinant Escherichia coli and high-density fermentation, which is applied in the direction of fermentation, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of difficult induction conditions and complicated mechanism of action, and achieve low price and low clinical morbidity high effect

Inactive Publication Date: 2012-10-10
FUJIAN MEDICAL UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

The main reason is that lactose itself can be used by bacteria as a metabolic energy source. Using lactose as an inducer has a complicated mechanism of action, and the induction conditions are not easy to grasp. Only after exploration and research can it be able to stably and efficiently induce recombinant Escherichia coli to express hIGF-1 through high-density fermentation

Method used

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  • Application of lactose to expression of hIGF-1 (Human Insulin-like Growth Factor-1) by high density fermentation of recombinant colon bacillus
  • Application of lactose to expression of hIGF-1 (Human Insulin-like Growth Factor-1) by high density fermentation of recombinant colon bacillus
  • Application of lactose to expression of hIGF-1 (Human Insulin-like Growth Factor-1) by high density fermentation of recombinant colon bacillus

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1 Lactose induced expression of hIGF-1 by high-density fermentation of E.coli DH5α / pET32a(IGF-1)

[0048] The previous work of the research group used the pET vector produced by Novagen to construct and preserve the recombinant engineered bacteria E.coli DH5α / pET32a(hIGF-1) containing the exogenous hIGF-1 gene, which was confirmed by gene sequencing and could express hIGF-1 under the induction of IPTG. 1[Liao Lianming, Yang Jian, Yu Changxi, Xu Ying. Prokaryotic expression and purification of human insulin-like growth factor-1, Chinese Journal of Biochemical Medicine, 2009, 30(4): 226-229]. Example 1 This engineering bacterium was used as an object, and lactose was used instead of IPTG to induce its high-density fermentation to express hIGF-1, so as to realize the application of lactose in recombinant Escherichia coli high-density fermentation and expression of hIGF-1.

[0049] 1 material

[0050] 1.1 Strains

[0051] The engineering bacteria E.coli DH5α / pET...

Embodiment 2

[0087] Example 2 Induction of E.coli BL by lactose 21 (DE3) / pET32a(hIGF-1) express hIGF-1 by high-density fermentation

[0088] Escherichia coli E.coli B and its derivative strains are more commonly used strains in the E. coli expression system. The acid production is relatively small, the protein expression is stable, and it is more conducive to the high-density fermentation expression of foreign genes. Therefore, the research group selected the strain E.coli BL 21 (DE3) replaced the original host strain E.coli DH5α for prokaryotic expression of hIGF-1. The recombinant plasmid pET32a(hIGF-1) containing hIGF-1 gene was extracted from the original engineering strain and then transformed into E.coli BL 21 (DE3) Competent cells, positive transformants were screened by ampicillin plate and confirmed by gene sequencing, and the engineering strain E.coli BL expressing hIGF-1 was obtained 21 (DE3) / pET32a(hIGF-1). It was then induced to express hIGF-1 in a high-density ferme...

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Abstract

The invention discloses an application of lactose to expression of hIGF-1 (Human Insulin-like Growth Factor-1) by high density fermentation of a recombinant colon bacillus and particularly relates to the application of the commodity lactose which exists in the form of an alpha-monohydrate to the expression of the hIGF-1 by the high density fermentation of the recombinant colon bacillus. The lactose can induce a pronucleus colon bacillus expression system which contains the exogenous hIGF-1 and is constructed based on a lactose operon principle to carry out high density fermentation expression of the hIGF-1; an expression product is subjected to a polyacrylamide gel electrophoresis analysis to obtain a molecular weight which accords with a designed theoretical value; and a protein western-blot analysis result prompts that an induced product has a human original IGF-1 antigen activity and shows that the lactose can be used as an induction agent in the high density fermentation of the recombinant colon bacillus to induce the expression of the hIGF-1. The application disclosed by the invention has the advantages of low expression cost and high expression efficiency; and the lactose has no toxins on a human body so that the application lays the foundation on the establishment of a safe and non-toxic hIGF-1 preparation process and the industrialized production becomes possible.

Description

technical field [0001] The invention belongs to the field of lactose application, in particular to lactose (molecular formula C 12 h 22 o 11 ) in the new application of hIGF-1 expressed by high-density fermentation of recombinant Escherichia coli. Background technique [0002] Human insulin-like growth factor-1 (human Insulin-like growth factor-1, hIGF-1) is an important growth-promoting factor in the human body, which has many physiological activities and potential medicinal value. In recent years, the more recognized indications of hIGF-1 in the treatment of clinical diseases include Laron dwarf syndrome, diabetes, insulin resistance syndrome and other persistent diseases, and good results have been achieved. Therefore, its clinical application value has been paid more and more attention by people, showing great application potential, and the preparation of hIGF-1 has become one of the hotspots of research and development at home and abroad. [0003] The development of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/19
Inventor 俞昌喜杨渐陈理廖联明许盈
Owner FUJIAN MEDICAL UNIV
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