High-expression semeglutide precursor recombinant engineering bacterium and construction method thereof

A technology of recombinant engineering bacteria and semaglutide, which is applied in the field of genetic engineering, can solve the problem of low fermentation expression and achieve high industrial application value and prospects

Active Publication Date: 2021-10-15
BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to solve the problem of low fermentation expression in the prior art, and to provide a new recombinant engineering bacterium that stably and highly expresses the semaglutide precursor and its construction method. The engineering bacterium prepared by the construction method of the present invention especially has Potential for high density fermentation

Method used

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  • High-expression semeglutide precursor recombinant engineering bacterium and construction method thereof
  • High-expression semeglutide precursor recombinant engineering bacterium and construction method thereof
  • High-expression semeglutide precursor recombinant engineering bacterium and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant Escherichia coli engineering bacteria that stably and highly express semaglutide precursor

[0037] (1) Synthesize the fusion polypeptide encoding genes shown in SEQ ID No.7 and SEQ ID No.8 respectively, and add Nde I enzyme cutting site, 3' end add stop codon and xho I restriction site;

[0038] (2) The gene fragments prepared in step (1) were respectively cloned into the expression vector pET-30a(+) by enzyme digestion. Nde I and xho Between the I enzyme cutting sites, thereby constructing two kinds of recombinant expression vectors pET-30a(+)-A1-GLP-1 and pET-30a(+)-A2-GLP-1;

[0039] (3) Transform the recombinant expression vectors obtained in step (2) into Escherichia coli BL21(DE3) by heat shock method, and select 8 clones after resistance screening, and select 8 clones from the two recombinant engineering bacteria, and name them respectively A1-GLP-1-1 to A1-GLP-1-8 and A2-GLP-1-1 to A2-GLP-1-8 were stored in glycerol....

Embodiment 2

[0041] Example 2: Fermentation of recombinant Escherichia coli engineering bacteria that stably and highly express semaglutide precursor

[0042] (1) Recovery of recombinant engineered bacteria

[0043] Take each of the 16 strains of glycerol bacteria preserved in Example 1, and insert them into 16 flasks containing 20 ml of sterilized LB medium according to the inoculation amount of 1 / 1000 respectively, and put them under the conditions of 37°C and 200rpm Cultivate overnight to obtain a recovery bacterial solution.

[0044] (2) Induced expression of recombinant engineered bacteria

[0045] Take the resuscitated bacteria solution in step (1) and put them into 16 flasks containing 50 ml of sterilized LB medium according to the inoculation amount of 1%, and cultivate them to OD at 37°C and 220rpm 600 When the value reaches 0.6-0.8, add IPTG with a final concentration of 0.5mM, and induce expression overnight at 37°C and 220rpm.

[0046] (3) Cell collection and HPLC expression...

Embodiment 3

[0050] Example 3: High-density fermentation of recombinant Escherichia coli engineering bacteria with stable and high expression of semaglutide precursor

[0051] Get the 16 strains of recombinant engineered bacteria constructed in Example 1 and carry out the secondary bacterial classification activation respectively, carry out high-density fermentation, in the fermentation process, after feeding starts, every 2h sampling measures OD 600 value, and draw the growth curve; after the fermentation, the cells were collected by centrifugation.

[0052] According to the fermentation results, A1-GLP-1 series recombinant engineering bacteria and A2-GLP-1 series recombinant engineering bacteria can both ferment to high OD 600 value, especially the A2-GLP-1 series of recombinant engineering bacteria, can reach OD 600 When the value is above 240, the biomass of inclusion bodies reaches above 300g / L. Taking A2-GLP-1-5 as an example, its fermentation OD 600 The value can reach 259, and t...

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Abstract

The invention provides recombinant escherichia coli capable of efficiently expressing a semeglutide precursor. Two different leader peptides and enterokinase restriction enzyme cutting sites are designed and added at the front end of a semeglutide precursor to construct a fusion polypeptide structure, a coding gene of the optimized polypeptide structure is inserted into a pET-30a(+) expression vector to obtain a recombinant expression vector, the recombinant expression vector is transformed into escherichia coli BL21(DE3), and the recombinant engineering bacterium capable of stably and highly expressing the simeglutide precursor is obtained. The recombinant engineering bacterium disclosed by the invention not only can stably and highly express the semeglutide precursor, but also is particularly suitable for a high-density fermentation method, and the expression quantity of a target protein can be further improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium highly expressing a semaglutide precursor and a construction method thereof. Background technique [0002] With the development of social economy and the gradual improvement of people's living standards, great changes have taken place in people's diet structure, which has also led to an increase in the incidence of obesity, and further led to a sharp increase in the number of diabetic patients. According to statistics, the number of diabetic patients in my country exceeds 100 million, and there are more than 150 million hidden pre-diabetic patients. Diabetes has become the third leading chronic disease. [0003] Diabetes is a complex chronic metabolic disease caused by the long-term interaction of genetic and environmental factors. It is a disease characterized by hyperglycemia caused by the lack of insulin secretion. It is divided...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C12N15/66C12N1/21C12R1/19
CPCC07K14/605C12N15/70C07K2319/50C07K2319/00C12N2800/22
Inventor 曹海燕连婕妮林兆生朱志伟王惠张世野
Owner BEIJING HUIZHIHENG BIOTECHNOLOGY CO LTD
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