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Self-organized Recombinant Hemoglobin of Vitiligo hyaline and Its Gene and Application

A technology of Vitiligo hyaline and hemoglobin, applied in the field of genetic engineering, to achieve the effect of improving the function of oxygen capture

Active Publication Date: 2018-02-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no study on the use of self-organized fragment recombination technology to improve the plasma membrane binding ability of recombinant Vitiligo hyaline hemoglobin

Method used

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  • Self-organized Recombinant Hemoglobin of Vitiligo hyaline and Its Gene and Application
  • Self-organized Recombinant Hemoglobin of Vitiligo hyaline and Its Gene and Application
  • Self-organized Recombinant Hemoglobin of Vitiligo hyaline and Its Gene and Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Expression of self-organized recombinant hemoglobin from Vitiligo hyaline in E.coli BL21 and its effect on the oxygen capture ability of the bacteria

[0038] Fermentation in the production of genetic engineering products is the main production method for the production of genetic engineering products with microorganisms, animal, plant and insect cells as host cells. Oxygen supply is one of the important factors affecting the fermentation process. Generally speaking, protein and antibiotic genes The production of engineered products places a higher demand on oxygen in the fermentation broth.

[0039] In the embodiment, use pGFPuv as the carrier of self-organized recombinant hemoglobin and its anaerobic promoter gene sequence (the pGFPuv vector is constructed based on the pUC19 vector), and use E.coli BL21 as the host cell produced by the genetic engineering product (host The cell E.coli BL21 is a kind of Escherichia coli cell), and the self-organized recombinant hemoglo...

Embodiment 2

[0071] In order to explore the influence of the self-organized module of the self-organized recombinant hemoglobin of Vitiligo hyaline on the stability of the host cell plasma membrane, the fermentation research of each transformant cell in Example 1 was carried out under relatively high oxygen supply conditions.

[0072] The specific operation is as follows:

[0073] The transformant cells loaded with pGFPuv-vgb9a, pGFPuv-vgb22a, pGFPuv-vgb and pGFPuv genes were cultured in LB medium with 200 micrograms per liter of ampicillin at 37 degrees Celsius and 240 rpm, and the OD value of the medium was From 0.7 to 0.9, transfer 3OD bacteria to 100 ml LB medium in a 250 ml Erlenmeyer flask containing 200 micrograms per liter of ampicillin, culture at 37 degrees Celsius and 180 rpm, and detect each transformant during the fermentation process The expression ability of the GFPuv protein in the cells was determined by measuring the GFPuv fluorescence signal intensity in the culture medi...

Embodiment 3

[0080] In order to explore the influence of the self-organized module of the self-organized recombinant hemoglobin of Vitiligo hyaline on the production of genetically engineered products in host cells, a higher oxygen supply condition was used to carry out the fermentation research of each gene carrier in the host cell E.coliBL21DE3 in Example 1 . The GFPuv protein in the pGFPuv gene vector can be self-expressed and IPTG-induced in E.coli BL21DE3 host cells.

[0081] Specific operation:

[0082] Add 1.0 micrograms of recombinant vectors pGFPuv-vgb9a, pGFPuv-vgb22a, pGFPuv-vgb, and pGFPuv to 100 microliters of E.coli BL21DE3 competent cells, mix well in ice bath for 30 minutes, incubate in a water bath at 42 degrees Celsius for 1 minute, and take it out immediately Place in an ice bath to cool for 2 minutes, add 800 microliters of LB liquid medium preheated at 37 degrees Celsius, incubate at 150 rpm at 37 degrees Celsius for 60 minutes, take 100 microliters of culture solutio...

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Abstract

The invention discloses self-organized recombinant vitreoscilla hemoglobin with self-organizing ability and application of a gene thereof in gene engineering product production and belongs to the technical field of gene engineering. The self-organized recombinant vitreoscilla hemoglobin is composed of a vitreoscilla hemoglobin module, a connecting peptide module and a self-organized module, amino acid sequence of the vitreoscilla hemoglobin module is SEQ ID NO.1, amino acid sequence of the connecting peptide module is (SEQ ID NO.2)n, n value refers to number of series-connection repeating times of the connecting peptide module, and n is an integer from 2 to 10; amino acid sequence of the self-organized module is SEQ ID NO.3 or SEQ ID NO.4. Compared with vitreoscilla hemoglobin, protein expressed by the gene has self-organizing ability, the self-organized recombinant vitreoscilla hemoglobin has higher plasmalemma combining performance, biomass and gene engineering product yield of host cells can be increased under the circumstance that dissolved oxygen in environment is insufficient, and production cost is lowered.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a self-organizing recombinant Hemoglobin of Vitiligo hyaline and the application of its gene in the production of genetic engineering products. Background technique [0002] With the application of cell biology and bioengineering technology in the fields of microorganisms, plants, and animals, genetic engineering technology can be used to produce, improve and even construct genetically engineered products with purpose functions or to produce target genetically engineered products. The production of genetically engineered products has made great progress. success. [0003] Combining genetic engineering technology with the cognition of the host cell's metabolic synthesis pathway, the use of genetic engineering technology can promote the production of host biomass, the promotion of target genetic engineering products, and the promotion of target secondary met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/805C12N15/31C12N15/63C12N1/21C12N1/19C12N5/10
CPCC07K14/805
Inventor 吕明杨少林吴家安周杰
Owner JILIN UNIV
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