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Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia

A technology of mycobacterium tuberculosis, ESAT-6, applied in the field of biology

Inactive Publication Date: 2006-09-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some people in China try to express ESAT-6 protein using the E. coli expression system. Although high-level expression can be obtained, the expressed recombinant ESAT-6 protein mainly exists in the form of inclusion body (inclusion body), and the protein has not been seen so far. Biologically active reports

Method used

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  • Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia
  • Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia
  • Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Construction and identification of recombinant expression plasmids

[0028] The plasmid pPIC9 was double digested with XhoI and EcoRI, the PCR product containing the α-2a interferon gene was double digested with XhoI and NotI, and the PCR product containing the esat-6 gene was double digested with NotI and EcoRI. Then it was ligated with T4 DNA ligase, transformed into E. coli Top10 competent cells, and the recombinant plasmid pPIC9-α2a-esat6 was extracted and identified. Then the recombinant plasmid was double digested with BamHI and SalI to recover the small fragment, and the plasmid pPIC9K was also double digested with BamHI and SalI to recover the large fragment, ligated with T4 DNA ligase, transformed into E. coli Top10, and the recombinant expression plasmid pPIC9K- α2a-esat6, enzyme digestion identification and DNA sequence determination, proved that the construction of the recombinant expression plasmid was completely correct.

Embodiment 2

[0029] Example 2. Electrotransformation of Pichia pastoris SMD1168 with recombinant expression plasmids

[0030] Pichia pastoris SMD1168 (pep4, his4) was inoculated into YPD medium, shaken at 30°C to OD600 of 1.3-1.5, centrifuged at 5000 r / min at 4°C for 5 min, collected the cells, washed with pre-cooled sterile water and 1mol / L The cells were washed once with sorbitol, and finally suspended with 200 μl of 1 mol / L sorbitol to obtain SMD1168 competent cells. Take 80μl of SMD1168 competent cells and mix with 5μg of recombinant expression plasmid digested with SalI single enzyme, transform by electroporation under the conditions of 1300v, 25μF, 200Ω, suspend with 1ml of 1mol / L sorbitol, spread on RDB plate, and culture at 30°C for 3 days. ~4d, more than 1000 transformants were obtained.

Embodiment 3、G418

[0031] Example 3. Screening of G418 highly resistant yeast transformants

[0032] More than 1,000 yeast transformant colonies grown on RDB plates were seeded on YPD plates containing G418 concentrations of 1.5, 3.0, and 4.0 mg / ml, cultured at 30°C, and G418-resistant strains were screened step by step. As a result, 28 strains were obtained on a plate containing 4 mg / ml G418.

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Abstract

The invention belongs to the biological technology domain, is concrete is in one tuberculosis difference bacillus Esat-6 protein in Bi Chishi yeast fusion expression method. He (Arab League sends) the esat-6 gene and person the -2a disturbance factorConnects, two linker manner enterokinase recognition sequence, will reorganize the fusion gene the DNA fragment insertion to express carrier pPIC9K, the electric shock inducts Bi Chishi in yeast strain SMD1168, will realize the Esat-6 fusion protein highly effective secretion expression. Obtain pure Esat-6 after the sparse water chromatographic analysis and the ionic exchange.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fusion expression method of Mycobacterium tuberculosis ESAT-6 protein. Specifically, the recombinant engineering strain of Pichia pastoris is constructed, the ESTA-6 fusion protein is efficiently secreted and expressed by shaking flask fermentation, and the ESTA-6 fusion protein is obtained by separation and purification. Background technique [0002] ESAT-6 protein is a secreted protein isolated from short-term culture filtrate of Mycobacterium tuberculosis. It consists of 95 amino acid residues and has a molecular weight of 9.9KD. ESTA-6 protein exists only in Mycobacterium tuberculosis and a few pathogenic mycobacteria, and all BCG vaccine strains lack this protein; more than 90% of non-pathogenic mycobacterial strains lack the esat-6 gene . Among the four secreted proteins of M. bovis, MPB59, MPB64, MPB70, and ESTA-6, only the ESAT-6 protein can distinguish BCG-immunized animal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/31C12N15/21C12N15/63C12N15/81C12N1/19C12P21/02
Inventor 宋大新赵志安王洪海刘林
Owner FUDAN UNIV
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