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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: 2005-04-06
FUDAN UNIV
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  • 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

[0028] Embodiment 1, Construction and Identification of Recombinant Expression Plasmids

[0029] Plasmid pPIC9 was double-digested with XhoI and EcoRI, the PCR product containing α-2a interferon gene was double-digested with XhoI and NotI, and the PCR product containing esat-6 gene was double-digested with NotI and EcoRI. Then connect with T4 DNA ligase, transform Escherichia coli Top10 competent cells, extract the recombinant plasmid pPIC9-α2a-esat6 and identify it by enzyme digestion. Then the recombinant plasmid was double-digested with BamHI and SalI and a small fragment was recovered, and the plasmid pPIC9K was also double-digested with BamHI and SalI and a large fragment was recovered, connected with T4 DNA ligase, transformed into Escherichia 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

[0030] Example 2, Electrotransformation of recombinant expression plasmid Pichia pastoris SMD1168

[0031] Inoculate Pichia yeast SMD1168 (pep4, his4) in YPD medium, shake culture at 30°C until OD600 is 1.3-1.5, centrifuge at 5000r / min at 4°C for 5min, collect the bacteria, and wash with pre-cooled sterile water and 1mol / L Wash once with sorbitol, and finally suspend with 200 μl of 1mol / L sorbitol to obtain SMD1168 competent cells. Take 80 μl of SMD1168 competent cells and mix them with 5 μg of the recombinant expression plasmid digested with SalI, transform by electroporation at 1300v, 25μF, 200Ω, suspend in 1ml of 1mol / L sorbitol, spread on the RDB plate, and incubate at 30°C for 3 ~4d, more than 1000 transformants were obtained.

Embodiment 3、G418

[0032] Example 3, Screening of G418 highly resistant yeast transformants

[0033] More than 1,000 colonies of yeast transformants grown on RDB plates were planted 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. Results 28 strains were obtained on the plate containing 4mg / 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, and the ESAT-6 fusion protein is efficiently secreted and expressed through shake flask fermentation, and the ESTA-6 fusion protein is obtained through separation and purification. Background technique [0002] ESAT-6 protein is a secreted protein isolated from the short-term culture filtrate of Mycobacterium tuberculosis, which 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 strains of BCG vaccine lack this protein; more than 90% of non-pathogenic mycobacteria strains lack the esat-6 gene . Among the four secretory proteins MPB59, MPB64, MPB70, and ESTA-6 of Mycobacterium bovis, only ESAT-6 protein can dist...

Claims

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Application Information

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