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Expression process of heat shock protein Hsp16.3 of mycobacterium tuberculosis

A Mycobacterium tuberculosis, expression method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as Hsp16.3 protein difficulties, and achieve the effect of broad-spectrum immune reactivity

Inactive Publication Date: 2008-10-22
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the great difficulty in obtaining large amounts of Hsp16.3 protein

Method used

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  • Expression process of heat shock protein Hsp16.3 of mycobacterium tuberculosis
  • Expression process of heat shock protein Hsp16.3 of mycobacterium tuberculosis
  • Expression process of heat shock protein Hsp16.3 of mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, Construction and Identification of Recombinant Expression Plasmids

[0042] The pProEXHTb vector was digested with BamHI and HindIII, and recovered with a gel recovery kit after 1% agarose electrophoresis; the Hsp16.3 coding gene was amplified from the chromosomal DNA of Mycobacterium tuberculosis H37Rv by PCR, and the PCR product was digested with BamHI and HindIII. After 1% agarose electrophoresis, it was recovered with a gel recovery kit; the two digested products were connected with T4DNA ligase, transformed into Escherichia coli DH5α, and the recombinant plasmid pProHspX was extracted. Further enzyme digestion identification and sequence analysis proved that the constructed recombinant expression plasmid was completely correct. The recombinant plasmid pProHspX was further transformed into Escherichia coli BL21 to obtain a genetic engineering expression strain of the recombinant protein Hsp16.3.

Embodiment 2

[0043] Embodiment 2, induced expression of recombinant engineering strain

[0044]The Escherichia coli BL21 strain transformed with the recombinant plasmid pProHspX was shaken overnight in LB medium at 37°C (containing ampicillin, final concentration 100 μg / ml); the next day, the bacteria were collected by centrifugation and placed in 1L LB medium at a ratio of 1:100 Continue shaking and culturing until OD600 reaches 0.6; add IPTG with a final concentration of 1 mM, and continue culturing for 4-6 hours. Take 1.5ml of the bacteria before and after induction, centrifuge, remove the supernatant, add 2×SDS sample buffer to the bacterial pellet, boil at 100°C for 5min, and detect with 12% SDS-PAGE. The results confirmed that the high expression strain of Hsp16.3 protein was obtained, and the expression level was as high as 200mg / L fermentation broth.

Embodiment 3

[0045] The western blot identification of embodiment 3 expression products

[0046] The SDS-PAGE gel after electrophoresis was stripped, and electrotransferred to the NC membrane under the condition of 200mA constant current for 2h; the transferred NC membrane was placed in 1% BSA-PBS blocking solution, shaken at 37°C for 1h, and washed with 0.05% Tween20-PBS (PBST) solution was shaken at room temperature and washed 3 times; 3ml of tuberculosis patient serum was used as the primary antibody, incubated at room temperature for 1h, and washed 3 times with PBST solution at room temperature with shaking; horseradish peroxidase-labeled goat anti-human IgG antibody ( Diluted with blocking solution (1:10000 fold) as the secondary antibody, incubated for 1 h, washed with PBST solution for 3 times with shaking at room temperature; developed color by chemiluminescence in a dark room. The results confirmed the specific expression of a protein with a molecular weight of about 19kDa.

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Abstract

The invention belongs to the biotechnical field, in particular relating to a method for high-efficient expression of Mycobacterium tuberculosis heat shock protein Hsp16.3 in colon bacillus. The method clones coding genes of the Hsp16.3 protein into a colon bacillus prokaryotic expression vector, transforms colon bacillus host bacteria and obtains the Hsp16.3 protein through IPTG inducement. The expression product is recombinant proteins with 6 His tags on the N end and with a molecular weight of approximate 19kD, and pure Hsp16.3 protein is obtained through further purification through affinity chromatography.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to protein expression, separation and purification, in particular to construction of Escherichia coli recombinant engineering strains, fermentation, induction of Hsp16.3 protein expression by IPTG, separation and purification to obtain Hsp16.3 protein. Background technique [0002] Tuberculosis (TB) is one of the important public health problems seriously affecting human health worldwide. In recent decades, the incidence of TB has been on the rise, and 2.5 million people die from TB every year. The number of people infected with Mycobacterium tuberculosis (MTB) in my country has reached 550 million, and about 130,000 people die from TB every year. [0003] Chronic infection and the persistence of the bacteria in the infected person are hallmarks of TB. MTB in chronic patients is prone to dormancy, which is the fundamental reason why TB treatment takes a long time and is difficult to cur...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P21/02C12R1/19
Inventor 范雄林石春薇付瑞玲
Owner HUAZHONG UNIV OF SCI & TECH
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