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Whole blood INF-gamma specific antigen protein and preparation method and application thereof

An INF-, antigen protein technology, applied in microorganism-based methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of high production cost, loss of protein activity, small molecular weight, etc., and achieve diagnosis in a short time. , high specificity, rapid diagnosis

Active Publication Date: 2013-03-20
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the small molecular weight of these tuberculosis-specific diagnostic proteins, it is difficult to directly isolate and purify them from the culture of Mycobacterium tuberculosis, and the production cost is high; although peptide synthesis can be used for production, the cost of peptide synthesis is very high , the cost of the clinical application kit thus established is greatly increased
In addition, the epitopes of the above antigens are obtained separately through genetic engineering technology. Due to the difference between in vitro and in vivo, the expressed products, as has been reported at home and abroad, are also prone to lose protein activity due to the formation of inclusion cells.

Method used

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  • Whole blood INF-gamma specific antigen protein and preparation method and application thereof
  • Whole blood INF-gamma specific antigen protein and preparation method and application thereof
  • Whole blood INF-gamma specific antigen protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The synthesis of the target gene of embodiment 1ESAT6-CFP10 fusion protein and the construction of expression vector

[0042] The DNA sequence of the artificially synthesized ESAT6-CFP10 was stored in DH5α, and then the DH5α and pET28b expression vectors were digested with NcoI and XhoI endonucleases respectively, and the target fragment and expression vector were recovered. 4 Under the action of DNA ligase, ligate at 4°C for 16 hours, transform into Escherichia coli BL21(DE3) by heat shock method, and culture at 37°C overnight. Pick colonies that grow well on the plate and have typical Escherichia coli characteristics and cultivate them in liquid LB medium. At the same time, add kanamycin with a final concentration of 30 μg / ml to the medium, and cultivate for 3 hours at 37°C and 220rpm , Extract the plasmid for enzyme digestion and sequencing identification, the enzyme map and sequencing map are in line with expectations. Vector construction diagram see figure 1 .

Embodiment 2

[0043] Example 2 Tandem expression of recombinant protein ESAT6-CFP10 Highly expressed in Escherichia coli

[0044] Pick colonies that grow well on the plate and have typical Escherichia coli characteristics and culture them in liquid LB medium, add ampicillin with a final concentration of 50 μg / ml and culture for 6 hours, add IPTG with a final concentration of 0.5 mM, 37 ° C, 200rpm, induced expression for 8 hours, collected bacteria, SDS-PAGE electrophoresis identification results see figure 2 .

Embodiment 3

[0045] Example 3 Purification of Recombinant Protein ESAT6-CFP10 Series

[0046] Protein purification was carried out according to Ni-NTA standard operating procedures. The brief steps are as follows. Take 50ml of the bacterial liquid and centrifuge to collect the engineered bacteria that induce expression, use ultrasonic waves to break the cells of the large intestine, centrifuge to get the supernatant, and the supernatant is in Tris (50mM, pH8.0) Dialyze in the buffer overnight, centrifuge to take the supernatant, directly load the sample on Ni-NTA resin equilibrated with Tris (50mM, pH8.0) buffer, elute with Tris (50mM, pH8.0) buffer after equilibration , and then eluted with 0-1M imidazole gradient, collected the eluted peaks, combined the eluted peaks, dialyzed overnight in Tris (50mM, pH8.0) buffer solution, and lyophilized the dialysate in a vacuum freeze dryer to obtain ESAT6-CFP10 freeze-dried powder, stored at -20--40°C for future use. SDS-PAGE identification of pur...

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Abstract

The invention relates to a whole blood interferon-gamma (INF-gamma) specific antigen protein prepared by a gene engineering method and a preparation method and application thereof, a nucleotide sequence for coding the protein, a recombinant vector and a recon. The antigen protein is a fusion protein obtained by connecting early secreting antigen target 6 (ESAT-6), cultufiltrate protein 10 (CFP-10) and MPB64 in any order through a connecting peptide, wherein the connecting peptide is a short peptide which does not influence the immunogenicity of the ESAT-6, the CFP-10 and the MPB64. The tuberculosis specific antigen quickly diagnoses tuberculosis, has high sensitivity and specificity, and has great significance for the early diagnosis of the tuberculosis.

Description

technical field [0001] The present invention relates to a whole blood INF-γ specific antigen protein prepared by genetic engineering method, its preparation method and application, as well as the nucleotide sequence encoding the protein, recombinant vector and recombinant. Background technique [0002] Tuberculosis is one of the important infectious diseases that are mainly transmitted through the respiratory tract and seriously endanger my country and the world. According to the World Health Organization, there are 1.7-2 billion Mycobacterium tuberculosis infections in the world, and at least 2 million people die from this disease every year (Xie Jianping, Methodology for Mycobacterium tuberculosis functional genome research, Microbiology Bulletin. 2001.28 (5): 92 -97). Therefore, methods of accurately screening infected persons play an extremely important role in the prevention and control and even the eventual elimination of tuberculosis. [0003] Existing diagnostic te...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12R1/19
Inventor 喻德华黄俊赵平峰陈银芳
Owner 武汉海吉力生物科技有限公司
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