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Detection kit for diagnosing tuberculosis

A detection kit and technology for tuberculosis, applied in biological testing, material testing products, hybrid peptides, etc., can solve the problems of high production cost, difficult separation and purification, loss of protein activity, etc., and achieve short diagnosis time and strong sensitivity. , high specificity effect

Active Publication Date: 2011-09-07
武汉海吉力生物科技有限公司
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  • 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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Embodiment 1

[0037] The synthesis of the target gene of embodiment 1ESAT6-CFP10 fusion protein and the construction of expression vector

[0038] 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, the target fragment and expression vector were recovered, and ligated under the action of T4 DNA ligase at 4°C for 16 hours. Transformed into Escherichia coli BL21(DE3) by heat shock method, cultured overnight at 37°C. 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

[0039] Example 2 Tandem expression of recombinant protein ESAT6-CFP10 Highly expressed in Escherichia coli

[0040] 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

[0041] Example 3 Purification of Recombinant Protein ESAT6-CFP10 Series

[0042] 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 later use. SDS-PAGE identification of puri...

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Abstract

The invention relates to a detection kit for diagnosing tuberculosis, comprising a recombinant antigenic protein, wherein the recombinant antigenic protein is the fusion protein obtained by connecting ESAT-6 (early secreting antigen target 6KD), CFP-10 (cultufiltrate protein 10KD), and MPB64 (Mycobacterium bovis 64KD) by connecting peptides according to an arbitrary sequence, wherein the connecting peptides are short peptides which do not influence the immunogenicity of ESAT-6, CFP-10 and MPB64. Preferably, the recombinant antigenic protein in the detection kit for diagnosing tuberculosis is the antigenic protein with an amino acid sequence which is SEQ ID: No.1. When the detection kit in the invention is used for diagnosing tuberculosis, the diagnosis time is short, the diagnosis is fast, the sensitivity is strong, and the specificity is high; and the detection kit has an important significance on early diagnosis of tuberculosis.

Description

technical field [0001] The invention relates to a detection kit for diagnosing tuberculosis, in particular to a detection kit for rapidly and specifically diagnosing tuberculosis. 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 techniques are very difficult for the diagnosis of Mycobacterium tuberculosis infection. In rec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/11C12N15/70C07K19/00
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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