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Mycobacterium bovis infection detection kit meditated by recombined fusion protein and method thereof

A technology of Mycobacterium bovis and fusion protein is applied in the field of Mycobacterium bovis infection detection, which can solve the problems of poor specificity, complex antigen components, and low sensitivity of serological detection methods, and achieve the effect of strong specificity and sensitivity

Active Publication Date: 2009-09-16
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the complex antigen components contained in PPD, some of the antigens are widely present in Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium avium and non-pathogenic environmental mycobacteria, resulting in poor specificity. False positives often occur during use
[0006] Therefore, there is an urgent need to develop a detection reagent and method that can improve the specificity and sensitivity of detection at the same time, which can not only overcome the low sensitivity of serological detection methods, but also have a higher detection rate than PPD-stimulated IFN-γ release test. specificity

Method used

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  • Mycobacterium bovis infection detection kit meditated by recombined fusion protein and method thereof
  • Mycobacterium bovis infection detection kit meditated by recombined fusion protein and method thereof
  • Mycobacterium bovis infection detection kit meditated by recombined fusion protein and method thereof

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Embodiment 1

[0027] Construction of embodiment 1 recombinant plasmid pET-E6-M63-H65

[0028] 1.1 Extraction of Mycobacterium bovis genomic DNA

[0029] Refer to the method described in the instructions of the Bacterial Genomic DNA Small Amount Rapid Extraction Kit (purchased from Beijing Biotech Gene Technology Co., Ltd.).

[0030] 1.2 Design of primers

[0031] Specific primers were designed according to the ESAT-6, MPB63 and HSP65 gene sequences of Mycobacterium bovis genomic DNA (accession number BX248333) in GenBank. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the sequences are shown in Table 1 (the underline is the enzyme cut point, the shaded place is Linker).

[0032] Table 1 PCR primer name, sequence and size of amplified product

[0033]

[0034] 1.3 PCR amplification of target gene and product recovery

[0035] Using Mycobacterium bovis genomic DNA as a template, LATaq DNA polymerase was used to amplify ESAT-6, MPB63 and HSP65 genes respect...

Embodiment 2

[0048] Example 2 Expression and Purification of Mycobacterium bovis Recombinant Fusion Protein rE6-M63-H65

[0049] 2.1 Induced expression, purification, SDS-PAGE and Western-blot analysis of recombinant fusion protein

[0050] Transform the recombinant plasmid pET-E6-M63-H65 prepared in Example 1 into BL21(DE3) competent cells, pick a single colony and inoculate it into 200 mL of LB medium containing a final concentration of 25 μg / ml ampicillin, and shake at 37°C Bed culture until OD600=0.7, add IPTG with a final concentration of 1mM, induce culture at 30°C, 160rpm shaker for 10h, centrifuge at 9000rpm for 10min to collect the bacteria, resuspend the bacteria in 60mL PBS (pH7.4), and break the bacteria by ultrasonication in an ice bath After crushing, the mixture was centrifuged at 12,000 rpm at 4°C for 30 minutes and the supernatant was taken, and the recombinant protein was purified according to the operation manual of His Link™ Protein Purification Resin (purchased from Be...

Embodiment 3

[0052] The establishment of embodiment 3 Mycobacterium bovis detection method

[0053] 3.1 Collection and culture of bovine whole blood to be tested

[0054] ①Collect 5ml of bovine heparin anticoagulated whole blood under aseptic conditions, transport it to the laboratory at room temperature (22±5°C) and culture it within 8 hours after blood collection. ② Add anticoagulant blood to 24-well tissue culture plate, 1.5ml / well, 2 wells for each cow to be tested, then aseptically add 50μl Tris-Cl (100mM, pH8.0, as negative control stimulus), 50μl Add the solution containing rE6-M63-H65 (20μg) into the corresponding well, mix thoroughly, and store at 37°C CO 2 Incubate in the incubator for 24h. ③ After incubation, carefully draw 400 μl of the upper layer of plasma and transfer it to a 1.5ml centrifuge tube (plasma can be stored at 2-8°C for 7 days, and at -20°C for several months), and draw 50 μl of plasma from each tube as the sample to be tested ELISA detection of bovine IFN-γ r...

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Abstract

The invention belongs to the field of immunodetection and relates to a mycobacterium bovis infection detection kit meditated by recombined fusion protein and a method thereof. The detection reagent comprises combined fusion protein rE6-M63-H65 used as a specific stimulation origin; the combined fusion protein can effectively stimulate sensitized peripheral blood lymphocyte cultured in vitro to release Gamma interferon (IFN-Gamma) at a high level. The cow IFN-Gamma release test established by using the detection reagent rE6-M63-H65 combined fusion protein as the stimulation origin overcomes the insufficiencies of serology detection method and the IFN-Gamma release test with PPD as the stimulation origin, thus enjoying very high sensitivity and specificity and distinguishing mycobacterium bovis infection from mycobacterium avium or non-pathogenic mycobacteria infection and even distinguishing the mycobacterium bovis infection from BGG immunity; therefore, the detection kit and the method of the invention can be effectively used to detect the mycobacterium bovis infection.

Description

technical field [0001] The invention belongs to the technical field of immunoassay. The invention relates to a kit and a method for detecting mycobacterium bovis infection. Background technique [0002] Bovine tuberculosis is a zoonotic chronic wasting infectious disease caused by Mycobacterium bovis and Mycobacterium tuberculosis. Cross-infection between humans and animals causes the disease to spread widely, so it has a very important public hygiene meaning. The World Health Organization (WHO) pointed out: "In countries where bovine tuberculosis exists, human beings are always threatened. Unless bovine tuberculosis is eliminated, the control of human tuberculosis will not be successful." At present, some relatively developed countries and regions, such as the United States, Bovine tuberculosis has been basically eradicated in Australia and Northern Europe. However, bovine tuberculosis is still one of the most common multiple diseases in my country. The results of two na...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
Inventor 朱鸿飞郝辉贾红侯绍华张泉丁家波
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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