Method for diagnosing bovine tuberculosis mediated by recombinant protein mixture and regent thereof

A technology of recombinant proteins and diagnostic reagents, applied in the field of immunoassays, can solve the problems of false negatives and unsatisfactory sensitivity, and achieve the effects of high specificity, stable content, strong specificity and sensitivity

Active Publication Date: 2012-09-26
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology

Then, use one or more of these recombinant proteins (also known as "cocktail method") as a coating antigen for enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA) detection, by detecting the corresponding Serological detection of bovine tuberculosis antibody level, although this method improves the specificity of detection to a certain extent, but its sensitivity is not ideal, especially in the early stage of infection and immunocompromised people often have false negatives

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  • Method for diagnosing bovine tuberculosis mediated by recombinant protein mixture and regent thereof
  • Method for diagnosing bovine tuberculosis mediated by recombinant protein mixture and regent thereof
  • Method for diagnosing bovine tuberculosis mediated by recombinant protein mixture and regent thereof

Examples

Experimental program
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Embodiment 1

[0037] The construction of embodiment 1 recombinant plasmid

[0038] 1.1 Extraction of Mycobacterium bovis genomic DNA

[0039] The culture of M.bovis ValleeⅢ strain (purchased from China Veterinary Drug Administration) was used, and the method was carried out according to the method described in the instruction manual of the Bacterial Genomic DNA Small Amount Rapid Extraction Kit (purchased from Beijing Bodatech Gene Technology Co., Ltd.).

[0040] 1.2 Design of primers

[0041] Specific primers were designed according to the ESAT-6, CFP-10, MPB70, and Ag85B gene sequences of M.bovis AF2122 / 97 genomic DNA (accession number BX248333) in GenBank. The upstream primers carried Bam H I restriction sites, and the downstream primers carried HindⅢ Restriction sites, primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the sequences are shown in Table 1 (protective bases and restriction sites are underlined).

[0042] Table 1 PCR primer name, sequence and size of...

Embodiment 2

[0057] Example 2 Expression and purification of recombinant proteins CFP-10 / ESAT-6, MPB70 and Ag85B

[0058] 2.1 Induced expression and purification of recombinant protein

[0059] Transform the recombinant plasmid PET-CFP10 / ESAT-6 prepared in Example 1 into E.coli BL21 (DE3) competent cells, pick a single colony and inoculate it into 10 mL of LB medium containing a final concentration of 25 μg / ml ampicillin, Shake culture at 200r / min at 37°C overnight, inoculate 1ml of the culture into 100ml LB medium containing ampicillin at a final concentration of 25μg / ml, and culture with shaking at 200r / min at 37°C until OD 600 When nm=0.6, add IPTG with a final concentration of 1 mM, and culture with shaking at 160 rpm for 10 h at 22°C. Centrifuge at 6000r / min for 10min to collect the bacteria, wash twice with 40mL PBS (pH 7.4), resuspend in 10ml PBS (pH 7.4), break the bacteria by ultrasonication in an ice bath, centrifuge the broken mixture at 12000rpm, 4℃ for 30min, and take it out....

Embodiment 3

[0063] Example 3 Activity Detection of Recombinant Proteins CFP-10 / ESAT-6, MPB70 and Ag85B

[0064] 3.1 Identification of cellular immune activity of recombinant protein

[0065] ①Use the traditional PPD intradermal allergy test and IFN-γ release test to screen bovine tuberculosis-positive cattle and 5 healthy cattle each, collect 5ml of heparin anticoagulant blood under sterile conditions, and transport it to the laboratory at room temperature (22±5°C) And cultured within 8h after blood collection. ②Add anticoagulant blood to 48-well tissue culture plate, 0.75ml / well, and aseptically add bovine PPD, poultry PPD, PBS (pH7.4), empty vector-tagged protein PET, and recombinant protein CFP-10 / ESAT-6 respectively , MPB70, Ag85B (recombinant protein is added in equimolar amounts, and the final concentration of PET is 10ug / ml) each 50μl, after shaking and mixing, 37 ℃ CO 2 Incubate in the incubator for 24h. ③ Carefully draw 200μl of the upper layer of plasma and transfer it to a 1...

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Abstract

The invention belongs to the field of immunodetection and provides a method for diagnosing bovine tuberculosis mediated by a recombinant protein mixture and a regent for diagnosing bovine tuberculosis mediated by the recombinant protein mixture; the reagent comprises the recombinant protein mixture used as specific irritant; animals infected by mycobacterium bovis can be simulated by the recombinant protein mixture so as to generate a DTH (delayed type hypersensitivity) reaction; compared with a PPD (purified protein derivative) intradermal allergic reaction test, a intradermal allergic reaction test established by regent provided by the invention has higher specificity; mycobacterium bovis infection can be distinguished from environmental mycobacteria infection; and therefore, the method and the reagent can be effectively used for clinical detection of the bovine tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of immunoassay. The invention relates to a novel kit and method for detecting mycobacterium bovis infection. Background technique [0002] Bovine Tuberculosis is a zoonotic chronic infectious disease mainly caused by Mycobacterium bovis infection, which can also be caused by Mycobacterium tuberculosis infection. It occurs in every country in the world, and the harm is very serious, which brings huge economic losses and trade restrictions to the animal husbandry. At present, about 50 million cattle are infected with tuberculosis in the world, and the annual loss is more than 3 billion US dollars. The disease can be transmitted to humans through unpasteurized milk and milk products, contact with contaminated aerosols or animal carcasses, which seriously threatens public health safety and human health, so it has very important public health significance. The World Health Organization (WHO) pointed out: "In co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/11C12N15/31C07K14/35
Inventor 鑫婷贾红朱鸿飞袁维峰侯绍华郭晓宇
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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