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Bovine tuberculosis detection reagent containing recombinant protein mixture

A technology of mixtures and reagents, applied in the field of immunodetection, which can solve the problems of false negatives and unsatisfactory sensitivity.

Active Publication Date: 2012-10-03
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

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

Method used

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  • Bovine tuberculosis detection reagent containing recombinant protein mixture
  • Bovine tuberculosis detection reagent containing recombinant protein mixture
  • Bovine tuberculosis detection reagent containing recombinant protein mixture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of recombinant plasmids PET-ESAT-6, PET-CFP-10 and PET-TB10.4

[0047] 1.1 Extraction of Mycobacterium bovis genomic DNA

[0048] The culture of M.bovis ValleeⅢ strain (provided by China Veterinary Drug Administration) was used according to the method described in the product manual of the Bacterial Genomic DNA Small Amount Rapid Extraction Kit (purchased from Beijing Bodatech Gene Technology Co., Ltd.).

[0049] 1.2 Design of primers

[0050] Design specific primers according to the CFP-10, ESAT-6 and TB10.4 gene sequences of M.bovis AF2122 / 97 genomic DNA (accession number BX248333) in GenBank, the upstream primers carry Bam H I restriction sites, and the downstream primers carry Hind Ⅲ Restriction site, the primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The nucleotide sequence of the primers is shown in Table 1 (the underlined are the protective bases and restriction sites).

[0051] Table 1PCR primer name, sequence and si...

Embodiment 2

[0058] Example 2 Expression and purification of recombinant proteins CFP-10, ESAT-6 and TB10.4

[0059] 2.1 Induced expression and purification of recombinant protein

[0060]The recombinant plasmids PET-CFP-10, PET-ESAT-6 and PET-TB10.4 prepared in Example 1 were respectively transformed into E.coli BL21 (DE3) competent cells, and a single colony was picked to inoculate to 10 mL containing the final concentration In LB medium with 25μg / ml ampicillin, culture overnight at 37°C with 200r / min shaking, inoculate 1ml of the culture into 100ml LB medium containing a final concentration of 25μg / ml ampicillin, and shake at 37°C at 200r / min 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. Collect the bacteria by centrifugation at 6000r / min for 10min, wash twice with 40mL PBS (pH7.4), resuspend in 10ml PBS (pH7.4), break the bacteria by ultrasonication in an ice bath, and centrifuge the mixture at 12000rpm at ...

Embodiment 3

[0063] Example 3 Activity Detection of Recombinant Proteins CFP-10, ESAT-6 and TB10.4

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

[0065] ①Use the traditional bovine PPD intradermal allergy test and IFN-γ release test to screen bovine tuberculosis-positive cattle and 5 healthy cattle respectively. Under sterile conditions, collect 10ml of heparin anticoagulant blood from each cattle, and store them at room temperature (22±20°C) within 8 hours. 5°C) to the laboratory. 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, recombinant protein CFP10, ESAT-6, TB10 respectively .4 (PET and recombinant protein are added in equimolar amounts, the final concentration of CFP-10, ESAT-6 and TB10.4 are all 10ug / ml) 50μl / well, after shaking and mixing, 37℃CO 2 Incubate in the incubator for 24h. ③ Carefully draw 200μl of the upper layer of plasma...

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Abstract

The invention belongs to the field of immunodetection and provides a reagent and a method for detecting bovine mycobacterial infection. The detection reagent comprises a recombinant protein mixture used as a specific stimulation source, wherein the recombinant protein mixture can be used for stimulating animals infected with bovine mycobacteria to generate a DTH (Delayed Type Hypersensitiity) reaction and stimulating peripheral blood lymphocyte to release IFN (Interferon)-gamma. The defects of the prior art are overcome by the detection reagent, and the detection reagent has the advantages of good biosafety, low cost and feasibility in standardized production and can be effectively applied to the clinic 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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IPC IPC(8): G01N33/569G01N33/531
Inventor 朱鸿飞贾红鑫婷侯绍华袁维峰郭晓宇
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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