Gene engineering preparation for detecting bovine mycobacterium infection

A technology of Mycobacterium bovis and genetic engineering, which is applied in the field of genetic engineering preparations for the detection of Mycobacterium bovis infection, can solve the problems of false negative and unsatisfactory sensitivity, and achieves high specificity, stable content, strong specificity and sensitivity. sexual effect

Active Publication Date: 2014-04-16
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

Method used

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  • Gene engineering preparation for detecting bovine mycobacterium infection
  • Gene engineering preparation for detecting bovine mycobacterium infection
  • Gene engineering preparation for detecting bovine mycobacterium infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of recombinant plasmids PET-CFP-10, PET-ESAT-6 and PET-TB27.4

[0046] 1.1 Extraction of Mycobacterium bovis genomic DNA

[0047] 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.).

[0048] 1.2 Design of primers

[0049] Design specific primers according to the CFP-10, ESAT-6 and TB27.4 gene sequences of M.bovisAF2122 / 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 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).

[0050] Table 1PCR primer name, seque...

Embodiment 2

[0057]Example 2 Expression and purification of recombinant proteins CFP-10, ESAT-6 and TB27.4

[0058] 2.1 Induced expression and purification of recombinant protein

[0059] The recombinant plasmids PET-CFP-10, PET-ESAT-6 and PET-TB27.4 prepared in Example 1 were respectively transformed into E.coliBL21(DE3) competent cells, and a single colony was picked and inoculated to 10 mL with a final concentration of 25 μg In LB medium containing ampicillin / ml, 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 at 22° C. with shaking at 160 rpm for 10 h. 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 12000rp...

Embodiment 3

[0062] Example 3 Activity Detection of Recombinant Proteins CFP-10, ESAT-6 and TB27.4

[0063] 3.1 Identification of cellular immune activity of recombinant protein

[0064] ① Use the traditional bovine PPD intradermal allergy test and IFN-γ release test to screen bovine tuberculosis-positive cattle and 5 healthy cattle, collect 10ml of heparin anticoagulant blood under sterile conditions, and transport it to the experiment at room temperature (22±5°C) room and cultured within 8 hours after blood collection. ②Add anticoagulant blood to 48-well tissue culture plate, 0.75ml / well, and aseptically add bovine PPD, poultry PPD, PBS (pH 7.4), empty vector-tagged protein PET, recombinant protein CFP10, ESAT-6, TB27. 4 (The three recombinant proteins are added in equimolar amounts, the final concentration is 10ug / ml) each 50μl, shake and mix well at 37°C CO 2 Incubate in the incubator for 24h. ③ Carefully draw 200μl of the upper layer of plasma and transfer it to a 1.5ml centrifuge ...

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Abstract

The invention belongs to the field of biological detection. The invention provides a gene engineering preparation for detecting bovine mycobacterium infection. The gene engineering preparation comprises a mixture of three recombinant bovine mycobacterium proteins; the protein mixture can stimulate bovine mycobacterium infecting animals to produce a DTH (delayed type hypersensitivity) reaction. The gene engineering preparation provided by the invention has the advantages of high specificity, bio-security, component accuracy, content stability, low cost, standardized production and so on. Allergic hypodermic tests established with the detection reagents can increase experimental sensitivity and specificity, and can distinguish bovine mycobacterium infection and avian mycobacterium or non-pathogenic mycobacterium infection, so that the gene engineering preparation can be effectively used for clinic detection of bovine tuberculosis. A primer for preparing the gene engineering preparation further is provided by the invention.

Description

technical field [0001] The invention belongs to the technical field of biological detection. The invention relates to a genetic engineering preparation 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. All countries in the world have occurred, and the harm is very serious, which has brought huge economic losses and trade restrictions to the animal husbandry. At present, 50 million cattle are infected with tuberculosis worldwide, and more than 3 billion US dollars are lost every year. 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 Organizat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11G01N33/68C12N15/70C12R1/32
Inventor 贾红鑫婷朱鸿飞袁维峰郭晓宇侯绍华
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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