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Bovine tuberculosis antigen specific gamma-interferon enzyme-linked immuno sorbent assay (ELISA) kit

A tuberculosis antigen and gamma interferon technology, applied in the biological field, can solve the problems of poor specificity, lack, and sensitivity to be improved, and achieve strong immunogenicity and immune protection, improved specificity and sensitivity, and high specificity. Effect

Inactive Publication Date: 2011-09-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Sichuan Agricultural University adopts the method of rHIS-CFP10 / ESAT6 fusion protein. Mycobacterium tuberculosis can secrete CFP10 / ESAT6. Mycobacterium bovis lacks the genes of these proteins. It is impossible to detect Mycobacterium bovis infection clinically, and the sensitivity needs to be improved.
The bovine gamma interferon antigen capture ELISA detection method established by Yangzhou University uses the Mycobacterium bovis antigen PPD as a stimulus, which is difficult to distinguish from environmental mycobacteria infections such as Mycobacterium avium in clinical diagnosis, and has poor specificity

Method used

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  • Bovine tuberculosis antigen specific gamma-interferon enzyme-linked immuno sorbent assay (ELISA) kit
  • Bovine tuberculosis antigen specific gamma-interferon enzyme-linked immuno sorbent assay (ELISA) kit
  • Bovine tuberculosis antigen specific gamma-interferon enzyme-linked immuno sorbent assay (ELISA) kit

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Experimental program
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Effect test

Embodiment 1

[0045] The construction of embodiment 1 genetically engineered bacteria

[0046] Specific primers were designed according to the published gene sequences of Mycobacterium bovis MPB83, MPB70; Mycobacterium tuberculosis ESAT6 and CFP10. The primer sequence is shown as SEQ ID No.1-8:

[0047] Table 1 Amplifies the primer sequences of MPB83, MPB70, ESAT6, CFP10 genes

[0048]

[0049]

[0050] Extract the genomic DNA of Mycobacterium bovis standard strain AF2122 / 97 and Mycobacterium tuberculosis H37Rv (preserved by our laboratory), use the genomic DNA as a template, and use the synthetic specific primer sequence to carry out PCR amplification. The total volume of the reaction system is 50 μl. The reaction system is as follows:

[0051] 10×PCR buffer 5μl

[0052] 10mM dNTPs 1.0μl

[0053] 10uM Primer 1 2.5μl

[0054] 10uM Primer 2 2.5μl

[0055] Ex-Taq enzyme 1μl

[0056] Double distilled water 36μl

[0057] DNA template 2μl

[0058] Total volume 50μl

[0059] React...

Embodiment 2

[0061] Expression and purification of embodiment 2 recombinant protein

[0062] 2.1 Induced expression of recombinant protein

[0063] A single colony of recombinant bacteria containing a positive recombinant expression plasmid was picked and inoculated in 5 ml LB liquid medium (containing 50 μg / ml kanamycin), and activated overnight at 37°C. Inoculate the activated bacteria solution into LB liquid medium containing 50 μg / ml kanamycin at a ratio of 1%, and shake at 225 rpm at 37°C until the logarithmic growth phase (OD600=0.6~0.8), and add the final concentration of 1mmol / L IPTG was cultured with shaking at 37°C for 4 hours on a constant temperature shaker to harvest the bacteria. Take out 1ml of bacterial culture at different times before induction and after induction, collect the bacteria by centrifugation at 7000rpm for 5min, discard the supernatant, add 2xSDS loading buffer, denature at 98°C for 10min, and use 15% polyacrylamide gel for SDS- PAGE analysis ( Figure 6-9 ...

Embodiment 3

[0066] Cloning and prokaryotic expression of the bovine IFN-γ gene of embodiment 3

[0067] 3.1 Isolation and culture of bovine lymphocytes

[0068] 1) Take 15ml of blood from a healthy cow and add anticoagulant sodium heparin.

[0069] 2) Dilute blood or plasma with an equal volume of PBS.

[0070] 3) Add 30ml of lymphocyte separation solution to the bottom of the centrifuge tube and warm up to room temperature.

[0071] 4) Use a Pasteur glass pipette to absorb 30ml of the diluted blood sample, and slowly spread it on the lymphocyte separation medium along the tube wall without disturbing the interface of the liquid layer.

[0072] 5) Centrifuge with a horizontal rotor at 2000rpm / min for 20min at a room temperature of about 20°C.

[0073] 6) After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation solution, and the top layer is plasma. Between the plasma layer and the separation fluid is a thin layer of dense buffy coat, containi...

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Abstract

The invention provides a bovine tuberculosis antigen specific gamma-interferon enzyme-linked immuno sorbent assay (ELISA) kit. The kit comprises a bovine gamma-interferon monoclonal antibody coated ELISA plate, enzyme-labeled bovine gamma-interferon monoclonal antibodies, and protein solution containing mycobacterium bovis MPB83 and MPB70 and mycobacterium tuberculosis ESAT6 and CFP10. The antibodies are captured and detected for different antigen surfaces of gamma-interferon respectively. A bovine tuberculosis antigen specific gamma-interferon ELISA detection method established by using the mycobacterium bovis recombinant proteins MPB83 and MPB70 and mycobacterium tuberculosis recombinant proteins ESAT6 and CFP10 is relatively stable; the specificity of the method is 96 percent, and the sensitivity of the method is 88.6 percent; and the specificity and the sensitivity of the gamma-interferon ELISA detection method for the bovine tuberculosis are greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, it relates to the cloning, expression and purification of four recombinant proteins of Mycobacterium bovis MPB83, MPB70, Mycobacterium tuberculosis ESAT6 and CFP10, and the determination of the optimal concentration of the four proteins as multiple combinations of diagnostic antigens, Expression of bovine IFN-γ recombinant protein, preparation and application of its monoclonal antibody. Background technique [0002] Tuberculin skin test (TST) has been used to detect bovine tuberculosis, but its sensitivity and specificity are low, and it is difficult to detect early bovine tuberculosis infection. IFN-γ, as the main indicator of Th1-type cellular immune effect, is significantly increased in the early stage of Mycobacterium bovis infection, which points out the direction for the development of effective detection reagents for early bovine tuberculosis. [0003] There are domestic resear...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535
Inventor 周向梅赵德明杨杨张奥邵安文鲁龙翔李丽好杨利峰尹晓敏王志刚王洋李泽盛
Owner CHINA AGRI UNIV
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