A bovine brucella elisa detection kit
A technology of Brucella bovis and detection kit, which is applied in the field of protein engineering to achieve the effects of good stability and repeatability, easy operation and low background value
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Embodiment 1
[0036] Example 1 Construction of recombinant expression vector of Brucella bovis outer membrane protein
[0037] 1. Primer design and synthesis
[0038] According to the Brucella bovis outer membrane protein gene sequence OMP19 (accession number L27997), OMP22 (accession number AY484562), OMP28 (accession number JQ865997) published on GenBank, the upstream and downstream primers were designed using primer5.0 software , Send the designed primers to Shanghai Shenggong Biological Engineering Technology Co., Ltd. for synthesis. The primer sequence is as follows:
[0039] F omp19 5’-cgc ggatcc ATGGGAATTTCAAAAGCAAG-3’,
[0040] R omp19 5’-ccg ctcgag TCAGCGCGACAGCGTCAC-3’;
[0041] F omp22 5’-cgc ggatcc ATGTTCAAGCGTTCTATC-3’,
[0042] R omp22 5’-ccg ctcgag CTAGAATTTGTAGTTCAG-3’;
[0043] F omp28 5’-cgc ggatcc ATGAACACTCGTGCTAGC-3’,
[0044] R omp28 5’-ccg ctcgag TTACTTGATTTCAAAAACG-3’.
[0045] Underlined are BamH I and Xho I restriction sites respectively.
[0046] 2. Amplificati...
Embodiment 2
[0052] Example 2 Induced expression and purification of the recombinant protein of the outer membrane of Brucella bovis
[0053] 1. Induction and expression of recombinant protein
[0054] The sequencing gene of Example 1 and the recombinant plasmids pCold-TF / OMP19, pCold-TF / OMP22, and pCold-TF / OMP28 with the correct reading frame were transformed into E.coli BL21(DE3) competent cells, respectively. Culture on LB medium containing 100μg / ml ampicillin. The bacterial solution containing the recombinant plasmid was inoculated into LB medium containing 100μg / ml ampicillin at the amount of 1%, and cultured with shaking at 37°C for about 2h, to OD 600 The value is 0.6-0.8, the inducer IPTG is added to the final concentration of 0.5mmol / L, and the bacteria are harvested after 24 hours of culture at 16°C. The bacterial solution was centrifuged at 10000g for 15min at 4℃, and then the pellet was resuspended in PBS buffer; ultrasonicated for 15min in an ice bath until the liquid became clear...
Embodiment 3
[0058] Example 3 Establishment of ELISA detection method for Brucella bovis
[0059] 1. Establishment of indirect ELISA method
[0060] Use NaHCO 3 (pH=9.6) The purified recombinant proteins rOMP19, rOMP22, and rOMP28 were diluted with buffer solution, and the concentrations were diluted to 1μg / ml, and then the recombinant antigens were mixed in a 1:1:1 volume ratio as the coating antigen. Add to each well Sample 100μl, and coat a 96-well ELISA plate at 37°C for 2h. Each well was blocked with 200 μl volume of 10% horse serum for 45 minutes, and washed with PBST five times. Negative and positive sera (China National Medical Products Administration) were diluted 1:50, 1:100, 1:200, 1:400, 100μl per well, incubated at 37°C for 30min, washed with PBST five times. Enzyme-labeled secondary antibody (2mg / ml) was diluted with PBST 1:10 000, 1:20 000, 1:40 000, 100 μl was added to each well, incubated at 37°C for 30 min, and washed with PBST five times. Substrate TMB 100μl / well protects ...
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