Human and animal brucella antibody immunochromatography test paper and preparation method thereof
A Brucella and antibody technology, applied in the field of immunological diagnosis and detection, can solve the problems of long detection time, difficult to popularize and use, and difficult to give detection results, and achieve the effects of long shelf life, high sensitivity and strong sensitivity.
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Embodiment 1
[0027] Example 1: Prokaryotic expression and purification of Brucella outer membrane protein BP26
[0028] 1.1 Design and synthesis of primers
[0029] With reference to the BP26 gene sequence of the Brucella melitensis reference strain 16M published on GenBank (accession number: U45996), according to the characteristics of the pGM-T cloning vector and the pET-28a prokaryotic expression vector, the design contains restriction internal Primer for the cleavage site of Dicer Xho I / EcoR I. The primer sequences are shown in Table 1-1.
[0030] Table 1-1 Primer sequences
[0031] Table 1-1 Primer sequences
[0032]
[0033] 1.2 PCR amplification of the target fragment
[0034] PCR amplification was carried out with Brucella 16M genome as a template, and the PCR reaction system is shown in Table 1-2. The reaction conditions were as follows: pre-denaturation at 94 °C for 5 min; 95 °C for 50 s, 58 °C for 45 s, 72 °C for 1 min, 30 cycles; extension at 72 °C for 7 min; 4 °C∞. Af...
Embodiment 2
[0065] Example 2: Prokaryotic expression and purification of Brucella outer membrane protein OMP31
[0066] 1.1 Design and synthesis of primers
[0067] With reference to the OMP31 gene sequence of the 16M Brucella sheep reference strain that has been published on GenBank, the 78bp signal peptide is removed. Primer for the cleavage site of Dicer Xho I / Nco I. The primer sequences are shown in Table 1-1.
[0068] Table 1-1 Primer sequences
[0069]Table 1-1 Primer sequences
[0070]
[0071] 1.2 PCR amplification of the target fragment
[0072] PCR amplification was carried out using 16M bacteria liquid of Brucella melis inactivated at 80°C for 30 minutes as a template, and the PCR reaction system is shown in Table 1-2. The following reaction conditions were used: pre-denaturation at 95°C for 4 min; 30 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min; extension at 72°C for 7 min; 4°C∞. After the reaction, the PCR product was analyzed on a 1.2% agarose gel, and...
Embodiment 3
[0104] Embodiment 3: the colloidal gold immunochromatography test strip of Brucella antibody in the preparation serum
[0105] 1.1 Preparation of colloidal gold
[0106] Clean the triangular flask for burning colloidal gold and then soak it in aqua regia overnight; take it out and rinse it with clean water 20 times, then wash it with distilled water for 3 to 5 times, then rinse it with ultrapure water for 3 times, and let it dry After drying, siliconize with 5% dichlorodimethylsilane in chloroform solution for 1 min, rinse with ultrapure water for 3 times after drying, and dry the triangular flask for gold burning.
[0107] Add 49ml of chloroauric acid with a concentration of 0.01% into a silanized amphorae, heat and stir on a magnetic stirrer, and quickly add 1ml of trisodium citrate aqueous solution with a concentration of 1.05% after the solution boils, and the color of the solution is about 5min The left and right sides turn into a stable purple-red color. After cooling, ...
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