Colloidal gold detection card capable of rapidly detecting Bh (Bartonella henselae) antibody and kit
A colloidal gold and detection card technology, applied in the biological field, can solve the problem that the overall biological detection does not have much advantage, and achieve the effect of fast detection speed and high sensitivity
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Embodiment 1
[0054] The amplification of the 17kDa class antigen protein gene of embodiment 1, Bh
[0055] Source of biological material: Bartonella henselae (Bh, B.henselae). The biological material came from a strain of Bartonella henselae from Peking cat, which was collected and preserved by the Vector Department of the Institute of Infectious Diseases, China Center for Disease Control and Prevention.
[0056] Cultivate the Bartonella henselae strain in chocolate plate medium (recipe: peptone 10g, beef powder 3g, sodium chloride 5g, defibrinated sheep blood 50mL, agar 15g, distilled water to 1L), and culture in the logarithmic growth phase of Bh The substance was placed in a 1.5mL centrifuge tube, vortexed and mixed to obtain a Bh suspension.
[0057] According to the 1584762-1585286 gene encoded by GenBank accession number CP020742.1 (login address:
[0058] https: / / www.ncbi.nlm.nih.gov / nuccore / CP020742.1?hl=en From=1584762&to=1585286) the 17kDa antigen-like protein gene sequence of ...
Embodiment 2
[0070] Embodiment 2, Bh 17kDa recombinant antigen protein design and synthesis
[0071] Take 1 μL of 15ng / μL -Blunt El Expression Vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd. -the vector in the Blunt E1Expression Kit, catalog number CE111-01); take 4 μL of the DNA of the purified PCR product obtained in Example 1, connect the vector to the PCR product according to the kit instruction manual, and add 50 μL of freshly thawed trans BL21 (ED3) competent, lightly flicked to mix, and ice-bathed for 30 minutes. Heat-shock in a water bath at 42°C for 30 seconds and ice-bath for 2 minutes, then take 200 μL of liquid to spread on the plate, and incubate overnight at 37°C.
[0072] Pick a small amount of overnight culture into a 50mL Erlenmeyer flask of LB liquid medium containing 60μg / ml ampicillin, and shake at 37°C until OD 600 = 0.6. Add 1.0mmol / L IPTG to the Erlenmeyer flask, culture with shaking at 37°C for 4 hours, take 10ml of the culture at 12000r / min,...
Embodiment 3
[0077] Embodiment 3, Bh 17kDa recombinant antigen protein purification
[0078] Take a clean Ni-NTA column (Converse Century, CW0894S), and add 2 mL of prepacked solution soaked in 20% ethanol in advance. After standing still for 30 minutes, let the liquid go, then add 0.5 mol / L imidazole aqueous solution to pass through the column, and then use one column volume of PBS with pH 7.4 to pass through the column.
[0079] The bacterial lysate prepared in Example 2 (the main components are: broken bacterial components, 50mM Tris-HCl pH7.4, 150mM NaCl, 1mM EDTA, 1vol% Triton x-100, 1vol% Sodium deoxycholate, 0.1vol% SDS, protease inhibitor mixture) through the column for 3 to 5 times to completely adsorb the target protein on the column. Use 0.2 mol / L imidazole aqueous solution and 0.01 mol / L PBS with a pH of 7.4 to pass through the column for 3 to 5 times to wash the foreign proteins. Pass through the column 3-5 times with 0.5 mol / L imidazole aqueous solution and 0.01 mol / L PBS w...
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