A kit for detecting porcine Japanese encephalitis virus antibody
A swine Japanese encephalitis and kit technology, which is applied to measurement devices, instruments, biological material analysis and other directions, can solve problems such as poor sensitivity, difficulty in achieving accurate diagnosis, etc., and achieve the effects of high precision, low use cost, and easy operation.
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Embodiment 1
[0045] Example 1 Construction of pGEX-6P-1-NS1 prokaryotic expression plasmid
[0046] 1. Primer design and synthesis
[0047] Using the gene sequence of the nonstructural protein NS1 of porcine Japanese encephalitis virus SA14-14-2 registered in GenBank as a template, specific primers were designed using Prime5.0 software (Table 1). The expected amplified length of the target fragment is 1245bp. Primers were synthesized by Beijing Sanbo Polygala Biotechnology Co., Ltd.
[0048] Table 1 Sequence amplification primers of porcine Japanese encephalitis virus NS1
[0049]
[0050] Note: The underline is the restriction endonuclease recognition sequence
[0051] 2. Amplification of the target gene
[0052] The plasmid pCMV-NS1 stored in the laboratory was used as a template for PCR amplification. The PCR reaction system is as follows:
[0053]
[0054] PCR reaction conditions: 94°C for 2min; 98°C for 10s, 55°C for 30s, 68°C for 1min, a total of 35 cycles, 68°C for 7min....
Embodiment 2
[0077] Prokaryotic expression and purification of embodiment 2 recombinant protein
[0078] 1. Transformation of recombinant expression bacteria
[0079] 2 tubes of competent cells BL21(DE3), thawed in ice until thawed. Add 2uL each of pGEX-6P-1-NS1 and empty vector pGEX-6p-1 to BL21(DE3), gently blow and mix with a pipette gun, and ice-bath for 30min; heat shock at 42°C for 60s, and ice-bath again For 5 minutes, take 30uL and spread evenly on the surface of LB solid medium containing ampicillin. Incubate overnight at 37°C and observe the growth of the colonies.
[0080] 2. Induced expression of target protein
[0081]Pick a single colony containing the recombinant plasmid pGEX-6P-1-NS1 and the empty vector pGEX-6p-1, and transfer them to 1 mL of Amp / LB medium, overnight at 37°C at 180 rpm; at a ratio of 1:50 Inoculate into 5mL of Amp / LB liquid medium, culture at 37°C with shaking at 180r / min until the logarithmic phase of growth, that is, the OD600 is between 0.4 and 0.6;...
Embodiment 3
[0103] Example 3 Establishment of Chemiluminescent Enzyme Immunoassay (CLEIA) and Condition Optimization
[0104] 1. Basic operation steps
[0105] With 0.05mol / L NaCO of pH9.6 3 -NaHCO 3 Dilute the recombinant NS1 protein with buffer solution to the working concentration, 100 μL per well, coat the polyethylene plate, 37 °C for 1 hour; wash the plate 4 times with PBS (PBST) containing 0.05% Tween-20 as the washing solution with a plate washer; Add 300 μL of 5% calf serum, block at 37°C for 1 hour, wash with PBST 4 times; add 100 μL of diluted serum to be tested for 1 hour at 37°C, wash the plate 4 times; add 1:10000 diluted HRP-labeled goat anti-pig IgG antibody, 100 μL per well, 37 ° C for 1 hour, wash the plate 4 times; add luminescent solution, mix A and B in equal volume (ready to use), 100 μL per well, react at room temperature for 3 minutes in the dark; use a chemiluminescence detector Luminescence intensity (relative light units, RLU) at 425 nm was measured.
[0106...
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