Border disease virus antibody detection ELISA kit
A sheep border disease virus and kit technology, applied in the field of molecular biology, can solve the problems of high background, vaccine immune impact, and high cost of virus production, and achieve good safety, high specificity and sensitivity, and good antigenicity. Effect
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Embodiment 1
[0013] Embodiment 1, the preparation of BDV recombinant EO protein
[0014] 1) According to the BDV gene sequence recorded in GenBank, design a pair of specific primers
[0015] The upstream primer is P1: GCCGAATTCATCGTAAGTAGGCGTGATTGC, (SEQ ID NO.1),
[0016] The downstream primer is P2: CCCAAGCTTGCTACGATCGAAGTTCAGTTT (SEQ ID NO.2).
[0017] The size of the amplified fragment is 365bp;
[0018] 2) The E0 gene of BDV was amplified by RT-PCR method, the RT-PCR product of BDV gene and the pET-32a(+) vector were simultaneously digested with Eco RI and HindⅢ, the target fragment was recovered, and T4DNA ligase was used to digest The two were connected, overnight at 16°C, and the next day, the connected product was transformed into competent cells BL21, and cultured in a 37°C incubator for 16-20h. Pick a single colony to expand culture, extract the plasmid, and use Sac I for enzyme digestion identification; sequence the positive recombinant plasmid, and obtain the positive recom...
Embodiment 2
[0020] Embodiment 2, the preparation of the indirect ELISA kit that detects sheep border virus antibody
[0021] 2.1 The preparation of the ELISA plate is as follows: use 0.5 mol / L carbonate buffer solution with pH=9.5 as the coating solution, dilute the recombinant BDV E0 protein prepared in Example 1 to 20 pg / mL, and add 100 ul / well to the ELISA reaction In the plate, coat overnight at 4°C, pat dry, block with 0.5% bovine serum albumin at 30°C for 2h, wash with PBST containing 0.1% Tween-20, pH 7.4, and pat dry.
[0022] The preparation conditions of the optimized ELISA plate are as follows: use 0.5mol / L carbonate buffer solution with pH 9.5 as the coating solution, dilute the recombinant protein to 20pg / mL, add 100ul / well to the Elisa reaction plate, and store at 37°C Blocked for 2h, coated overnight at 4°C, patted dry, and blocked with 0.5% bovine serum albumin (BSA) for 2h at 30°C.
[0024] The ELISA kit includes the purified recombinant ...
Embodiment 3
[0025] Embodiment 3, ELISA kit sample detection comparison
[0026] Use the indirect ELISA detection kit established by BDV E0 protein and the BDV antibody detection kit from Svanova to detect sheep serum samples at the same time; and compare the detection results of the two methods, the results are as follows:
[0027] Table 1. Swedish svanova test results (positive random sample serum)
[0028]
[0029]
[0030] Table 2. Swedish svanova test results (negative random sample serum)
[0031]
1
2
3
4
5
6
7
8
virus hole
control well
virus hole
control well
virus hole
control well
virus hole
control well
A
0.087
0.076
0.235
0.129
0.302
0.251
0.166
0.101
B
0.082
0.063
0.291
0.201
0.165
0.146
0.192
0.155
C
1.109
0.236
0.282
0.179
0.222
0.153
0.271
0.181
D
1.090
0.175
0.317
0.212
0.189
0.172
...
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