A kind of j subgroup avian leukemia immune colloidal gold antibody detection test strip
An avian leukemia and antibody detection technology, applied in biological tests, viruses/phages, viruses, etc., can solve the problems of false negative antigen variation of nucleic acid molecule detection methods, inapplicability to farms, and time-consuming virus isolation and identification. Strong, easy to operate and carry
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Embodiment 1
[0068] Cloning and sequencing of embodiment 1ALV-Jgp85 gene
[0069] Design primers based on the existing ALV-J proviral genome DNAgp85 envelope protein gene, the forward primer is 5'-GC GGATCC ATCAAGAACGGAACAACACG-3', containing a BamHI restriction site (underlined part), its gene sequence is shown in SEQ ID NO.2; the reverse primer is 5'-GCGCGC AAGCTT GTCCCCACAAATCAAGAAAATA-3', containing a HindIII restriction site (underlined part), its gene sequence is shown in SEQ ID NO.3.
[0070] Using the above pair of primers to carry out PCR amplification using the DNA of subgroup J avian leukosis virus as a template, wherein the selected subgroup J avian leukosis virus is NX0101 (ALV-J, DQ115805.1);
[0071] The PCR reaction conditions are: 95°C for 5min, fully denatured and then enter the cycle system: 95°C for 40s, 65°C for 1min, 72°C for 1min, and after 35 cycles, extend at 72°C for 10min.
[0072] The PCR product was electrophoresed in 1% agarose gel, the results are shown i...
Embodiment 2
[0077] Expression and purification of embodiment 2gp85 protein
[0078] The positive BL21 bacteria containing the recombinant plasmid after sequencing were inoculated in LB liquid medium containing Kan (final concentration of 10 μg / ml) (commercially available conventional medium containing 1% (w / v) Tryptone, 0.5% (w / v) YeastExtract, 1% (w / v) NaCl, 0.1mg / mlKanamycin), shake (200rpm) at 37°C, cultivate to OD600=1.0, add IPTG with a concentration of 500mMol until the final concentration of IPTG in the whole medium is 1mmol / L at 37°C for 3 h, and at the same time set BL21 bacteria transformed with uninduced recombinant plasmids as a control.
[0079] The SDS-PAGE analysis shows that the detection steps are as follows: collect the cultured engineering bacteria liquid, centrifuge at 5000rpm for 5min, discard the supernatant, add standard PBS3ml to the precipitate according to the ratio of 100ml initial medium, add standard PBS to the precipitate to obtain resuspension solution, r...
Embodiment 3
[0102] Activity detection of embodiment 3gp85 protein
[0103] The above purified His-gp85 protein was used to coat the ELISA plate, and the biological activity of the protein was detected by enzyme-linked immunosorbent assay. The chicken serum tested positive by the ALV-J antibody detection kit produced by IDEXX Company was used as the positive control, the healthy SPF chicken serum was used as the negative control, and the HRP-labeled goat anti-mouse IgG enzyme-labeled secondary antibody was used according to the instructions. The results showed that the ratio of the OD value of the positive control to the negative control was greater than 2.1, indicating that the protein had good biological activity and antigenicity.
[0104] The specific steps of ELISA are as follows:
[0105] 1) Coating: Take the purified recombinant SU protein, dilute it with normal saline to the optimal concentration, coat the reaction plate, 100 μl / well, overnight at 4°C;
[0106] 2) Washing: shake o...
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