Method for diagnosing avian leukosis virus subgroup J of sicken chicken flocks
A technology of avian leukaemia, diagnostic method, applied in the biological field, capable of solving problems such as false positives
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Embodiment 1
[0046] Example 1: The construction of the gp85 gene prokaryotic expression vector PGEX-6P-1 / gp85 lays the foundation.
[0047] 1) Virus culture and virus cDNA extraction, culture chicken embryo fibroblasts, when the cells grow to 80% monolayer, inoculate the virus, amplify the virus, and extract the cell DNA as a template.
[0048] 2) Amplification of the target fragment. In this experiment, the S1 and S2 primers were used to carry out PCR amplification of the virus sequence integrated into the host genome, in order to amplify 960 bp including the complete gp85 gene of the virus, and the reaction system was 100 μl. The reaction conditions were pre-denaturation at 94°C for 4 minutes, followed by 30 cycles. One cycle is: denaturation at 94°C for 45 seconds, annealing at 57°C for 45 seconds, extension at 72°C for 1.5 minutes, and finally extension at 72°C for 10 minutes, and the reaction product is stored at 4°C.
[0049] 3) Prepare 1% low-melting point agarose, mix the PCR reac...
Embodiment 2
[0056] Example 2: The identification of the PGEX-6P-1 / gp85 recombinant plasmid is achieved by linking the gp85 gene to the vector.
[0057] The prepared double-digested purified product was ligated, and a total of 10 μl reaction system was ligated overnight at 16°C. The ligated product will be used to transform Escherichia coli competent cells DH5α. The connection volume is as follows:
[0058] 3 μl of purified gp85 gene with sticky ends
[0059] 1.5 μl of purified sticky-end PGEX-6P-1
[0060] 10×ligase buffer 1μl
[0061] T4DNAligase 1μl
[0062] Water 3.5μl
[0063] Competent cells were prepared by the calcium chloride method. Pick out the frozen strain DH5α with a sterile inoculation loop and streak on the LB plate, and at the same time, streak on the LB plate containing ampicillin as a control, and cultivate overnight in a 37°C incubator. On the next day, a single colony that grew well was picked and inoculated in 5ml LB liquid medium with shaking at 37°C overnight....
Embodiment 3
[0064] Embodiment 3: The method for diagnosing J subtype avian leukemia for diseased flocks, not only includes the detection process of the gp85 fragment of the virus strain cloned by specific primers, but also includes the detection process of NRAMP1 gene;
[0065] The gp85 fragment recognizes the specific viral receptor on the target cell membrane, called the outer membrane protein
[0066] NRAMP1 gene is an immunity-related candidate gene for disease resistance, which is mainly expressed specifically in phagocytes, neutrophils and peripheral blood cells, and affects the innate immunity of animals;
[0067] Include the following steps:
[0068] 1) Virus cultivation and propagation;
[0069] 2) extraction of proviral DNA;
[0070] 3) PCR reaction to amplify the target gene fragment;
[0071] 4) PCR reaction product electrophoresis detection;
[0072] 5) Gel recovery of PCR reaction product;
[0073] 6) Enzyme digestion identification of PCR reaction products;
[0074] 7...
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