Vitellus immune globulin for preventing prawn virus, tis preparing method and use thereof
A yolk immunoglobulin, virus disease technology, applied in the direction of antiviral immunoglobulin, egg-derived immunoglobulin, antiviral agent, etc. The effect is weakened, etc., to achieve the effect of improving disease resistance, safe use, and direct effect.
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Embodiment 1
[0036] Preparation of Crawfish Primary Cells
[0037] Take healthy crayfish without WSBV infection, wash the body surface, soak in 0.01% potassium permanganate solution for 5 minutes, rinse with sterile water, and disinfect the junction of the carapace and abdomen with alcohol cotton balls. Draw 2-3mL anticoagulant (0.14mol / L NaCl, 0.1mol / L glucose, 30mmol / L citric acid, 26mmol / L sodium citrate, 10mmol / L EDTA, pH4.6) with a sterile syringe, and collect blood from the sinus , anticoagulant and hemolymph were mixed and centrifuged, centrifuged at 1500r / min for 10min, the supernatant was discarded, the precipitate was rinsed twice with L-15 medium, and washed with whole culture solution (15% calf serum, 100U / ml penicillin and 100 μg / ml streptomycin in L-15 medium), gently and uniformly resuspend lymphocytes, inoculate into culture flasks or culture plates, culture overnight at 27°C, and set aside.
Embodiment 2
[0039] Target gene cloning
[0040] The VP28 gene was amplified by PCR method, and the sequencing results showed that the gene contained 615 nucleotides, and the homology of sequence fragments of WSSV from different sources in GeneBamk was 100%.
Embodiment 3
[0042] protein induced expression
[0043] The results of SDS-PAGE electrophoresis showed that the strain carrying the recombinant plasmid pGEX-2T produced a specific protein band at the molecular weight of 24KDa, 26KDa, and 28KDa respectively, and the expression amount accounted for more than 40% of the total bacterial protein. , while no corresponding protein band was produced in the control strain.
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