The Construction And Application Of The Immune Monitoration Method Of The Efficacy Of Anti-swine Fever Virus Vaccine

Swine fever is a kind of contagious disease, which always causes huge economiclosses to the pig farming. Pig immunization with swine fever vaccine is taken as acommon means to control classical swine fever. However, vaccine from differentmanufacturers or different batches often display different immune effects. In order todetermine the exact amount of classical swine fever antibodies producing andevaluate different immune effects after vaccines injection, an effective method toaccess the efficacy of classical swine fever vaccine should be established. The E2protein is a major epitope inducing neutralizing antibody, and which is also the maintarget protein in various researches on vaccines. Therefore, in this study, a propermethod to access the efficacy of classical swine fever was established, using E2protein as the central target.The gene from2455bp to3016bp of the open reading frame of Shimen swinefever virus genome was amplified by PCR and then cloned into the prokaryoticexpression vector PET-32a, thereby constructing the prokaryotic expression plasmidnamed PET32a-mE2. Transforming the Prokaryotic expression plasmids intoEscherichia coli BL21, SDS-PAGE electrophoresis analysis showed that mE2proteinwas mainly in Escherichia coli existing as inclusion bodies, at37°C with0.1mMIPTG expression induced by3hours. The mE2protein was denatured with8M ureatreatment, purified by Ni-NTA column, and re-natured with6M urea,4M urea,2Murea,1M urea,0.5M urea, PBS gradient dialysis re-naturation. The concentration ofthe protein is152ug/ml determined by BCA Protein Assay Kit. The purified protein isused as coating antigen to establish an indirect ELISA for detecting antibody againstclassical swine fever virus, and then determine the optimum reaction conditions.OPDcolor15min in dark with room temperature, then terminate the reaction with2Msulfate. Measuring the OD450values with a microplate reader at last. When the testedserum OD450values are greater than0.222, identified as positive serum. The repetitionof experiment group with the indirect ELISA method showed a good repeatability result. PRV, PCV2positive serum was specificity measured, the results showed thatthe excellent specificity of this method. We exerted this method to measure92serumsamples and these results were compared with an IDEXX swine fever antibodydetection kit detecting results. The coincidence rate was84%. We applied this methodto compare a manufacture of swine fever live vaccine deriving from spleen with threemanufactures of swine fever live vaccine deriving from cells to immune120pigletsand these immune effects was also examined. It is found that the effects of swinefever live vaccine deriving from spleen were better than the effects of live vaccinederiving from cells and the positive rate of swine fever live vaccines deriving fromcells which were produced by three different manufactures was basically same, but itwas also revealed that the antibody titers were different.In conclusion, the above results showed that this indirect ELISA establishedcould be used to monitor the effectiveness of the immune swine fever vaccine and thismethod could act as the foundation for exploiting classical swine fever antibodydetection kits.