Construction Of Real-time PCR Method Of African Swine Fever Virus VP72Gene And The Research Of VP73Protein

African Swine Fever Virus (ASFV) is a strong infectious virus. ASFV can cause acute, fatal infectious disease. The mortality rate of ASFV often approaches100%in domestic pigs. African Swine Fever (ASF) can cause huge economic losses on the pig industry. Therefore, it is important to construct an effective way of ASF assay. In this study, we successfully established an efficient real-time quantitative PCR assay and prepared a polyclonal antibody against major antigenic epitope region of VP73protein, and which build the foundation for the immunological research on ASF VP73protein.In order to construct Real-time Quantitative PCR assay for detection of ASFV, I was based on23strains of gene sequence which encodes ASFV structural protein p72in GenBank, and designed the primers and probes. The PCR reaction conditions were optimized by using different annealing temperature, different Mg2+concentrations, different primers and probe concentrations.The Real-time PCR system can automatically generate standard curve. The repeatability, sensitivity and specificity were tested, and wild boar samples were detected by this assay. The results showed the optimal annealing temperature is60℃, the optimal mg2+concentration is4mmol/L, the optimal primers and probe concentration are0.8μmol/L and0.3μmol/L respectively. The coefficients of variation of repeatability test were less than1.3%. The sensitivity tests can detect10copies plasmids. The specificity was tested by detecting five other swine viruses and ASFV plasmid, and only detection of ASFV plasmid appears amplification curve. In conclusion, the constructed Real-time Quantitative PCR assay is rapid, sensitive and specific assay for detection of ASFV.The constructed recombinant prokaryotic expression vector pET32a-VP73was trans-formed into E.coli BL21. The major antigenic epitope of VP73protein was expressed stably and efficiently by IPTG induction. The SDS-PAGE results showed the expression yield of recombinant major antigenic epitope region of VP73protein was highest when final concentration of IPTG is1.0mmol/L and incubation for5h, and with the molecular weight approximately42kD. The protein was identified as major antigenic epitope region of VP73protein by SDS-PAGE and Western Blot. The purified major antigenic epitope region of VP73protein was injected to New Zealand rabbits, and the serum was collected before and after injection. Antibody titer was determined by indirect ELISA. The specifi-city of this protein was determined by indirect ELISA whose primary antibody are ASF positive serum, rabbits serum before and after injection and positive serum of CSF, PRRS, Pig Pseudorabies. The result showed that the titer of antiserum reached up to1:1024000, and reacted with VP73major antigenic region protein. The polyclonal antibody builds the foundation for immunology research of VP73and serological diagnosis of ASF.