Establishment Of Indirect ELISA For Atypical Swine Fevervirus And Preliminary Study Of E0E2 Fusion Protein

Atypical porcine pestivirus(APPV)is the main pathogen of piglet type A-II congenital tremor(CT).The clinical symptoms are systemic or local muscle spasm and contraction accompanied by ataxia,causing newborn Piglets died because they were unable to suck milk,causing huge economic losses to some pig farms.Because APPV has very limited replication and low efficiency in cell lines suitable for separating CSFV and other pestiviruses,and researchers have not yet obtained a virus that can be passaged stably.Therefore,there is currently no internationally recognized specific diagnostic method and no effective drug or vaccine for APPV to treat or prevent APPV infection,which brings difficulties to the prevention and control of the disease.This study aims to establish an ELISA method for rapid and accurate detection of APPV serum antibodies,which provides an effective tool for clinical rapid detection of APPV-induced congenital tremor in piglets;the recombinant protein E0,E2 and E0E2 fusion of APPV were obtained through prokaryotic recombinant expression and purification.After immunizing animals,the protein is tested and analyzed for its immunogenicity,which lays the foundation for the study of APPV gene recombinant subunit vaccines.1.Use gene cloning technology to construct recombinant expression plasmids p ET-E0 and p GEX-E0;use fusion PCR and cloning techniques to construct recombinant fusion expression plasmid p ET-E0E2.E.coli BL21 was transformed,induced expression,and purified by affinity chromatography to obtain four recombinant proteins E0-His,E0-GST,E2-His and E0E2-His.2.The purified renatured E0 recombinant protein was immunized to New Zealand white rabbits,blood was collected after 4 immunizations,the serum was separated,and the ammonium caprylate sulfate precipitation method was used to obtain high titer E0 rabbit polyclonal antibodies.3.With the E0-GST recombinant protein as the coating antigen,after optimization of conditions,an indirect ELISA method for detecting APPV E0 antibody was successfully established.Using the established indirect ELISA method,400 serum samples of pigs of different stages were tested,and the positive rate was 82%.4.Immunize BALB/c mice with E0,E2 and E0E2 recombinant proteins,and use indirect ELISA and CCK-8 to measure lymphocyte proliferation,detect humoral immunity and cellular immunity,and compare and analyze their immunogenicity.As a result,compared with the control group,all three recombinant proteins can induce higher levels of humoral and cellular immunity in immunized mice.Among them,the E0E2 fusion protein has the strongest immunogenicity,indicating that the E0E2 fusion protein linked by a flexible peptide is An ideal candidate target for subunit vaccines.