Study On Enzyme-linked Immunosorbent Assay For Antibody And Virus-like Particles Vaccine Of Porcine Sapovirus

Sapovirus(Sa V) belong Caliciviridae of Sapporo-like virus genus, which is a single-strand RNA virus. Humans and animals can cause acute viral gastroenteritis and diarrhea by the fecal-oral transmission. Porcine Sapovirus(Po Sa V) can cause Piglets diarrhea and is widespread in the world. In recent years, few studies research on the Sa V. A new study show that Sa V can infect a variety of animals, for Sa V different genotypes of human origin and animal origin are similar in the genetic and antigenic characteristics, and have been found Sa V recombinant strains from people and porcine source. It is indicated that Sa V have interspecies transmissibility to humans causing a potential threat of public health. Therefore, it is an urgently task that the research combat higher viral infection for more convenient ELISA diagnostic methods and better immunogenicity,safety, preventive vaccines.According to the study indicate that VP1 sequence can be divided into S and P region for Po Sa V major capsid protein components. To get related to porcine Sapovirus capsid protein genes that Sa V(CH430 strain) RNA as a template, RT-PCR amplification of VP1, S, P gene cloned into p ET-30 a vector, and converted into E. coli BL21(DE3). Purified Po Sa V of VP1 and the S and P region of the protein, respectively, S proteins were HRP labeled by optimizing ELISA coating concentration and closure time, other conditions closed successfully established Po Sa V double antigen sandwich ELISA provides the basis of serological diagnosis. While purify of VP1, P, S protein and immunize rabbits and Piglets were prepared two animals hyperimmune serum for the study of the immunogenicity and protein detection provides an experimental basis.Because VP1 is assembly required district for Po Sa V virus-like particles(VLPs). According to the baculovirus expression system, the VP1 protein-coding genes are inserted in the p Fast BacTM HTB vector, transformed competent cells constitute DH10 BACTM positive recombinant bacmid. Recombinant bacmid sf9 transfected cells, third generations of recombinant baculovirus RNA, DNA was amplified by PCR to identify the exogenous gene is not lost. Using the TCID50 method confirmed the recombinant virus titer. It is successfully expressed in insect cells by confocal microscopy and Western-blot tests proved exogenous gene, relative molecular mass 58-61 k Da. After protein concentration treatment, size and morphology of VLPs were simulated natural virus structure by EM.Meanwhile, the VLPs immunized piglets, detected by the established of double antigen sandwich ELISA. According to virus challenge test, piglets immunized group can effectively suppress viral replication in the intestine, while the control group not inhibit virus replication. Therefore, we demonstrated VLPs could stimulate the body produce neutralizing antibodies and block the tissues of body and virus surface antigen binding prevents virus adhesion receptors on target cells and intrusion of cells. We hope that VLPs could developed neutralizing antibodies high titers in a short time. In this study, Sa V-VLP vaccine for the prevention and treatment of Po Sa V has good prospects. We can put it as one of the new vaccine against the Po Sa V, which construction the foundation for the study of genetic engineering and Po Sa V subunit vaccine.