Gene Clone And Prokaryotic Expression Of Structural Protein VP1of Porcine Kobuvirus And Development Of ELISA For Detection Of Antibody To VP1

Porcine kobuvirus (PKV) is a member of the genus kobuvirus, family Picornaviridae. Some papers reported that PKV is potentially associated with porcine acute gastroenteritis. Swine infection with the virus are found in most countries all of the world since2007. Acute gastroenteritis is a major cause of death for young children and young animals. The relationship between PKV and swine gastroenteritis is not clear, but the virus was found in clinic an adaption to the host. Pigs at the different ages can be infected with PKV, which were co-infected with other enterovirus. It may be a disease-induced factor, has potential harm to modern pig industry.At present we have some difficulties to do research on the virus, because PKV cannot be cultured in vitro. Only RT-PCR and serological techniques were used for the virus detection of clinical samples and epidemic investigation. PKV analysis has not been reported. In this study, we cloned and expressed PKV VP1gene, and established an indirect ELISA method for PKV VP1antibodies detection. It will promote the serological diagnosis and surveillance of PKV.1.Gene Clone and Prokaryotic Expression of Structural Protein VP1of Porcine kobuvirusThe antigen epitopes information of VP1protein of PKV XD strain was analyzed by the molecular biology software DNAstar and ABCpred. The results showed the fragment between785aa to1038aa in VP1has strong antigenicity and hydrophilicity. The primers were designed based on this fragment. The fragment of VP1gene of XD strain was amplified by RT-PCR, then it was cloned into the prokaryotic expression vector to construct recombinant plasmid pET-32a-XDVP1for the inducible expression in E.coli (DE3). The high-level expression of soluble recombinant protein VP1was obtained under28℃of induced temperature,0.005mmol/L of IPTG final concentration for3hours. The positive swine serum to PKV was identified by western blotting, which was first co-incubated with lysate of Eschericha coli to absorb non-specific reaction. 2. Development of ELISA for Detection of Antibody to VP1An indirect ELISA method for the detection of PKV VP1was established with the purified recombinant protein VP1. Optimal operating conditions of the ELISA reaction were tested. The results showed that0.4μg/mL was for the optimal coating concentration of the recombinant protein;1:50for the test serum;1:4000for HRP-labeled goat anti-pig IgG;37℃of temperature and30min incubation for the antibody reaction. The threshold for the positive serum was OD value0.35. The ELISA method showed good specificity and no cross-reaction with the serum to porcine epidemic diarrhea virus and positive transmissible gastroenteritis virus.720porcine serum samples from8pig farms were collected and detected with the established method. The results showed that the VP1antibody positive from33.33%to98.89%, average positive was81.25%, which indicated that the positive rate of PKV was various in different pig farms.Conclusions:The VP1indirect ELISA can be used in PKV sera-diagnosis and clinical monitoring.