Expression Of Major Nonstructural Proteins Of Porcine Sapovirus And Screening Of Proteins Interacted With VP2

Sapoviruses(SaVs) are members of the family Caliciviridae and cause acute viral gastroenteritis in human and animals. They are spread by faecal-oral transmission, and cause mild diarrhea or acute diarrhea accompanied by nausea, vomiting and abdominal pain symptoms. In recent years, the study found that SaVs have been isolated from humans, swine, cattle, dogs, cats, bats, mink and sea lions. Porcine Sapoviruses(PoSaVs) have been first identified in the United States in 1980. To date, PoSaVs have been reported from several countries of the world and represent an important cause of threat and economic loss. In China, Po SaVs were first identified in fecal specimens collected from diarrheic piglets. Recent research has shown that some PoSaVs have been not only detected in symptomatic pigs, but also in asymptomatic pigs, which is the potential threat to the pig industry production. So the study of PoSaVs biological characteristics and the exploration of the virus function causing diarrhea especially in weaning piglets plays a key role for preventing the disease to reduce the economic loss and safeguard human health. This study we obtained VPg, 3C, 3D, p70 and VP2 recombinant proteins of PoSaV CH430 and their polyclonal antibodies by using prokaryotic expression system, and preliminarily screen twenty three known proteins interacting with the VP2 protein by using the yeast two hybrid system.VPg, 3C, 3D, p70 and VP2 genes of PoSaV CH430 were obtained by one-step RT-PCR and were subcloned to pET-30 a to construct recombinant expression plasmids pET30a-VPg/3C/3D/p70/VP2 in this study. The pET30a-VPg/3C/3D/p70/VP2 was transformed into the express bacterium E.coli BL21(DE3) and expressed by induction of IPTG. SDS-PAGE identification showed that the VPg, 3C, 3D, p70 and VP2 proteins were highly expressed and the molecular weight were 22, 23, 67, 81 and 26 kDa. They mainly exist in the form of inclusion body. Highly purified VPg/3C/3D/p70/VP2 proteins were obtained by an affinity chromatography using Ni Sepharose 6 Fast Flow purification system after induction by IPTG. Western-blot showed that these proteins had good antigenicity. Then, injected into rabbit for polyclonal antibodies against VPg, 3C, 3D, p70, VP2 proteins. Western-blot demonstrated that polyclonal antibodies can identify the corresponding proteins and can be used in follow-up studies.This study we constructed a cDNA yeast two-hybrid library with porcine intestinal mucosal epithelial tissue and transformed it into Y187 yeast competent cells. pGBKT7-VP2 was also constructed as yeast two-hybrid bait vector and then was transformed into Y2 HGold yeast competent cells to identify its toxicity and autonomous activation. Y2 HGold yeast cells with pGBKT7-VP2 and Y187 yeast cells with cDNA library were co-cultured, then hybrid bacteria liquid cultured in SD/-Ade/-His/-Leu/-Trp/X/A medium to screen positive clones. Positive clones were further identified by sequencing. Using BLAST to analysis the homology of the positive clones. The results indicted that VP2 was well expressed in the yeast strain Y2 HGold, pGBKT7-VP2 bait does not autonomously activate the reporter genes. Finally, there are twenty three kinds of protein interacting with the VP2 protein. This study will provide some new ideas and direction for further investigation on the function of structural protein VP2 of porcine sapovirus.