Establishment Of Cell Lines Expressing Porcine Rotavirus NSP4Stably And Subcellular Localization Of NSP4

Rotavirus （RV） is a highly contagious virus, it is the most common cause ofdehydrated diarrhea in infants, young children and various young animals. Porcine rotavirus（PoRV） is one of the main pathogens causing diarrhea in piglets. This virus can mainlyinfect weaning piglets before and after. The piglets diarrhea was attack rapidly after the PoRVinfection and usually co-infection or secondary infection with other enteric bacterias andviruses, and most piglets finally died of dehydration and malnutrition. Recent years, porcinerotavirus obtained widespread attention for the reason of it can cause serious damage instock farming. Studies have shown that rotavirus can across the species barrier leading tointerspecies transmission and heterologous infection. It has proved that porcine rotavirus canpotentially infect human, so porcine rotavirus has the potential threat to human health and hasan important significance in Public Health. Rotavirus non-structural protein4（NSP4）,encoded by rotavirus genome segment10, is a viral enterotoxin with pleotropic functions inviral morphogenesis and pathogenesis. From the experiments, we obtained following results:（1）VP6gene was amplified from PoRV by RT-PCR and ligated to cloning vectorpMD-19T to construct correct standard plasmid. The reaction system and the reactionconditions were optimized and the standard curve was developed successfully. A SYBRGreen Ⅰ real-time PCR method to detect porcine group A rotavirus was established. Thedata shown this method had high specificity and good repeatability. The detectionsensitivity can reach a minimum of10copies/μL, which is100times higher than generalRT-PCR and at the range from1.0×109to1.0×102copies/μL shows a good linearrelationship. Good reproducibility was obtained because the intra-coefficient variation wasless than1%, and the inter-coefficient variation was less than2%. Use this method, we testedthe PoRV proliferation in ZYM-SIEC02cells, results showed that the ZYM-SIEC02cellscan be used as the PoRV target cells.（2）NSP4gene was amplified from PoRV by RT-PCR and ligated to expression vectorspEGFP-N1and pEGFP-C1to construct recombinant plasmids NSP4-EGFP and EGFP-NSP4. After the determination of RT-PCR, double enzyme digestion and sequencing, therecombinant plasmids were transfected into ZYM-SIEC02cells, we screened the stablizedexpressed cell lines. The cell lines were identified by RT-PCR, and about528bp fragmentswas obtained, this result was consistant with expected result and suggested that NSP4couldbe expressed stablely in mRNA level.（3）The endoplasmic reticulum localization vector pDsred-ER was transfected intoZYM-SIEC02cells, we screened the stable expression cell lines. The recombinant plasmidsNSP4-EGFP and EGFP-NSP4were transfected into the cells expressing the endoplasmicreticulum localization vector. After24hours, we observed the cells under a fluorescencemicroscope. The results showed that the NSP4-EGFP fusion protein mainly localized in theendoplasmic reticulum, and only a small part was located in the cytoplasm besidesendoplasmic reticulum; EGFP-NSP4fusion protein was mainly localized in the endoplasmicreticulum, and only a small part was located in the nucleus.In this study, we established a SYBR Green Ⅰ real-time PCR method to detect porcinegroup A rotavirus, use this method, we tested the PoRV proliferation in ZYM-SIEC02cells.We established the cell lines expressing NSP4stably and we studied the subcellularlocalization of NSP4in ZYM-SIEC02cells. These results provided new references for furtherstudies about the pathogenesis of PoRV non-structural protein NSP4.