| Porcine Rotavirus (PRV) is nonenveloped, segmented, double stranded RNA viruses classified in the family Reoviridae. Porcine rotaviruses are the major cause of diarrhea in the piglets. Porcine rotavirus has been reported widely distributed in the world and caused pig producers serious loss. So, it is much important to study an effective way to restrain the infection of PRV.Taking reference of GenBank PRV JL94strains sequences, we designed a pair of specific primers to clone the complete length of VP4gene DN30209strain of PRV. Expanding product was constructed into a recombinant plasmid VP4-pMD18-T successfully. By gene sequencing and analysis, the results showed that the VP4gene complete length of DN30209was2331bp, compared with JL94strains, nucleotide homology was99.7%, amino acid homology was99.5%. The VP4gene was connected with prokaryotic expression vector pGEX-6P-1, constructed and screened the positive prokaryotic expression vector recombinant plasmid, marked as pGEX-6P-1-VP4. Following, it was transformed into Rosetta competent cell and induced by1PTG, recombinant VP4proteins of112kDa was got and in the form of inclusion body. The purified protein was used as antigen to immunize the white rabbit, and then, rabbit anti-VP4protein polyclonal antibody was prepared, and its biological activity was characterized through the indirect ELISA or IFA or Western Blot assay. The results showed that:the titer of polyclonal antibody was approximately103, moreover, the polyclonal antibody, which has good antigen-antibody reactivity with PRV.Lipid raft micro-domains in the plasma membrane play an important role in many biological processes of many viruses. Cholesterol and sphingolipids are critical structural components of lipid rafts. In our study, in order to demonstrate whether cholesterol is required for cell infection by Porcine Rotavirus (PRV), and whether PRV infection was associated with lipid rafts. The pharmacological agents of methyl-beta-cyclodextrin (MBCD) was used to deplete cholesterol in MA104cells and then IFA or Real Time PCR or Western Blot was performed to assay multiplication of PRV. The results showed that cellular membrane cholesterol depletion by the agent significantly inhibited infectivity of PRV strains DN30209. Moreover, the inhibition could be retrieved by addition of exogenous cholesterol. These studies suggest that cholesterol is critical for PRV infection, which is likely to be mediated by a lipid-raft-dependent pathway. |
