Cloning Of Porcine GBP1and GBP2Genes And Investigation Of Its Anti-Viral Effect On PRRSV And PRV Propagation

Interferon-inducible guanylate binding proteins(GBPs), also named p65GTPase, blongs to interferon-inducible GTPase super family. It is an important interferon-inducible genes(ISGs). Since the family of interferon-inducible guanylate binding proteins discovered in1979, it was found that the family have an important effect on cell growth and anti-pathogens. At present, reseacrhes on the family maily concentrate on human and mouse. Even though the family are testified to be able to inhibit virus replication, but the specific reasons are abscure. At moment, there are few researches on porcine GBPs’biological functions and the reports on porcine GBPs only focus on inhibiting Salmonella typhimurium replication. In addition, there are no reports about whether porcine GBPs can inhibit virus replication or not. PRRSV and PRV causing huge losses to the pig industry and the diseases caused by PRRSV and PRV are difficult to be prevented and treated. This reseacrh can provied a new method for treatting the disease caused by PRRSV and PRV. The research studies the PRRSV and PRV which have different type of nucleic acid to make sure whether the family of porcine interferon-inducible guanylate binding proteins can inhibit the PRRSV and PRV or not.The main contents as fowllow:1. The Cloning and sequence analysis of the porcine GBP1and GBP2genesThe RNA of PK-15cells were used as the template and the porcine GBP1and GBP2gene sequence were amplified via RT-PCR. The results of sequencing analysis demonstrated that sequences of porcine GBP1and GBP2gene family were respectively1773bp and1776bp, and their cDNA encoded respectively590aa and591aa residues. GBP1and GBP2had a nucleotide homology of77.31%, an amino acid homology of69.20%. Phylogenetic tree analysis exhibited that there were differences among the amino acid sequence of different species; porcine GBP1and GBP2had the closest relationship with human. It indicates that porcine GBPs family were highly conserved in evolution.2. The protein structure prediction of porcine GBP1and GBP2The results of protein structure prediction revealed that porcine GBP1and GBP2have the typical structures of the family of interferon-inducible guanylate binding proteins,which consist of the N-terminal combine GTP globular structure domain, the C-terminal alpha helix and connection among regions.3. The eukaryotic expression and subcellular localization of porcine GBP1and GBP2The eukaryotic expression plasmid of pCAGGS-HA-GBP1and pCAGGS-HA-GBP2were transfected into PK-15cells and Marc-145cells. Via Western Blot assay, it was found that porcine GBP1and GBP2protein could be expressed in PK-15cells and Marc-145cells correctly. By Indirect Immunofluorescent Assay(IFA), it was discovered that porcine GBP1and GBP2were most located in the cytoplasm.4. The influence of porcine GBP1and GBP2on PRRSV and PRRSV propagation The eukaryotic expression plasmid of pCAGGS-HA-GBP1and pCAGGS-HA-GBP2was transfected into PAM cells and Marc-145cells which were then infected with PRRSV(MOI=0.5). By relative quantitative PCR and Virus plaques assay, we detected that porcine GBP1and GBP2can inhibit the propagation of PRRSV. Different doses of the pCAGGS-HA-GBPland pCAGGS-HA-GBP2transfect Marc-145cells, then infected with PRRSV(MOI=0.5).By Virus plaques assay, we detected that porcine GBP1and GBP2can inhibit the propagation of PRRSV and it has dose dependent.The eukaryotic expression plasmid of pCAGGS-HA-GBP1and pCAGGS-HA-GBP2was transfected into PK-15cells which were then infected with PRV(MOI=1). By relative quantitative PCR and Virus plaques assay, we detected that porcine GBP1and GBP2can inhibit the propagation of PRV. Different doses of the pCAGGS-HA-GBP1and pCAGGS-HA-GBP2transfect PK-15cells, then infected with PRV(MOI=1). By Virus plaques assay, we detected that porcine GBP1and GBP2can inhibit the propagation of PRV and it has dose dependent.5. The influence of PRRSV or PRV infection on the porcine GBP1and GBP2The eukaryotic expression plasmid of pCAGGS-HA-GBP1and pCAGGS-HA-GBP2were transfected into PK-15cells and Marc-145cells, and then the cells were inoculated with the PRRSV and PRV respectively. Through Indirect Immunofluorescent Assay(IFA), it was found that porcine GBP1and GBP2were also located in the cytoplasm. Therefore, the inoculation of the PRRSV and PRV could not change the subcellular localization of porcine GBP1and GBP2.The influence of PRRSV infection and PRV infection on the expression of the procine GBP1and GBP2gene was investigated via ralative quantitative PCR. It was discovered that the expression was activated significantly by PRV infection in PK-15cells, implying that the porcine GBP1and GBP2might have some defensive effects on PRV infection. However, it was noticed that the expression of porcine GBP1and GBP2wasn’t activated significantly by PRRSV infection in PAM cells.