ABSTRACT
To understand the genetic origin and variation of porcine epidemic diarrhea virus (PEDV) prevailing in Henan province,a total of 22 PEDV positive samples from suckling piglets with severe diarrhea were collected from 16 pig farms and amplified by RT-PCR.ORF3 genes were then cloned into pMD18-T vector and sequenced.The result of sequencing showed that the length of ORF3 genes all were 675 bp which encoded 224 amino acids.The strains in this study had eight amino acid substitutions when compared with CV777 strain.The nucleotide homologies of the 22 strains were 95.9％-100％ and amino acid homologies were 96.4％-100％.By comparing with those of European strain CV777 and vaccine strain truncated CV777,the nucleotide homologies were 97.1％-97.9％ and 94.8％-95.4％,respectively.Based on the phylogenetic relationship of ORF3 genes,the PEDV field strains and PEDV reference strains could be divided into two groups,and all the field strains identified in this study belong to group 2.It suggested that prevalent PEDV strains in this study seem to be closely related to Henan isolates in previous years,domestic strains,Janpanese strains,American strains as well as Korean strains but differ genetically from European strains or vaccine strains used in China.This experiment analyzed the ORF3 gene sequence features in prevailing of Henan region in recent years,which provides a new support of epidemiology study and protein function research of PEDV ORF3 gene.
ABSTRACT
Objective:To study immunosuppression mediated by the porcine FcγRⅢ in porcine reproductive and respiratory syndrome virus ( PRRSV ) infection to pulmonary alveolar macrophages ( PAMS ).Methods: In this study pulmonary alveolar macrophages cells were treated with containing 200 TCID50 PRRSV,lipopolysaccharide (LPS) (100 ng/ml) and purified mouse anti-pig FcγRⅢIgG (550 μg/ml) separately,simultaneously,PAM cells treated with purified mouse anti-pig FcγRⅢIgG (550 μg/ml) was infected by 200 TCID50 PRRSV ,untreated PAM cells as the control group.Each group were post-cultured 12,24,36,48,60,72 h, the cells and the supernatant were collected.The dynamic variation of PRRSV RNA copies in inoculation group were detected by using real-time fluorescence quantitative PCR method.mRNA level of IFN-αand TNF-αin each group were detected by using relative fluorescence quantitative PCR.Results:The result showed that mRNA level of IFN-αwas improved during PRRSV infection to PAMS 12-24 h,and mRNA level of IFN-αwas inhibited during 36-72 h,then mRNA level of IFN-αrecovered normally; mRNA level of TNF-αwas increased slightly post-infection 12-72 h.IFN-αand TNF-αmRNA levels of PAM cells treated with LPS were both up-regu-lated,using the purified mouse anti-pig FcγRⅢ IgG to treat the PAM cells,selective activation of porcine FcγRⅢ in the PAM cells down-regulated significantly mRNA levels of IFN-αand TNF-α.PRRSV infection assay mediated by selective activation FcγRⅢof the PAM cells inhibited antiviral cytokine ( IFN-αand TNF-α) mRNA levels.Conclusion:The results show selective activation of FcγRⅢinhibited significantly mRNA levels of the antiviral cytokine IFN-αand TNF-αof host cells,and innate antiviral immune response to PRRSV infection.
ABSTRACT
Duck IL-18 gene was amplified from plasmid pGEM-DuIL-18 by PCR. The PCR product digested with Pst I and Xho I was inserted into eukaryotic express vector pcDNA3.1(+) to generate an recombinant expression plasmid pcDNA3.1/DuIL-18 (pDuIL-18), and transformed into Escherichia coli JM109. The recombinant colonies were identified by restriction enzyme digestion, PCR and sequencing. DNA sequence confirmed the correct sequence of the recombinant eukaryotic expression plasmid pDuIL-18 in the reading frame and the ligation part. After the transfection of pDuIL-18 into Cos7 cells, duck IL-18 mRNA was expressed in Cos7 cell. The SDS-PAGE analysis showed that the expressed duck IL-18 protein had molecular weight of 23 000 D. The results of methyl thiazolyl tetrazolium (MTT) assay showed that duck IL-18 protein expressed in Cos7 cell could induce significantly transformation of duck T lymphocytes. Immunoenhancement effect of recombinant expression plasmid pDuIL18 on avian influenza vaccine was observed by proliferation response of the T lymphocytes from spleen. It can obviously enhance the cell-mediated immune response.