Effect Of PPRV H Protein On PPRV Multiplication

Peste des petits ruminants(PPR) is a severe, highly contagious, Office Internationale des Epizooties(OIE) notifiable disease of sheep and goats. PPR was first reported in China in July, 2007 and is currently endemic in most of China since December, 2013, and place a huge disease burden on agriculture. Therefore, to strengthen the basic and applied research of Peste des petits ruminants virus(PPRV) is imperative. The H gene of PPRV was expressed transiently in CHS-20 cells and the effect on PPRV multiplication was studied in order to provide certain basis for improving the vaccine production.A fragment of 788 bp of PPRV N gene was amplified and cloned into pMD 19-T cloning vector. The real time PCR assay was performed using SYBR premix Ex Taq. The standard curve was plotted and the specificity, sensitivity and reproducibility of the developed assay were assessed. The generated standard curve showed linearity over the entire range of quantification and the developed assay showed good specificity and sensitivity. The lower detection limit, based on plasmid copy number, achieved was 2.82 copies/μL and was 1000 times more sensitive than conventional PCR assay.The whole coding sequence of H gene of PPRV was amplified using RT-PCR and cloned into eukaryotic expression vector pEGFP-C3. Then pEGFP C3-PPRV H plasmid was used to transfect the CHS-20 cells. RT-PCR, fluorescence observation and Western blot analysis were used to detect H gene expression, in mRNA and protein levels. The results showed that the H protein was successfully expressed in CHS-20 cells.The CHS-20 cells were transfected with the recombinant plasmid and then infected with PPRV Nigeria 75/1 after 48 h.After 72 h, the virus titre were analysed by the relative quantitative RT-PCR. Results showed that compared with the untransfected cells and the cells transfected with pEGFP-C3 plasmid,the titre of PPRV in cells transfected with pEGFP C3-PPRV H was higher.And the data has the significance of difference.The overexpression of PPRV H promoted the PPRV multiplication.