Research Of PPRV Full-length Gene Cloning And The Interactions Between H Protein And SLAM Receptor

Peste des petits ruminants(PPR)is an acute infectious disease caused by Peste des petits ruminants virus which belongs to paramyxovirus(Paramyxoviridae),measles virus genera(Morbillivirus).It is characterized by fever,diarrhea,stomatitis,and pneumonia,with high morbidity and mortality.The outbreak of PPRV in China appeared in Tibet region in 2007.Within the following two years,the outbreak spread around China,including Xinjiang,Inner Mongolia autonomous region,Liaoning,Shandong,Hunan,Anhui,Guangxi,Yunnan,and Guizhou.Therefore,prevention and control of PPR is under a severe condition in China.It is imperative for us to develop effective products for the disease prevention and control.To date,however,the mechanism underling the pathogenicity of PPRV is unclear.Thus,it is critical to establish the PPRV reverse genetics platform for PPRV infectious clone related research works and the development of a new carrier vaccine.In order to construct PPRV infectious clone,two universal reverse genetic carriers were conducted for negative chain segment RNA virus by reconstruction of pVAX1 and pBluescript ? II KS(+)vector,named them pBKS-HDV and pVAX1-HDV vector.In addition,the most commonly T7 RNA polymerase promoter and virus genome cDNA were inserted into the pBKS-HDV to conduct a pBKS-PPRV N75/1 vector,as well as pVAX1-PPRV N75/1 which was conducted by inserting CMV promoter and the virus genome cDNA into the PVAX1-HDV vector.A serial of pHelper plasmid were also constructed based on PPRV cDNA,such as PCI-N,PCI-P,and PCI-L.In order to get the full-length cDNA gene,a serial of micro genetic plasmids were constructed using eGFP report gene.They are pKS-LGT,pKS-HAM-TGL,PTVT-LGT,and PTVT-HAM-TGL.Virus rescue condition was optimized by mixing the plasmids and the pHelper,in which way to indentify the validity of the pHelper plasmid.The rate of virus rescue was highly increased.Additionally,a receptor cell lines,stable expression of goat SLAM,was established laid a foundation for reverse genetic rescue of PPRV via RNA polymerase.To understand the effects of H protein on virus pathogenicity,a cartoon of SLAM receptors of people,dogs and goats interaction with H protein from PPRV N75/1 clone strain,PPRV N75/1 standard strain and Tibet 07 strain was draw via bioinformatics methods.The interaction sites between loci and the differences of interaction sites were analyzed according to the Cartoon.To confirm the accuracy of the results,plasmids with different H protein extracellular regions were constructed,the truncated extracellular region coming from the PPRV N75/1 strain and Tibet 07 strain,as well as the extracellular region of goat SLAM receptor.It was confirmed that the H protein of PPRV N75/1 strain and Tibet 07 strain can interact with goats SLAM receptors by Co-IP experiment.To further understand the underlying mechanism of interaction between the receptors and the virus,precisely localize the site of H protein interaction with goat SLAM receptor should be performed via PPRV infectious clone.