Establishment Of Vero Cell Line Stable Expressing Goat BST-2 Protein And Its Effect On PPRV Proliferation

PPR is a viral infectious disease characterized by fever,stomatitis,diarrhea and pneumonia,which is caused by Peste des Petits Ruminants Virus.Peste des Petits Ruminants Virus belongs to genus Morbillivirus,family Paramyxoviridae.PPR mainly infects goats and sheep.The morbidity and mortality rate of PPR is up to 90%,so the Ministry of agriculture of China has listed it as a class one animal epidemic disease.PPRV is also a kind of RNA virus with membrane.Its replication and budding mechanism are not fully explored.BST-2 is an natural immune limiting factor that is induced by interferon in the cell and it is expressed in mammalian mature B cells,T cells,monocytes,macrophages,and dendritic cells.In order to study its mechanism in PPRV replication and budding mechanism,we carried out the research.Firstly,a pair of specific primers were designed according to the gene sequence of goat BST-2?GenBank Accession No : XM₀18051380.1?in GenBank.The target gene was amplified by PCR using pUC-gBST-2 plasmid as template.Then the target gene was cloned into the plasmid pLOV-GFP-3×Flag.The recombinant plasmid pLOV-gBST-2,outer membrane plasmid pMD2.G and packaging plasmid psPAX2 were cotransfected into 293 T cells by calcium transfection,and a recombinant lentivirus containing gBST-2 gene was obtained.The recombinant lentivirus containing gBST-2 gene were used to infect Vero cells and the cell line was screened through puromycin screening.The cell line was named Vero-gBST-2.The fifth,tenth,fifteenth generation of Vero-gBST-2 cells were identified by Western blot,PCR and RT-PCR.The results showed that Vero-gBST-2 cell lines could express gBST-2 protein stably and the cell line was continuously subcultured to the 15 generation.Secondly,to explore the effect of this gBST-2 protein on the proliferation of PPRV in Vero cells.The one-step growth curve of PPRV was drew and compared with that in normal Vero cells,the results showed that the proliferation of PPRV was clearly inhibited in Vero-gBST-2 cells.Subsequently,the expression of N protein was detected with Western blot.The results showed that the production of virus particles has been significantly decreased due to the presentation of gBST-2 protein.The results of laser con-focal microscopy detecting results showed that gBST-2 protein mainly located on the cell membrane of the Vero cells where many PPRV particles exist,indicating that the decrease in the amount of PPRV in infected cells is due to the restriction of the gBST-2 protein.In conclusion word,we have constructed a cell line stable expressing gBST-2 protein,with the help of Vero-gBST-2 cell line,we proved that BST-2gene could inhibit the proliferation of PPRV and limit its budding process.The results of these would provide a good platform for further exploring the effect of goat BST-2 on the restriction of PPRV.