Establishment Of Stable Expression SLAM/V Cell Line By Lentivirus-mediated

Peste des petits ruminants(PPR),also known as goat plague and stomatitis-pneumonia syndrome,is an important infectious disease for domesticated and wild small ruminants and its pathogen is PPR virus(PPRV).At present,the disease is still spreading in the world,resulting in significant economic losses.Signaling lymphocyte activation molecule(SLAM)is a major natural receptor of the host for PPRV,The hemagglutinin(H)of the virus binds to the host SLAM receptor and allows the virus to adsorb and invade the host cell.The expression of Signaling lymphocyte activation molecule(SLAM)protein into cells can increase the sensitivity of cells to PPRV.It is reported that the binding of V domain,at N-terminal of SLAM receptor,with H protein start measles virus infection,and PPRV and measles virus belong to Morbolivirus.In this study,we used the lentivirus expression system to establish a Vero cell line stably expressing goat SLAM/V to improvethe the sensitivity of cell to PPRV for the separation of the wild virus of PPRV.The research mainly includes the following five parts:1.SLAM/V gene was amplified by RT-PCR from the RNA of peripheral blood lymphocytes of goat,then was ligated into p GEX-6P-1 and SLAM/V protein and GST tag fusion protein was successfully expressed.The molecular weight of the fusion protein is about 39 k Da,mainly included in inclusion.It was proved by Western-blot that the purified fusion protein had good reactivity with goat anti-human SLAM polyclonal antibody.2.The expression vector p EGFP/SLAM/V was constructed and transfected into Vero cells and the cells were fluoresced at 24 h,36 h and 48 h after transfection with Vero cells.The results showed that the SLAM/V gene could be transfected into Vero cells and expressed.The expression of the target gene was the highest at 36 h after transfection.The expression of SLAM/V protein in Vero cells was confirmed by Western-blot.3.The expression vector p DEST/SLAM/V was constructed by homologousrecombination method.Witch was transfected 293 FT cells with p LP1 vector,p LP2 vector and p LP/VSVG vector.Harvest the lentivirus witch was packaged SLAM/V gene and without replication ability.Then Vero cells were infected by the lentivirus.The transgenic cells were screened by 2.5 μg/m L brucellin S.The transgenic positive cells were screened by the ratio of dilution witch were identified by PCR and Western-blot using β-actin as a control.The two cells were identical in activity,and the subcloned cells expressed SLAM/V protein.The transfected subclones were identified by indirect immunofluorescence.Vero cells were not fluorescent and SLAM/V protein was integrated into subcloned Vero/SLAM/V cells.4.The cell growth curves of VS cell line and Vero cell line were produced respectively.Vero cells grew slightly faster than Vero/SLAM/V cells,but they could reach to the same order of magnitude.There was no significant difference on cellular morphology between the two kinds of cell lines,except that the long axis of Vero/SLAM/V cell is slightly shorter than Vero cell.The PCR target band of corresponding size can still be amplified After 50 generations of passage,which demonstrates that the SLAM/V gene was integrated into the genome of Vero/SLAM/V cell line and can be stable inherited.5.Vero/SLAM/V cell and Vero cell were inoculated with PPRV vaccine Nig/75/1venom,syncytial cell lesions can be observed on the second day.Vero/SLAM/V cell line appear larger and more Syncytium after PPRV infection with an obvious characteristic lesion;after 3 generation of virus transmission,collection time of SLAM/V cells toxicity was shortened.Real-time RT-PCR method was employed to analyze the copy number of PPRV in different sample,Wherein the relative copy number of the viral PPRV in the Vero/SLAM/V cell can increase by about 100 s,and it indicated that Vero/SLAM/V cells significantly enhanced the replication of small ruminants virus.In this study,SLA/V gene transfected positive subcloned Vero cell lines were successfully constructed.