The Research On Newcastle Disease-Virus Like Particles

As a efficient investigation method, Virus-Like Particles (VLPs) technique has play an important role in the study of character, polymorphism, vaccine of many viruses. So the application of VLPs technique will help to discover the budding mechanism, develop novel, efficient and safe vaccine of NDV.We constructed the mammalian cells expressing vector pGFP- -M, pGFP- -HN and pGFP- -F based on the structure gene of F48E9 strain of NDV,which is correlative with the formation of ND-VLPs.Then the recombinant plasmid pGFP- -M, pGFP- -HN, pGFP--F and pGFP--NP were co- transfected into BHK21 cells , some particles like NDV can be observed by electronic microscopy in collected 72h-post co-transfected cells and culture media,but we couldn't draw a conclusion that it's just ND-VLPs. To increase the efficiency of co-transfection, pGFP--IVK pGFP- -HN and pGFP'-F were transfected into BHK21 cells respectively, and screening with G-418 selective media, the result show that we have ot the stable cell line harbouring the recombinant plasmid pGFP--M, pGFP--HN, pGFP"-F and pGFP- -NP respectively.But expression was not proved in transfected cells and culture media analyzed by SDS-PAGE,indirected mmunity fluorescence and RT-PCR. The cloning and selection are undergoing now.To investigate the possibility of construction of NDV-like particle in insect cell or out of cell, we sub-cloned successfully the M, F and HN gene of NDV into recombinant baculovirus transposition vector pFASTBACl, and transformed them into DH10BAC cells, by scanning plaque of white and blue and identification of PCR, the recombinant baculovirus shuttle vector Bacmid-M, Bacmid-F and Bacmid-HN were obtained. The recombinant baculovirus shuttle vector then were transfected into Sf9 insect cells respectively and the recombinant baculovirus rBacmid-M, rBacmid-F and rBacmid-HN were got. SDS-PAGE analysis showed that M and F gene have been expressed in Sf9 cells infected with recombinant baculoviruses.Two kinds of expression system have been adopted to study the budding and assembly of ND-VLPs in this research. One is the mammalian cells expression system ,the proteins M, NP, F and HN expressed in mammalian cells can be used to study the detail interaction mechanism of1 NDV in cells; another is baculoviruses expression system with advantange of forgein protein expression in large quantity, so we can purify the expressed proteins and assembly ND-VLPs in vitro.