Rabies virus (RV) belongs to a member of the genus Lyssavirus of the Rhabdoviridae family. RV is highly neurotropic and causes severe progressive encephalitis. There are no effective treatment methods currently, almost100%of death upon onset of clinical. Rabies virus encoded five proteins, of which G protein plays an important role in determining viral host range, neurotropic virulence, immunogenicity and interaction with the receptors molecules on the surface of host cells. Viral receptors acts as a gateway and can specific combine with virus, mediates virus invasion of susceptible host cells and startes its replication process, is the portal which virus invade the target cell. The nicotinic acetylcholine receptors which located in the postsynaptic membrane and the neural cell adhesion molecules which located in the presynaptic membrane play an impotant mediateing role when RV invade neuromuscular junction. The nerve nutriment receptor P75as a receptor of RV, can binding with the G protein of RV and then retrogradely transported in the cytoplasm.In this study, we extraction of total RNA from NA cells which infection with Flury after one hour, the NCAM, P75and nAchR gene were cloned from these total RNA and then subcloned into the multiple cloning sites of prokaryotic expression vector pET-32a(+). The recombinant expression plasmid pET-NCAM-711, pET-P75-513and pET-nAchR-525were successfully constructed, the recombinant expression plasmids were recepctively transformed into E.coli BL-21for inducting expression by IPTG. SDS-PAGE analysis found that recombinant NCAM-711, nAchR-525and P75-513protein respectively expressed52,46and45KD fusion protein, and confirmed that NCAM-711and nAchR-525mainly erpressed as a form of inclusion body, however, the P75-513product is confirmed as a form of solubility. Respectively purification recombinant protein via Ni-NTA resin, and then the purified NCAM-711, nAchR-525and P75-513protein were used as antigens to immunize mice, finally, the polyclonal antibodies against the three proteins were obtained.Due to the quantity of three receptors on the cell surface expression is very low on the natural conditions, it’s hard to reach detetion standards because of the reactivity is very weak by the conventional method of indirect immunofluorescence (IFA) and western blotting (WB), so we constructed the eukaryotic expression plasmid NCAM-1818-3.0, P75-1284-myc and nAch.R-1509-myc of the three receptors respectively. Transfection BHK-21cells and obtain the cells of overexpresssion NCAM, P75and nAchR protein.Using the normal NA cells and the BHK-21cells or the cells protein which transfection with the eukaryotic expression plasmid after24hours as samples, using indirect immunofluorescence and western blot method to detect the reactivity between receptors of cells surface and the polyclonal antibody preparation. IFA result showed that NCAM, P75and nAchR polyclonal antibody can binding with the receptors and show specific fluorescence. Western blot result showed that polyclonal antibody of NCAM and P75can reaction with the NCAM and P75protein of transfection, While nAchR polyclonal antibody can binding with the nAchR protein of NA cell or BHK-21cell of transfection, and we can see the specific fluorescence via IFA, but we can’t detectable nAchR protein on the surface of cells by western blot.Application the three polyclonal antibodies detetion the expression changes of the three receptors protein after infection RC-HL24hours in the NA, BHK-21and Raw264.7by flow cytometry. The result showed that the expression of threee receptors protein up-regulation about2times in the NA and BHK-21cells, while the expression raised even more significant, up-regulation8,9and6.3times respectively in the Raw264.7cells.On this basis, we detetion the expression changes of the three receptors mRNA after infection RC-HL24hours in the NA, BHK-21and Raw264.7by real-time fluorescent quantitative PCR. The result showed that the mRNA of NCAM, P75and nAchR have varying degrees of increase after infection RC-HL24hours by real-time fluorescent quantitative PCR. The expression of mRNA of NCAM, P75and nAchR up-regulation79.3,28.2and8.7times in NA cells;3.5,1.9and1.8times in BHK-21cells and10.97ã€9.1and3.5times in Raw264.7cells respectively, relative to before infection.We successfully obtained the three polyclonal antibodies via amplification the genes of three receptors and construct prokaryotic expression vector and then expression and purification protein, the three polyclonal antibodies can reaction with the receptors protein on the cells surface, the expression of the three receptors were up-regulation after infection RV which treated with the polyclonal antibodies, and the mRNA of receptors also up-regulation after infection RV, and consistent with the results of protein levels. These results suggested that the three receptors play an important role during the infection process. We hope to clarify the mechanism of virus invasion cells point of view from receptors, and provide a certain amount of research basis and reference datas. |