| Rabies is an important zoonosis infectious disease caused by rabies virus(RABV) which can infect all warm-blooded animal and cause a fetal encephalomyelitis. Rabies seriously damage the central nervous system,the fatality rate is as high as 100%. According to the WTO Report,there are nearly 60,000 deaths each year in the world, most of them occurred in the developing world, especially in Asia and Africa. Our country is a high incidence of rabies region, annually thousands people die from rabies in recent years and the number of deaths still have a rising trend. The morbility and mortality of rabies are second only to India in the world. Therefore, the prevention and treatment of rabies is imminent and tough.RABV is an enveloped, nonsegmented, negative-stranded RNA virus, belongs to Lyssavirus genus of the family Rhabdoviridae, mainly encode five structural proteins including nucleoprotein(N), phosphoprotein(P), matrix protein(M), glycoprotein(G) and polymerase(L)proteins. RABVs are bullet shaped virus particles, the appparent lipid bilayer membranes surface contain G protein spikes, which mainly involved in mediating cell membrane fusion,receptors combination and inducing host to produce neutralizing antibody, furthermore, it also associated with the virulence and pathogenicity of the virus. M locates inside of the membrane,connected nuclear capsid to enveloped, mainly involved in the progeny virus assembly, budding and the formation of the virus. Its internal encapsulated N, P, L protein and virus genome RNA form neucleocapsid, namely RNP,is the temple of RABV transcription and replication. N is mainly associated with the transcription and replication of RABV, closely combined to genome RNA form N-RNA compounds in the process of virus replication, protect virus nucleic acid from hydrolyzed, made the nuclear capsid in the helical symmetry structure of transcription required. The interferon antagonist P protein plays an important role in the successful replication of virus in host cells. L protein is of great importance to the catalytic effect in the transcription and replication process of RABV.RABV particles released from infected cells could carry many host proteins,some of which may play an important role in viral replication. it may due to virus and host cell membrane fusion and carried proteins to the surface of enveloped, it also may due to interact with N, P, L and carried proteins to the inside of enveloped. So the identification of host proteins associated with virus proteins is of great significance to understand the interaction between RABV and host and the molecular mechanism of viral replication from the molecular level, and providesstrong theoretical basis to develop reasonable targeted treatment reagent of rabies.In the present study, after virus culture and purification by sucrose density gradient ultra centrifugation, a proteomic approach was used to analyze the protein composition of purified RABV particles(attenuated vaccine strains of SRV9). Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking(14%),molecular chaperone(12%), cytoskeleton(24%), signal transduction(8%), transcription regulatory(12%), calcium ion binding(6%), enzyme binding(6%), metabolic process(2%),ubiquitin(2%) and other(14%). Of these,four proteins(β-Actin, β-Tubulin, Cofilin and Hsc70)were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism RABV replication.Then we abstained the span of N and P gene from the RABV CVS-11 strains, cloned them to p Zero Back/blunt vector, then took P gene sub-clone to the prokaryotic expression vector of p ET-32 a. Construction of prokaryotic expression recombination plasmid was converted to a prokaryotic expression strain BL21(DE3), by IPTG induction, there is a 54 k D recombination P protein mainly expressed in the form of inclusion body by SDS-PAGE electrophoresis analysis.Under the condition of degeneration, using Ni-NTA affinity chromatography to purify high purity of the recombination P protein. After immunization of mice, we obtained the anti-P polyclonal antibody, which titer can reach 1:32000 by the detection of indirect ELISA, and verified good specificity by IFA and Western Blot, and can identify the RABV CVS-11 strains P protein expression in the BHK cells. The polyclonal antibody can be used to screen the stable cell lines of expression P protein. Our study lay the foundation to further study of P protein related function.We also took N and P gene clone to eukaryotic expression vector p NTAP-B of TAP system,which can expression of N protein, P protein with amino terminal SBP-CBP tandem affinity tag.The recombination plasmid was transfected into BHK-21 cells and used G418 to screen the stable cell lines of expression target protein. By RT-PCR, IFA and Western Blot to verify the stability cell lines, which expanded respectively culture and harvest cell extracts, purify the protein complexes through two steps of affinity purification, and screening host proteins interaction with N and P protein, the elution to SDS-PAGE electrophoresis and siver stain, mass spectrum analysis, after removed the control background, we respectively got 41, 21 host proteins interaction with N and P protein.Screening of stable cell lines to test the target protein localization in cells by IFA, we found that N protein mainly located in the nucleus of cells, while P protein mainly located in thecytoplasm of cells, and the results were verified by extraction proteins in cytoplasm and nucleus of the stable cell lines by Western Blot analysis. Moreover, IFA were conducted to test the stable cell lines of target protein expression whether can affect RABV proliferation. We found that the expression of heterologous N protein and P protein can suppress RABV proliferation, and the inhibition effect of SRV9 is stronger than CVS-11.Based on the above studies, we analysised the proteomic of RABV particles, detected 50 host proteins, and TAP method combined mass spectrum analysis to screen, respectively 41 and 21 host proteins interaction with N and P protein. Compared with these proteins, 9 and 6 host proteins exist in the RABV particles,respectively. We also found that N protein mainly located in the nucleus of cells, while P protein mainly located in the cytoplasm of cells, meanwhil, N and P proteins expressed in stable cell line can suppress RABV proliferation, and suppress SRV9 proliferation strength is greater than CVS-11.The study identified proteomic of RABV particles and host proteins interaction with RABV N and P protein, laid a foundation to the further study of RABV replication and its infection mechanism. |
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