Expression, Purification And Characterization Of RV Matrix Protein In Baculovirus-infected Insect Cells

Rabies is a kind of zoonotic diseases caused by rabies virus, which could induce hydrophobia, photophobia, depression, progressive paralysis and eventually death in human and most warmed-blood animals. The genome of RV encodes five structure protein: nucleoprotein(N), phosphoprotein(P), matrix protein(M), glycoprotein(G)and large protein(L) in that order from the 3′ to 5′ end of the genome. Among those viral proteins, the G protein is known to determine the pathogenicity of RV. Many studies have shown that RV pathogenicity in adult mice is altered by amino acid substitutions in the G protein. Those substitutions affect biological properties of virus,such as cell-to-cell spread, membrane fusion, and apoptosis-inducing ability. While the mechanism by which the G protein determines viral pathogenicity is becoming increasingly clear, little is known about the contribution of viral proteins other than the G protein to pathogenicity. However,In 2006, Shimizu K et al used the reverse genetic technology to replace the M gene of RV strain Ni-CE, which has been established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells,causes nonlethal infection in adult mice, with the M gene of RV strain Nishigahara that kills adult mice after intracerebral inoculation.Then, they proved that the chimeric CE(NiM) strain, which has Nishigahara M gene in the genetic background of Ni-CE strain, could kill adult mice after intracerebral inoculation. This research opened a new chapter in depth study of pathogenicity of RV. Thus, Further investigated the effects of M protein on RV pathogenicity in multiple perspectives has a dramatic signification to clarify the pathogenic mechanisms of RV and control the epidemic of RV.In this study, in order to explore the effects of M protein alone on RV pathogenicity, we carried out the following test:Construction and identification of recombinant baculovirus expressing M protein of RV. Test content: a. Amplified the M gene of RV strain BD06 by RT-PCR; b. M gene sequences were inserted into a baculovirus vector plasmid pFast Bac I andconstructed the recombinant plasmid pFast Bac I-M; c. The corrected recombinant plasmid pFastBac I-M were transformed into DH10 Bac E.coli competents to construct recombinant shuttle plasmid Bacmid-M, which were identified by PCR; d.For obtaining the recombinant baculovirus AcMNPV-M, the recombinant shuttle plasmid Bacmid-M were transfected into Sf 9 cells which were mediated by lipofectamine 2000. then, Using electron microscope observed the cytopathic supernatant and identified the shape of the recombinant baculovirus; e. Identification of recombinant baculovirus expression by Western Blot assay with mice monoclonal antibody anti-His tag, rabbit anti-RV positive serum and canine anti-RV positive serum.Purified the recombinant M protein and preparation of its polyclonal antibody.Experiment content: a. Using a nickel iron affinity chromatography purified the recombinant M protein; b. Purification of recombinant M protein were immunized rabbit, one week after each immunization, collecting blood from ear vein and separating blood serum; c. Analysed the cross-reactivity of the rabbit anti-M polyclonal antibody with M protein from RV strain aG, PV2061, ERA, SRV 9,CVS-24 and BD06 by Western Blot; d. The rabbit anti-M polyclonal antibody were tested by FAVN assay.Preliminary study on biological characteristic of M protein. In order to explore the effect of M protein on latency period, the incidence and mortality in mice after intracerebral inoculation or intramuscular inoculation with RV strain CVS-24 and BD06, respectively. The specific test procedure is as follows: a. The experimental mice were randomly divided into intracerebral inoculation group and intramuscular inoculation group, the two big groups were set up high-dose and low-dose poison attack groups, respectively; b. Purification of the recombinant M protein mixed with an equal volume oil adjuvant which immunized the four groups of mice at 0d, 7d, 14 d,by intraperitoneal inoculation, respectively. Meanwhile, setting the same amount of dialysate solution group and Ac MNPV group as control; c. Collecting blood from the tails and separating serum on 5 day after the third immunization, the immunogenicity of recombinant M protein were analysed by Western Blot assay; d. One week after the third immunization, the two groups of mice were intracerebral inoculation and intramuscular inoculation with RV strain CVS-24 and BD06, respectively; after challenge, the mice were continuously observed in 14 days, recording the latencyperiod, the incidence and the number of deaths.Through the above tests, the following results were obtained:1. The recombinant baculovirus Ac MNPV-M were successfully obtained and it have a typically form of wide baculovirus observed through electron microscope. The Sf 9 cells which were infected by the recombinant baculovirus AcMNPV-M could express an approximately 25 kD protein that were identified by Western Blot assays with mice monoclonal antibody anti-His tag, rabbit anti-RV positive serum and canine anti-RV positive serum.2. Purified recombinant M protein under the denatured conditions had a single band in SDS-PAGE. The rabbit anti-M polyclonal antibody had an good ability to cross-react with the M protein of rabies vaccine strain and pandemic strain, an single band about 25 kD were appeared in the identification of Western Blot assay,respectively. Otherwise, the FAVN test results showed that anti-M rabbit polyclonal antibody has no rabies virus neutralizing capacity.3. The mice were challenged with RV assay results showed that: whether intracerebral inoculation with 3 LD50 or 50 LD50 RV strain CVS-24, the latency period,the incidence and the mortality of mice in different groups had no signification difference. After intramuscular inoculation 100 LD50 RV strain BD06 in hind legs, all the mice in AcMNPV-M group were dead in 11 days, while the mortality rate of mice in AcMNPV group and dialysis solution group both were 80%. However,after intramuscular inoculation 3 LD50 RV strain BD06 in hind legs, all mice in three groups were survived.Through the above findings,the following conclusion were obtained:1. The recombinant baculovirus AcMNPV-M were successfully obtained and the M protein that expressed by recombinant baculovirus Ac MNPV-M infected Sf 9insect cells had a good reactgenicity, which laid the material foundation for further study on the other biological properties of M protein.2. The recombinant M protein could be successfully purified by nickel iron affinity chromatography under the denatured conditions and it had a good immunogenicity. The prepared rabbit anti-M polyclonal antibody have a good reactogenicity. M protein have no ability to induce virus-neutralizing antibody in body.3. The effect of M protein separate from RV on latency period and the incidenceof mice which were challenged with high-dose or low-dose CVS-24 by intracerebral inoculation has no signification difference. No signification effect on the latency of mice which were challenged with different dose BD06 by intramuscular inoculation,but the M protein could be trend to enhance the incidence of mice after the latency period. The results prompted that the individual immunization with M protein in mice may effect the rabies virus infection and pathogenicity.Innovation of thesis:1. Using the baculovirus expression system successfully expressed the M protein of RV strain BD06, established a method of purification and prepared rabbit anti-M polyclonal antibody, which provided a material foundation for the further study on the biological characteristics of M protein.2. This study explored the effect of M protein on RV pathogenicity and provided some basic data for further study on the pathogenic mechanisms of RV.