Eukaryotic Expression Of Matrix Protein Gene Of Rabies Virus And Intracellular Localization Of The Protein

Rabies is an important zoonosis between human and animals, which is caused by rabiesvirus and mainly hurts the central nervous system of the host. Once the clinical symptoms ofillness are appeared in the host, the death rate will be almost100%.Rabies virus belongs to the Rhabdoviridae, lyssavirus, and negative-strand RNA viruses.Rabies virus can transcript and translate five structural proteins, such as nucleoprotein（N）,phosphoprotein（P）, matrix protein（M）, glycoprotein （G）and transcription protein（L）.The matrix protein of rabies virus is the shortest and muti-functional one among the fivestructural proteins. The protein form the connection between RNP and glycopro tein instructure,and associate with the release of the rabies virus and viral virulence in Function. Inrecent years, there are much reports on matrix protein in the west countries, but there is littlereport in China.Adenovirus expression system is one of the eukaryotic expression system has beenwidely applied in recent years,as it can be right for heterologous protein expression andmodification,And the operation is simple, the use is security.Therefore it has been widelyused in the field of veterinary research.The purpose protein expression of recombinantadenovirus has been widely used for vaccine research pathogen detection technology researchand in areas such as viral structural protein function research.This study intends to construct non-replicative human adenovirus5serotype expressionof RV matrix protein.，Clear recombinant virus growth characteristics and express foreignproteins. locate the expressed matrix protein in the affected cells，And lay a foundation forfurther studies on rabies virus matrix protein.In this study, the matrix protein gene of BD06was cloned by RT-PCR using the BD06RNA as template, which was isolated from mice brain tissue infected by rabies virus.Subsequently, the matrix protein gene of BD06was inserted into the PMD18-T vector. Afterverified by sequencing, open reading frame of rabies virus MP gene of BD-06strain wascloned into the multiple cloning sites of shuttle vector of adenovirus expression system toconstruct the recombinant shuttle plasmid pacAd5-BD06-M. Then the linearizedpacAd5-BD06-M and linearized backbone plasmid pacAd59.2-100were cotransfected intoHEK-293AD cells mediated by FuGENE transfection reagent. After CPE appearance, the cellcultures were collected and identified by electronmicroscopy, RT-PCR, and western blot.Virus titer was measured in HEK-293AD cells. The results of restriction enzyme digesting and sequencing proved that recombinant plasmid pacAd5-BD06-M was constructedcorrectly; The recombinant adenovirus rAd5-BD06-M showed typical structurecharacteristics of wild-type adenovirus under electronic microscope. The specific band with alength of about600bp of matrix protein of rabies virus was observed by RT-PCR. Westernblot showed that rAd5-BD06-M and the polyclonal antibody of M protein of rabies virusBD-06strain were able to occur the specific reaction and produce a specific band. The resultsabove revealed that the matrix protein gene of BD06was expressed successfully in theEukaryotic cell HEK-293AD through recombinant adenovirus plasmid rAd5-BD06-Mcotransfection. The even titer of rAd5-BD06-M was about1×10^7.0CFU/mL, which was alittle lower than that of the wild type adenovirus. the Kunming mice （n=10） immunized withrecombinant adenovirus rAd5-BD06-M at106TCID50by way of Introperitoneal injection。The neutralization antibody titers of the sera respectively separated28d were determined byfluorescence antibody virus neutralization test. The results show the serum does not havevirus neutralizing activity.Indirect immunofluorescence analysis was carried out using by the Polyclonal antibodyagainst rabies virus matrix protein prepared by our laboratory. Using different strains ofrabies virus（including higher titer JX08-45CC, Attenuated rabies virus SRV9, and strongrabies virus BD06） to infect BHK-21cells for36hours, the results showed that the matrixprotein was mainly located in cytoplasm part and on the nuclear membrane of the BHK-21cells. But in the HEK-293AD cells only infected by recombinant adenovirus for24hours, thematrix protein was mainly located in the whole cells, including the Nucleoplasm.Above all, in this study, recombinant replicable-defective adenoviruses using for expressBD06matrix protein were successfully constructed and identified in term of morphologicallyand molecular biologically. When different strains of rabies virus were used to infect BHK-21cells, the matrix protein was mainly located in cytoplasm part and on the nuclear membraneof the BHK-21cells. However, in the HEK-293AD cells only infected by recombinantadenovirus, the matrix protein was mainly located in the whole cells, including theNucleoplasm. In the point of view, this research laid a foundation for further studies on rabiesvirus matrix protein.