Development Of Monoclonal Antibody Against The Structure Proteins Of Rabies Virus And Establishment Of Serodiagnostic Method

Rabies is an acute human and animal infectious disease caused by rabies virus (RV), Rabies mainlycaused progressive encephalomyelitis, which is almost100%of fatality in animals and human. China isa high incidence zone of rabies. With the increasing number of pet dogs and cats, the prevention andcontrol of rabies face a more serious situation, so it is of great significance for prevention and control ofrabies to strengthen monitoring the serum antibody levels of dogs and cats. In order to eliminate thepotential threats of animals which did not have enough antibody to protect, it is so urgent to establish akind of rapid detection methods used to deteced the serum antibody levels of rabies virus.To generate monoclonal antibodies against the structure proteins of RV, the purified RV was usedas antigens for immunization of BALB/c mice. The spleen cells of the BALB/c mice were fused withthe SP2/0cells. Six hybridoma cell lines named1G10、3F7、3F8、5C8、5A11and6B10respectively,which were screened and obtained by indirect ELISA with RV as an antigen. Then，it is found that the3F8is against the matrix protein of rabies virus by ELISA and Western Blot，the6B10is against theglycoprotein，and the other is against the nucleoprotein.The subtype of1G10is IgG2a,the3F7and3F8is IgG1, the5C8、5A11and6B10is IgG2b. The results of IFA indicated that6B10can recognize RVand3F8can not。According to the antigen epitope of G protein and the sequence of G gene from Genbank, threepairs of primers were designed for subcloning the G gene; the three subunits were amplified by PCR.Then the subunits were cloned into pET-32a (+) expression vector successfully. The subunit proteinswere induced by IPTG.The results by Western blot and SDS-PAGE showed that three of the subunits ofG protein expressed well. The results by Western Blot showed the6B10can react with the secondsubunit, between the212and317Amino acids.The M gene of rabies virus Flury LEP strain was amplified by PCR and cloned into prokaryoticexpression vector pGEX-6P-1. The resultant construct pGEX-RV-M plasmid was transformed intoBL21(DE3) and protein expression was analyzed by SDS-PAGE, The results showed that the M proteinwas efficiently expressed, which were mainly soluble, and purified with the affinity chromatography.The results of Western blot indicated that the recombinant protein M showed good immunogenicity. Theindirect ELISA method was established with the purified recombinant protein M, and a total of95serum samples were detected by the method and the indirect ELISA method based on the recombinantprotein P respectively. The results showed that compared with the commercially available ELISA kitcoating RV as antigen, the indirect ELISA method based on recombinant protein P had a highercoincidence rate, and it can replace the ELISA method based on RV.