Preparation Of Recombinant Antigen Of Rabies Virus And Development Of ELISA Method For Rabies Antibody Detection

Rabies is a fatal central nervous system zoonosis caused by rabies virus. It gets a great reputation for its highest mortality which is 100% in unvaccinated patients. Rabies virus is a RNA virus belonging to the genus of the Rhabdoviridae family. All worm-blood animals can be infected by rabies virus with different sensitivity. Bites by rabid animals generally inoculate virus-laden saliva through the skin into muscle and subcutaneous tissues. Wild animal is a major reservoir of rabies virus. Dog and cat play a key role in the course of human rabies distribution. According to the reports from WHO in 1997, 35 000-50 000 people died of rabies each year and most of these deaths happen in developing countries, and Asia is the most serious area of rabies epidemic. In the last several years, more than 80% of rabies cases happened in Asia. Because of the weakness of animal rabies prevention, human rabies has become seriously prevalent in China and become an important public healthy problem now. These problems make people recognize if the antibody of immune animals has produced enough antibody to resist rabies virus, so it is very important to carry out antibody surveillance after immunization. Serological detection of RV antibody is important diagnosis methods and antibody surveillance tools. But the problem of RV antibody detection method is the lack of sensitive, specific antigen.The glycoprotein (G) and the nucleoprotein (N) are the two main components of the five structure proteins of rabies virus, which can stimulate protective immune response after inoculation of animals. To develop a new type of serological detection antigen to assess antibody titration of immune animals , the following experiments have been carried out and a series of useful results and data were gained.Recently, recombinant baculovirus have become widely used as vectors to express heterologous genes in cultured insect cells and insect larvae, Inmost cases, the recombinant proteins are processed, modified, and targeted to their appreciate cellular locations, where they are functionally similar to their authentic counterparts. In this research , We constructed three recombinant transfer plasmids (pFast-G(without TMR), pFast-Gm and pFast-N), all the recombinant transfer plasmids were introduced into E. coli DHlOBac cells which included a shuttle vector,Bacmid. By site-specific transposition, different target genes were integrated into Bacmid, and the recombinant shuttle vector were constructured , named Bac-G% Bac-Gm and Bac-N. The cultured Sf9 cell were directly transfected with recombinant bacmid DNA, and the pure recombinant baculovirus was obtained, named rAcV-G, rAcV-Gm and rAcV-N, We get recombinant baculovirus were identified through electron microscope asssy, PCR analysis revealed that target genes were correctly inserted into baculovirus genome under the control of polyhedron promoter.Indirect ELISA examine the supernatant of the cultured Sf9 cell infected rAcV-G, rAcV-Gm and rAcV-N, results reveal that recombianant proteins can react with canine anti-RV positive serum. Recombinant baculovirus of rAcV-G, rAcV- Gm and rAcV-N infected the single layer Sf9 cell, discovering through immunity fluorescence examination , rAcV-G, rAcV-Gm and rAcV-N infected the insect cell can react with the canine anti-RV positive serum occurring particularity fluorescence reaction, apply canine anti-RV positive serum to detect recombinant proteins by Western-blot after recombinant baculovirus infected the cultured Sf9 cell , the result shows that the G, Gm and N recombianant proteins can react with canine anti-RV positive serum antibody, the Mr of expression product is 67 KDa(G), 54 KD(Gm) and 57 KDa(N).As well, a method to conjugate fluorescence reagent TITC to anti-RV IgGwas developed, and the antigenicity of low virulent strain SRV9 were compared with the recombinant protein as the detective antigen. All theparameters effected I-ELISA test were carefully optimized, and as a basis, the I-ELISA test to detect the anti-RV antibody was created. The result shows that all the recombinant baculovirus (rAc-Gnu rAc-G and rAc-N) that contained the RV Gm,G and N gene can express the RV Gm, G and N protein after infection the sf9 insect cell, and the recombinant protein shows less antigenicity compared with low virulent strain SRV9. So the standard procedures of I-ELISA test included the following steps, the wells were coated with SRV9 infected cell antigen denatured by formalin diluted 10 time in carbonate/bicarbonate buffer pH 9.6, by incubation at 37°C, 2h or 4"C, 24h; and the blocking condition is 5% BSA in PBS for lh at 37°C; the first antibody of dog serum was diluted 100 time in sample diluent solution of CD, and the second antibody of HRP —IgG or HRP-SPA-IgG of dog were diluted 1:3000 or 1:1600 with 10% FBS in PBS respectively; all the optimized incubated time of sample, the second antibody and the substrat solution are 30 min. The values of P/N over 1.744 were considered as positive.This work is basis for development of RV diagnosis and antibody detection kit.