Comparison Of Indirect ELISA Methods Based On The Four Recombinant Protein Of Rabies Virus

Rabies is an important zoonotic disease, which is seriously harmful to people's health. In China, more than a thousand cases infected with rabies through dogs, cats and other animal vectors each year and the death rate ranks the third among the various infectious diseases. Enzyme-linked immunosorbent assay (ELISA) for their short experimental period, simple operation, easy standardization, etc, has been widely used in the clinical detection of rabies virus (RV). Most of the the commercial ELISA kits use the whole RV as a diagnostic antigen, and this study is to find the alternative antigen to instead of the whole RV as a diagnostic antigen. The four recombinant proteins (M,N,G,P) which were used as diagnostic antigen were compared comprehensively with the whole RV as a diagnostic antigen systematically to determine the best diagnostic antigen. In this thesis, we mainly focused on the following aspects.First, the cloning of RV M gene and prokaryotic expression:RT-PCR amplified the M gene of RV Flury LEP strain, then it was cloned into the prokaryotic expression vector pET-32a (+), and got the recombinant plasmid pET-32a-M.The recombinant plasmid pET-32a-M was transformed into the host strain E.coli Rosetta.We got the pET-32a-M recombinant protein after inducing with IPTG. The SDS-PAGE showed the recombinant protein was about42ku, and expressed in the form of inclusion body. The Western Blot analysis showed the recombinant protein had good reactogenicity.Second, the establishment of the indirect ELISA which used the recombinant protein pET-32a-M and rabies virus as coating antigen:pET-32a-M recombinant protein was purified by gel purification method, and the purified protein was used as a diagnostic antigen to establish the indirect ELISA. After optimizing the reaction conditions.We determined the best concentration of coating antigen was the amount of6μg/mL, the best dilution of serum was1:200and the reaction time was30min, the best blocking reagent was10%fetal bovine serum, the best dilution of HRP-protein A was1:2000, the reaction time was60min,15min for optimum coloring. The RV purified by PEG precipitation was used as diagnostic antigens to establish the indirect ELISA. After optimizing the conditions,we determined the best concentration of coating antigen was the amount of0.25μg/mL, the best dilution of serum was1:200and the reaction was30min, the best blocking reagent was10%fetal bovine serum,the best dilution of HRP-protein A of dog was1:2000, the reaction time was60min,15min for optimum coloring. After the repeatability of intra-experiment and inter-experiment, we established the indirect ELISA which used the recombinant protein and rabies virus as diagnostic antigens.Third, the comparison of the indirect ELISA methods:We used the indirect ELSIA methods which used the whole RV and the four recombinant proteins as diagnostic antigens to detect291clinical serum samples respectively, the results were analyzed with statistical methods,the results between the method with RV and the methods with M, G and P protein had apparent difference (P<0.05), but the results between the method with RV and the method with N protein had not significant difference (P>0,05).We used the method with N protein and the commercial ELISA kit to test45clinical serum samples, the results showed that the coincidence rate was as high as97.78%.