Eukaryotic Expression Of Glycoprotein,Phosphoprotein And Matrix Protein Of Rabies Virus And Their Preliminary Applications

Rabies is an acute zoonotic infectious disease caused by rabies virus(Rabies Virus,RABV)infection.After the onset of infection,when there are significant neurological symptoms,almost 100% of death.Rabies remains a serious threat to human health in developing countries.At present,the function and pathogenic mechanism of the main structural proteins of rabies virus are still unclear.Having a large amount of information about the interaction between viral proteins and host proteins will help to reveal the mechanism of the pathogenic process of viruses on the host.However,the lack of appropriate monoclonal or polyclonal antibodies is a serious impediment the pace of our research,the development of applicable antibodies has become a prerequisite for relevant research.This study first performed codon optimization on the original sequences of the three structural protein genes of the RABV CVS strain provided by the NCBI using JCAT software.the rare codon is replaced by the HEK preference codon according to the preference for the HEK codon without changing the amino acid sequence,but the host preference codon is finally used as much as possible,taking into account elements such as GC content and avoiding enzymatic hydrolysis.the 5' and 3' ends of the synthesized gene sequence were designed with BamHI and EcoRI digestion sites,respectively,and Flag labels were introduced at the 5' ends to facilitate subsequent protein purification.Not listed a termination codon was introduced at the 3' end in the case of a redundant sequence and the codon adaptation index(CAI)was optimized to increase protein expression.After translation by DNAMAN software,the amino acid sequence homology was confirmed to be 100%.The RABV G CAI rose from 0.22 to0.79,the RABV M CAI rose from 0.21 to 0.65,and the RABV P CAI rose from 0.22 to 0.70 and the CAI was improved after sequence optimization,which was more beneficial to the expression of target protein in HEK..Gene sequence optimized eukaryotic expression vector was transfected into 293 T cells to achieve recombinant protein expression.the RABV M,P,G protein was successfully purified by Anti-Flag affinity gel after purification and concentration,andthe expression levels of the protein were respectively;the recombinant protein was combined with the firs adjuvant to immunize Balb/C mice according to reasonable immune procedures,and serum was collected to prepare polyclonal antibodies.The Western Blot proved that Flag-RABV M?P?G multi-antibodies could detect purified RABV M?at 23 KD?40KD?52KD successfully P?G fusion protein as well as viral protein expression in infected cells.On the basis of determining the good antigenicity of proteins,the RABV M?P and G expressed by eukaryotic cells were selected as coated protein antigens to establish an indirect ELISA diagnosis method.optimizing the reaction conditions and establishing a critical value of yin-yang sex to evaluate the specificity,repeatability and sensitivity of indirect ELISA methods.The results showed that the optimal amount and time of inclusion of antigen were 500?g? 37 ? 2 h;,and the optimal dilution and action time of the positive serum to be tested were 1:32000,37?1,and the best sealing solution and sealing time were 5% skim milk,37 and 1,respectively h;optimal dilution and action time of the second antibody were 1:1000,37?1 h,The optimum reaction time of substrate was 9 min.The critical value of yin and yang was established at 0.202.The established indirect ELISA diagnosis method has no cross-reaction to canine parvovirus,canine coronavirus,canine distemper and canine adenovirus positive sera,and the specificity is good.The titer of polyantibodies against RABV M,P,G protein prepared in this study is 1/128000.