Establishment And Preliminary Application Of An Indirect ELISA Method Based On Porcine Getah Virus E2 Protein

Getah virus（GETV）is an arbovirus belonging to the Alphavirus genus of the Togaviridae family.Its hosts are mosquitoes and a variety of vertebrates,mainly causing disease in pigs and horses,and recently discovered that it can also cause disease in blue foxes.After horses are infected with GETV,symptoms such as lymphadenopathy,edema,rash and fever will appear;after pigs are infected with GETV,piglets will develop arthritis,paralysis,diarrhea,etc.The infection and incidence of blue foxes are related to eating pork products;after blue foxes are infected with GETV,symptoms of fever and anorexia will appear,and there will also be Neurological symptoms appear,and eventually death occurs.Serological detection methods are currently more reliable GETV detection methods,such as hemagglutination inhibition test（HI）,complement fixation test（CFT）,neutralization test（NT）and indirect immunofluorescence（indirect immunofluorescence）.These methods are often used to detect GETV,but these methods are complicated and time-consuming.Therefore,a fast,accurate,sensitive and specific detection method is very important.The E2 protein of GETV is a very important structural protein among all its proteins,and has good antigenicity.In this study,a prokaryotic expression plasmid was constructed using the E2 gene sequence,and a large amount of E2 protein was prepared to establish an indirect ELISA method for the detection of GETV.The build process is divided into the following two parts:1.Cloning and expression of GETV E2 gene and preparation of polyclonal antibodyIn this study,referring to the E2 gene sequence of GETV GS10-2 strain（Gen Bank accession number:EU015070.1）from Gen Bank,a pair of primers that can completely amplify the E2 gene sequence was designed according to the sequence,and Eco R I and Xho I were added for restriction enzyme digestion.site.The E2 gene sequence was amplified by PCR,and the E2 gene sequence was ligated with the prokaryotic expression vector p ET-28a with ligase after double enzyme digestion.After subsequent sequencing identification,the p ET-28a-E2recombinant plasmid was successfully constructed.Then,the plasmid was transferred into BL21（DE3）expressing bacteria after a series of optimized conditions,and finally the E2 protein with a molecular weight of 46k D was successfully expressed by SDS-PAGE after induction with0.1m M IPTG concentration at 37℃for 6h.The highly expressed E2 protein was purified and concentration determined,and then mixed with Freund’s adjuvant in equal proportions to prepare polyclonal antibodies,which were analyzed with the E2 protein antigen by Western blot to prove a specific reaction.2.Establishment and preliminary application of GETV indirect ELISA methodThe purified E2 protein was used as the coating antigen,and the prepared polyclonal antibody was used as the primary antibody serum.After a series of optimizations,the optimal indirect ELISA reaction conditions were determined:8μg/m L antigen was coated overnight at4℃,and the serum to be tested was 1:100.Dilution,2.5%nonfat milk powder blocking solution for 1h,antigen-antibody action for 60min,enzyme-labeled secondary antibody 1:5000 dilution for 60min,color development time 25min.Positive serum samples for swine fever virus（CSFV）,porcine reproductive and respiratory syndrome virus（PRRSV）,porcine circovirus type 2（PCV2）,and pseudorabies virus（PRV）were detected using the established method,and the results were all Negative,indicating that the established method has good specificity.The critical value of this method was determined by calculating the mean（x?）and standard deviation（SD）of 50 negative sera,when OD₄₅₀≥x?+3S=0.415 was positive,and when OD₄₅₀≤x?+2S=0.332 Negative,in between is judged suspicious.When the GETV positive serum was diluted by 1:2560,the detection result was still positive,indicating that the sensitivity of the method was good.The inter-assay and intra-assay repeatability results were also good,with the coefficient of variation within 10%.The established method can be used for the detection of GETV.Using this method,300 serum samples collected from some pig farms in Jilin Province were stored in the laboratory for testing,and 47 samples were positive for GETV,with a positive rate of 15.67%.In conclusion,this study successfully established an indirect ELISA method for the detection of GETV,and proved that the established indirect ELISA method can be used for sample detection,which provides a basis for the detection of GETV.