Establishmengt Of GETV C Protein Elisa Detection Method,Screening Of Vaccine Candidate Strains

Getah virus(GETV),is a member of the family Armoridae,which is mainly transmitted by blood-sucking insects,especially mosquitoes,between arthropods and vertebrates.Ever since its discovery in Malaysia in 1955,it has been widely distributed in many countries and regions Mainland Eurasia;serological surveys have shown that the virus is infected in humans and many other animals.GETV can cause abortion in pregnant sows,diarrhea in piglets and even death;the clinical symptoms of horses are mainly fever,dermatitis and joint swelling;there are no reports of clinical onset in the population,but there are high anti-GETV virus antibodies in the population,which are generally considered to be a human orcovirus.GETV can infect rabbits,mice,guinea pigs and hamsters under laboratory conditions,and can cause the death of milk mice under 9 days of age.Our laboratory isolated GETV from Mainland pigs in China for the first time in 2016,but the related research and investigation is relatively lagging poor.There is no clear data and efficient method for the diagnosis and hazard prediction of epidemic disease.Its transmission and distribution in China,as well as clinical infection rate,incidence,hazards and other indicators were uncertain.Based on the above theory,this study first established the indirect ELISA detection method of C protein IgG antibody,and established SYBR GreenI fluorescence quantitative detection method to provide support for vaccine evaluation in subsequent animal trials.Inactivated vaccines were prepared to screen vaccine candidates and evaluate their immunogenicity and efficacy in order to provide materials for subsequent vaccine development.1.The Establishment and Application of Indirect ELISA Detection Method for C Protein IgG AntibodySpecific primer amplification Cap,based on GETV gene(GenBank)sequence A prokaryotic expression vector(pET28a-Cap)was constructed for protein purification and identification,purified Cap protein as coated antigen,optimized ELISA reaction conditions by chessboard titration.Then the tests of specificity,sensitivity,repeatability.The indirect ELISA detection method of GETV C protein IgG antibody was successfully established.A total of 119 samples of suspected GETV infected pigs collected in the field were used for testing.The positive rate of clinical samples was 59.6%(59/99),experimental results showed that the ELISA test method was successfully established and the clinical materials were tested.Enrich the way GETV virus is detected,to provide technical means for its epidemic detection.2.Establishment and Application of GETV SYBR Green I Fluorescence Quantitative Detection MethodThe conserved region of pig source GETV NSP3 gene in reference GenBank,designed and synthesized primers,estimated amplification length 126 bp.The cDNA of reverse transcription after RNA was extracted as template for PCR amplification,and then ligated to prepare standard plasmids.According to the conventional method,the standard plasmid was used as the template to optimize the reaction conditions,and the standard curve was established,and then the specificity,sensitivity,repeatability test and sample detection were carried out in turn.The minimum detection limit of this method can reach 224 copies,the coefficient of variation(CV)is 0.3 1%-0.91%,which only peaks for GETV nucleic acid,and the specificity and repeatability are good,which provides a technical means for rapid and quantitative detection of GETV.3.Screening of GETV inactivated vaccine candidate strains and evaluation of immune efficacyBy comprehensive analysis of amino acid sequence,genetic evolution,comparison of the biological characteristics and isolation region of 8 GETV clinical isolates,three strains and one subculture strain were selected for vaccine candidate screening and immune effect evaluation.TCID50/mL,106.8 virus proliferation titer by immunizing KM mice,7 d after conception,Determination of serum specific IgG antibodies and neutralizing antibodies,Statistics of litter size and embryo protection in pregnant rats,Anatomy and recording of histopathological changes,Detection of tissue viral load,Maternal antibody protection rate,Finally,GETV-V1 strains were selected as seed strains.Flow cytometry to detect changes in peripheral blood lymphocyte subsets and cytokine expression,CD4 cell subsets increased significantly,the expression of IL-10 and TGF-β increased significantly,mediating Thl biased immune responses,induce effective specific humoral immunity against GETV.This study provides data support for evaluating the immune efficacy of vaccines in pigs,For the development of inactivated vaccines to prevent GETV infection,For the study of GETV pathogenesis and comprehensive prevention and control to provide a reference basis.This study provides data support for evaluating the immune efficacy of vaccines in pigs,provides candidate strains for the development of inactivated vaccines that can prevent GETV infection,and provides a theoretical reference for the study of pathogenesis and comprehensive prevention and control.