Preparation Of The Monoclonal Antibody Against Envelope Protein Of JSRV And Establishment Of Double-antibody Sandwich ELISA

BACKGROUND: Jaagsiekte sheep retrovirus(JSRV)is the pathogen of ovine pulmonary adenocarcinoma(OPA),which is a contagious lung tumor in sheep.The OPA occurs in many regions of the world.Antibodies to JSRV have not been detected in infected sheep and therefore,the routine serological tests are not available for diagnosis.JSRV cannot yet be propagated in vitro,therefore the routine diagnostic methods,such as virus isolation,are not available for diagnosis.The diagnosis of OPA mainly relies on pathological anatomy,histopathology and immunohistochemistry examinations.OBJECTIVE: To establish a double antibody sandwich ELISA method of detecting JSRV envelope protein that provides technical support for establishing reliable laboratory method for the diagnosis of OPA in individual animals.METHODS:1.To analyze JSRV envelope protein(Env)on the basis of biological information The secondary structure of JSRV Env was predicted by the DNAStar,SOPMA,PSIPRED and Predictprotein softwares.The hydrophilicity,exposed surface,flexibility and antigenic propensity of JSRV envelope protein was predicted by Protean software.The B-cell epitopes of the envelope protein were predicted using the Bcepred,IEDB and ABCpred softwares.Combining the analysis results of Env amino acids of en JSRVs,ex JSRVs and ENTVs,the regions rich in epitopes and the dominance epitopes areas which were in the VR3 hypervariable region of ex JSRV and en JSRV envelope protein were screened2.The proteins in regions rich of epitopes of JSRV Env and the dominance epitope area of the VR3 hypervariable region of Env of ex JSRV and en JSRV,were expressed and purified by prokaryotic expression system3.The mouse anti-JSRV Env polyclonal antibody and rabbit anti-JSRV Env51-394 polyclonal antibody were prepared by the purified JSRV Env and JSRV Env51-394.And the specificity of polyclonal antibody was evaluated by Western blot and immunohistochemistry assay.4.The mouse anti-JSRV cytoplasmic tail(anti-JSRV CT)monoclonal antibody was prepared by synthesis peptide JSRV Env572–615.And the specificity of monoclonal antibody was evaluated by Western blot and immunohistochemistry assay.5.Using the prepared monoclonal antibody and polyclonal antibody were respectively as detection antibody and capture antibody,a double antibodies sandwich ELISA method to detect JSRV envelope protein by sheep peripheral blood leukocytes was established.And the method was preliminary application in the detection of JSRV.RESULTS:1.The predictive dominance epitopes of JSRV Env is: 54 ~ 67,90 ~ 109,118 ~ 165,179 ~ 194,201 ~ 215,236 ~ 250,273 ~ 284,295 ~ 310,309 ~ 332,335 ~ 356,363 ~ 381,461 ~ 476,540 ~ 546,572 ~ 596 and 600 ~ 615 amino acids.2.The JSRV Env and the 51-394 amino acids fragment of JSRV Env were expressed and purified,and the prepartion of mouse anti-JSRV Env polyclonal antibody and rabbit anti-JSRV Env51-394 polyclonal antibody were successfully manufactured.The results of Western blot and immunohistochemical experiments showed that the two polyclonal antibodies not only specifically recognize denatured complete envelope protein,but also have immune reaction with natural intact Env.It demonstrated that the prepared antibody in this study as capture antibody of double antibody sandwich ELISA could be used to detect the intact Env and have better performance on capturing the JSRV Env3.The results of Western blot and immunohistochemical experiments showed that the prepared anti-JSRV CT monoclonal antibody could identify both denatured and non-denatured JSRV envelope protein antigen,and the prepartion of mouse anti-JSRV CT antibody were successful.Using anti-JSRV CT monoclonal antibody,a Western blot experiment of sheep peripheral blood leukocytes was carried out,comparing with the results of immunohistochemical experiment,we confirmed that two cases of OPA were detected by using anti-JSRV CT monoclonal antibody.The results showed that the monoclonal antibody had higher specificity and applicability.As detection reagent of JSRV,the monoclonal antibody could be conducive to the establishment of detection methods of OPA.4.We successfully established the double antibodies sandwich ELISA method to detect JSRV Env.The detection results of the vaccines of sheep common diease(included brucellosis,braxy,lamb dysentery,enterotoxemia,struck,capripox,pests des petits ruminants and foot-and-mouth disease,mycoplasma ovipneumoniae,ENTV and en JSRV)by the developed method were negative.The minimum detection concentration of extraction protein of infected lung tissue is 0.034 mg/m L.The inter-and intra-variation coefficient of detection result are 3.112%~6.801%,and1.380%~4.763%,respectively.For the packaged plate stored at 37 °C for 7 days,the detection results of positive samples by the developed method were still positive.Compared with the packaged plates stored at 37 °C for 0 to7 days,the detection results of positive samples showed no significant difference(P > 0.05).The study results showed that the method has outstanding specificity,stability,sensitivity and rapidity,and application value in clinical detection of JSRV in live animals.CONCLUSIONS: In the study,we predicted the dominant epitope areas of JSRV Env,and expressed and purified the JSRV Env,and the 51-394 amino acids fragment of JSRV Env with the regions rich in epitopes of JSRV Env,.Based on the anti-JSRV Env51-394 polyclonal antibody and anti-JSRV CT monoclonal antibody,we established the double antibodies sandwich ELISA method of detection JSRV Env.