| Porcine reproductive and respiratory syndrome,which is also referred as blue ear disease,is one of the most important infectious diseases in pigs and it causes great economic loss in swine industry every year.The disease is caused by porcine reproductive and respiratory syndrome virus(PRRSV).Its typical clinical symptoms such as reproductive failure in sows,high fever and respiratory distress could be found in infectious pigs.Pig infected with highly pathogenic PRRS V strains usually shows more severe clinical signs,lung lesions and abnormal host immune responses.According to the difference in genome homology,PRRSVs are now divided into two species:PRRSV1 and PRRSV2.The diagnosis of the disease mainly relies on pathogenic diagnosis and serological diagnosis.In this study,the glycoproteins of PRRSV(GP3,GP4 and GP5)were expressed in E.coli system,and specific polyclonal antibodies against GP3,GP5 and monoclonal antibody against GP4 were obtained by mice immunization,which could be good for studying the function of PRRSV structural proteins and developing diagnostic methods.At the same time,an indirect ELISA method for detecting PRRSV specific antibodies by coating different polypeptides was established and optimized the reaction conditions,which provides a potential for rapid PRRSV diagnosis.1.Development of antibodies to PRRSV glycoproteinsIn this study,specific primers for PRRSV glycoproteins GP3,GP4 and GP5 were synthesized and the genes were amplified by RT-PCR from viral RNA.The prokaryotic expression plasmids pET32a-PRRSV-GP3,pET32a-PRRSV-GP4 and pET32a-PRRSV-GP5 were constructed and transformed into BL21 competent cells.Recombinant proteins were expreesed by IPTG induction and subjected to ultrasonic disruption.The supernatant and the cell pellet after centrifugation were subjected to SDS-PAGE analysis respectively.The result showed that the target proteins were expressed,and their molecular weight were about 40KD,30KD and 30KD,respectively.Western blot result shows that all three recombinant proteins could be recognized by anti-His monoclonal antibody.Recombinant GP3 and GP5 proteins could also be recognized by porcine positive antiserum to PRRSV.Female BALB/c mice at 6-8 weeks old were immunized with the purified pET32a-PRRSV-GP3 protein,pET32a-PRRSV-GP4 protein and pET32a-PRRSV-GP5 protein mixed with adjuvant respectively After three immunizations,the specific antibody titers of the mice sera were measured.The results showed that all three different proteins could induce specific antibody in mice.The mouse with the highest antibody titer against GP4 protein was boosted again three days before the euthanization.The spleen was then fused with mouse myeloma cells.The supertanant of hybridomas were screened with MARC-145 cells infected by PRRSV by IFA.The positive hybridoma was subcloned.Finally the monoclonal antibody against PRRSV GP4 protein was obtained and named as PRRSV-GP4-1F11.2.Establishment of peptide ELISA antibody detection methodAn indirect ELISA method for detecting PRRSV antibodies was established by using a synthetic polypeptide as a coating antigen.The method was optimized in polypeptide coating concentration,serum dilution,blocking solution and blocking time,primary antibody and HRP-labeled sheep Anti-pig IgG incubation time,and incubation time of TMB substrate.The specificity and the repeatability of the ELISA assay were evaluated after optimization of all conditions.The experimental results showed that the method could detect PRRSV-positive sera but not other sera against common porcine viruses such as CSFV,PRV,PEDV.In addition,the intra-assay and inter-assay coefficients of variation of the method is less than 10%.133 samples of pig sera were tested by the established indirect ELISA method.The positive rate for GP3/4-ELISA was 78.6%,and the positive rate for GP5-ELISA was 81.6%.These results demonstrate that different coating proteins may have some differences in the detection of PRRSV antibodies.It could be used as a reference for evaluating vaccine efficacy or infection. |
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