Construction And Protective Immunogenicity Of A Recombinant Prrsv Expressing ORF2 Of Porcine Circovirus Type 2

Porcine reproductive and respiratory syndrome virus(PRRSV)and porcine circovirus type 2(PCV2)are very two important pathogens that have coursed huge economic losses in swine production in worldwide.In this study,a recombinant PRRSV expressing ORF2 of porcine circovirus type 2 was constructed,and then the swine was immunization with rTJM/Cap and challenged with PCV2.We studied the immune protection effect of recombinant virus rTJM/Cap.The results show that:the live recombinant virus rTJM/Cap can provide force protection against PCV2.In addition,an indirect ELISA for detecting antibody to PRRSV was developed.This method has laid the foundation for the development of PRRSV whole virus indirect ELISA kits and would become an effective method for serological detection of PRRSV.The contents of this paper contain three parts as following:1.Construction and identification of a recombinant PRRSV expressing ORF2 of porcine circovirus type 2Porcine reproductive and respiratory syndrome virus(PRRSV)and porcine circovirus type 2(PCV2)are very two important pathogens that have coursed huge economic losses in swine production in worldwide.In this study,a vector pCMV-TJM containing the full-length cDNA clone of PRRSV attenuated strain TJM-F92 was firstly constructed by PCR method.Then a gene sequence containing Afl ?/Mlu ? restriction enzyme sites and a transcription regulatory sequence for ORF6(TRS6)was inserted between ORF7 and 3'UTR,yielding an expression vector pCMV-TJM-TRS.Subsequently,a plasmid pCMV-TJM-Cap was constructed by cloning of PCV2 ORF2 gene into the unique sites Afl ?/Mlu ? of pCMV-TJM-TRS plasmid DNA.Then three recombinant PRRSV,rTJM,rTJM/TRS and rTJM/Cap,were rescued by transfection of pCMV-TJM,pCMV-TJM-TRS and pCMV-TJM-Cap into Marc-145 cells,respectively,and confirmed by the genome sequence,restriction enzyme digestion,Western Blot and IFA.They all had the molecular markers which was different from the parent virus.The growth characteristics of the rescued viruses were similar to that of parent virus.rTJM/Cap co?Ld also express efficiently PCV2 Cap protein in Marc-145 cells.At passage 8,it still had PCV2 ORF2 gene which examined by RT-PCR.It indicated that the full-length cDNA clone of PRRSV attenuated strain TJM-F92 and recombinant PRRSV rTJM/Cap expressing PCV2 Cap protein were successfully constructed.It made an important foundation for studying on the pathogenic mechanisms of PRRSV and PRRSV-PCV2 vaccine in the future.2.Protective immunogenicity of recombinant PRRSV expressing ORF2 of PCV2 against PCV2In this study,the immunization protection experiment was performed by the use of 28-30-day-old piglets immunized with recombinant virus rTJM/Cap live vaccine(106 TCID50/ml)and inactivated rTJM/Cap vaccine by BEI.The result showed that piglets inoculated with rTJM/Cap live vaccine were able to induce the production of high levels of PCV2-specific ELISA antibodies and neutralizing antibodies,but no significant in cellular immune response.After challenged with PCV2,the relative daily weight gain(RDWG)of rTJM/Cap live vaccine group was higher compared with the challenged control group.Significant reduced PCV2 viremia and viral load in lymph node were observed in rTJM/Cap live vaccine group in which the clinical symptoms and histopathological damages of lung and lymph node were significantly lower than those of the challenged control group.The PCV2 viremia,histopathological damages in lymph node and the measured antigen of inactivated rTJM/Cap vaccine group were similar to that of the challenged control group.It demonstrated that the recombinant virus rTJM/Cap live vaccine,to some extent,was capable of providing protection against PCV2 infection,which laid an important foundation for the development of new PCV2 vaccine.3.Development of an indirect ELISA for detecting antibody to PRRSVAn indirect ELISA for detecting antibody to porcine reproductive and respiratory syndrome virus was developed based on the coating antigen of PRRSV from differential centrifugation.The optimal conditions were in detailed below:the coating concentration was 2?g/ml,the serum dilution was 1:200,the coating antigen condition was 4? for 12h,the sealing solution was PBST containing 1%BSA,the sealing condition was 37? for 2h,the reaction condition of serum was 37? for 1h,the dilution of HRP-SPA was 1:20000,the reaction condition of HRP-SPA was 37? for 45min,the reaction condition of TMB was 37? for 20min without light.The cut-off value of ELISA was determined by detecting PRRSV negative sera.When the serum samples with S/P ratios more than 0.369 were considered as positive,those with S/P ratios less than 0.276 were considered as negative,and the others were suspicious.This method possessed high specification for no crossing reaction with other standard positive serums,such as FMDV,CSFV,PRV gB,PRV gE,PEDV and PCV2.This method possessed high repeatability for the CV%of intra-batch repeatability test and inter-batch repeatability test was all under 10%.The diagnostic sensitivity,specificity and accuracy of the PRRSV ELISA were 100.0%,88.2%and 92.7%,compared with those of IDEXX ELISA kit.It has laid the foundation for the development of indirect ELISA kits for detection of antibody against PRRSV.