Preparation And Identification Of Monoclonal Antibodies Against The Capsid Protein Of Porcine Torque Teno Virus Type1

Torque Teno viru（sTTV） as a emerging virus is first isolated from a patient with post-transfusionhepatitis. TTV has also been detected in several animal species. Analysis of complete genomicsequences of PTTV, two distinct genogroups have been identified in swine: PTTV1and PTTV2. PTTVare now widespread throughout the world with prevalences ranging from30%to100%. The pathogenicrole of PTTV is unclear. However, recent report suggests that the association of PTTV infection withporcine circovirus2(PCV2) infections that causes postweaning multisystemic wasting syndrome(PMWS). Meanwhile, there are reports suggest that PTTV may paly a role in the porcine reproductiveand respiratory syndrome (PRRS), porcine dermatitis andnephropathy syndrome (PNDS) and porcinehepatitis E (PHE). In view of its highly epidemic and potential synergistic pathogenicity, it is necessaryto study the virus. PTTV has not been isolated in the cells in vitro. So far, the diagnosis of PTTV1infection entirely depends on detection of viral DNA by conventional PCR assays. In this study, PTTV1recombinant Cap (PTTV1-rCap) protein was expressed using prokaryotic expression system and themonoclonal antibodies (MAbs) against the capsid protein was prepared by PTTV1recombinant Capprotein, meanwhile the MAbs were identified using the antigen of PTTV1-Cap protein expressed in theeukaryotic transient expression system.This paper offers some convenient tools for epidemiology survey,serological diagnostic methods and epitope analysis of the virus.In this study, the structural protein gene of PTTV1-ORF1was amplified by PCR and cloned intoprokaryotic expression vector pGEX-6P-1to express truncated fusion protein. ThepGEX-TTV1-ORF1-C plasmid was transformed into the cells of Rosetta (DE3). PTTV1-rCap proteinexpression was analyzed by SDS-PAGE and Western blot. The results showed that PTTV1-rCap proteinwas efficiently expressed with MW64.0kDa as inclusion body form. Western-blot analys is showed thatfusion protein could react with anti-GST monoclonal antibody. As PTTV1-rCap is the major structureprotein of vrius, so it is an ideal target antigen. The expression of PTTV-rCap recombinant proteinprovides a foundation for establishing serological diagonistic method for the virus.BALB/c mice were immunized with the purified PTTV1-rCap protein. Eight hybridomas couldstably secrete MAb against the PTTV1-rCap protein were screened after three cycle clone, whichtermed as1H4,1E9,1E11,4G12,5C5,7B8,8C2,8G2. All MAbs belonged to IgG1subclass and lightchain. The supernatant titers of MAbs were from1:400to1:3200, and ascite titers were from1:4.0×105to6.4×106. Seven MAbs could react with PTTV1-rCap antigen by Western blot using anti-GST MAb asindicator. While, one of MAb showed negative, it possibly belongs to the conformation epitope. TheseMAbs which to the PTTV1-rCap were obtained in the study, it could be applied in the serologicaldiagnostic method and antigen epitope analysis of the virus.For further identification of the MAbs, the MAbs were detected by immunoperoxidase monolayerassay (IPMA) method in293T cells which were transfected with recombinant eukaryotic expressionvector of pcDNA3.1containing full-length and partial ORF1gene, respectively. The result shows that all MAbs could react positively with the transient expression of full-length PTTV1-rCap.This studyprovide a useful tool for further evaluating the immunogenicity of recombinant plasmids DNA andestablishing the IPMA diagnostic method and epitope analys is for the virus.IPMA method is widely used in the detection of porcine disease with the advantage of highspecificity and sensitivity, and the result can be judged easily. In this study, an IPMA was developed fordetection of antibodies against PTTV1. The eukaryotic expression vector of pcDNA3.1containingfull-length ORF1gene was transfected in293-T cells and the cells which as the antigen was fixed byparaformaldehyde. A total of100serum samples from provinces of Heilongjiang, Jilin, Hebei, Shanghai,Shandong were detected by the PTTV1-IPMA. The positive rate was82%for PTTV1, indicating thathigh prevalence of PTTV in China pig herds.