| Porcine torque teno virus was also known as transfusion transmitted virus,was a member of Anelloviridae,genus Iotatorquevirus.Many scholars have carried out TTV epidemiological investigation since porcine TTV was confirmed in1999and revealed evidence of porcine TTV infection in pigs as early as1985in spanish pig farms and TTV infection was worldwide.Although there was no report for the pathogenicity of swine TTV,its extensive epidemic and relationship with other diseases made it not be ignored.In view of the domestic diagnosis of porcine TTV infection entirely depends on detection of viral DNA by PCR.,In the present study, we first expressed and purified the partial gene of ORF1capsid protein of TTV2in Escherichis coli. furthermore, the indirect ELISA assay based on the purified recombinant TTV2-ORF1protein was development and privided serological detection means and the foundation for the establishment of a standardized test kit and vaccine.1Cloning of the ORF1gene of porcine Torque teno virus2and its expression in Escherichia coli BL21According to the pubilished ORF1gene sequence of porcine Torque teno virus2in Genbank,two pairs specific primers were designed and synthesized.Two fragments respectively,1206bp and855bp were amplified from DNA extracted from the tissue by PCR and cloned into the pMD18-T vector and identified by nucleotide sequencing.Then the two recombinant plasmids and expression vector pcold were digested with Xhol和HindⅢ restricition enzyme,and the digested two gene fragments were subcloned to expression vector, then were respectively transformed into the E.coli BL21for expression under induction of IPTG. The expressed products were identified by SDS-PAGE electrophoresis and Western blot. The results of the SDS-PAGE electrophoresis and Western-blotting revealed that the two fragments had been expressed solubly in E.coli,and the size of the two proteins were approximately52KD and39KD.2Purification of the recombinant proteins and preparation of polyclonal antibodyTwo recombinant proteins were inclusion bodies form,therefore we need to use sonication and purified by nickel column.Two kinds of purified proteins were respectively used to immune the New Zealand white rabbits.Antigens were respectively mixed with Freund s complete adjuvant in the first immunization and taking blood from the arterial of ear as a negative control.Antigens were respectively mixed with Freunds incomplete adjuvant in the later immunization interval two weeks,four times immunizations were done.Seven days after the last immunization,blood were taked from the rabbits’ heart,carrying out western-blotting detection,clear bands were present in the corresponding position,antibody titer was1:12800by indirect ELISA.3Establishment of indirect ELISATo establish an indirect ELISA method for detecting porcine TTV2antibodies,purified protein was used as the coating antigen and the reaction conditions were optimized as follows:the best concentration of antigen was1.65μg/ml,the optimal dilution of serum was1:160, the best coated liquid and blocking liquid were respectively carbonate buffer and2%skimmed milk powder,coating antigen for4℃overnight,blocking37℃2h, serum sample and HRP labeled goat anti rabbit IgG(1:4000)being respectively incubated at37℃1h and37℃30min. Determined by statistical analysis of the critical value was0.248.The specificity was proved well by the crossing test with swine serum positve to other pathogens repeated tests confirmed the variation of the test results were less than10%and showed the ELISA assay had a good repeatability.352serum samples from two different farms were tested and the positive rate were42.7%and7.5%,respectively. |
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