| Torque teno virus (TTV) is a small non-enveloped single-stranded circular DNA virus, which was first discovered in1997in Japan.Since then numerous TTV strains that infect non-human primates,porcine, bovine,ovine,canine,feline,avian and wild animals have been identified.TTSuV is widely distributed in swine populations, and it is imperative to analysize its antigenicity. The ORF1gene of TTSUV2were amplified and cloned into prokaryocyte expression system, recombinant protein with biological activity were expressed and its immunogenicity was analysized. The main experiments and results are as follows:1The objective of this study is to investigate the prevalence of porcine TTV species (TTSuV1and TTSuV2).The study was conducted in7different intensive pig farms of Guangdong province in China. A total of159swine sera were collected,and then the virus were tested using Nest-PCR. The results demonstrated that the TTSuV1and TTSUV2infection rates were54.72%and34.22%respectively, and the co-infection rate was29.56%. We designed PCR primers based upon the conserved region of the two viruses for amplifying the two virogenes. By blast anlysis with our cloned TTsuVs sequences in NCBI database, we found that our isolated TTSuV1ORFland TTsuV2ORF1, have high homologousto to those known TTSuVs sequences,72-75%and88-97%respectively; while have big gaps with the TTV which infecting other species like feils, canis, tupaia, tamarin, douroucouli and homo in the nucleotide sequence. To sum up, TTSuVs had been widely distributed in Guangdong’s pig populations. This study laid the foundation for further research of the prevalence situation, serologic diagnostic methods and to prepare for safe and valid vaccine. 2We designed primers and established a PCR method based on TTSuV2-GDIMAI.After running a PCR amplification,cloned the product to vector and sequenced.We were acquainted with the sequence by predicting and analyzing antigenicity,signal peptide, transmembrane domain, hydrophobicity and secondary structure of TTSUV2ORF1protein and then we desected TTSUV2ORF1to two part named ORF1-VP1and ORF1-VP2separately,bonded them to vector pET-21b and got two protein by Escherichia coli expression system respectively.Highly purified products were obtained after protein purification and recovery. Expression protein were confirmed by Western blot analysis by using positive serum samples which detected through nest-PCR method.The results showed those expressed products of ORF1-VP1and ORF1-VP2were inclusion body protein and infection of TTSuVs called antibodies in pig’s blood indeed. Both two pieces of protein had immunity activity.Our research is significant meaningful to futher research of TTSUV2on bioinfomatic characteristics.3A indirect Elisa assay was developed to detect the specific IgG antibody of TTSUV2in swine serum samples by using the protein of TTSuV2ORF1-VP1and ORF1-VP2as antigens,we use the results to analyze and filter the antigenic epitope of TTSUV2ORF1.Results showed that a number of negative samples which were detected by Nest-PCR method performed high levels of antibodies,on the contrary,some positive samples presented a low antibodies levels. Expressed protein TTSUV2ORF1-VP1and ORF1-VP2have good antigenicity from the experiment,and the results showed that the neutralizing antigen may be located within the protein ORF1-VP1and ORF1-VP2.Results of commercial ELISA kit for detection of CSFV、PRRSV、PRV、HCV in the same213samples were84.44%、86.22%、45.33%and68.42%。Our research support the hypothesis that TTSUV2might function as co-factors in other diseases, and laid a foundation for screening and indentifying the virus antigenic epitopes and Elisa detection method. |
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