Cloning, Expression And Bioinformatics Analysis Of Swine TTV1-ORF1 And TTV2-ORF1 Gene

Torque teno virus(TTV) is a single-strained, circular DNA virus first reported in1997 by a Japanese scientist Nishizawa. TTV, also named as transfusion transmitted virus, was discovered in serum sample from a patient with unknown post-transfusion hepatitis via the method of representational difference analysis(RDA). Ever since the discovery, scientists from around the world have conducted epidemiological and etiological studies on TTV in various kinds of animals, revealing that both TTV1 and TTV2 are potential pathogens of Postweaning Multisystem Wasting Syndrome(PMWS), meanwhile opening reading frame 1(ORF1) might encode the nucleocapsid protein. As a regular detection method of TTV, polymerase chain reaction(PCR)shows high accuracy but is difficult to be rapidly and widely applied to livestock farms. However, serum ELISA can be used for rapid and epidemiological study of TTV infections. In our study, the whole genomes of porcine TTV1-ORF1-XJ and TTV2-ORF1-XJ were cloned and inducing expressed respectively, laying a foundation for applying porcine TTV recombinant protein to indirect ELISA detection method. The main results are as follows:1. Based on two pairs of both forward and reverse primers which were designed according to the whole genome sequences published on Gen Bank, porcine TTV1-ORF1-XJ and TTV2-OFR1 were amplified through the method of nested PCR,resulting in two fragments of 1917 bp and 1878 bp, which were verified to match the reference sequences. The two genes were cloned into the p EASY-T1, and named p TTV1-ORF1-XJ、p TTV2-ORF1-XJ, respectively.2. After sequencing, bioinformatics prediction including physicochemical properties, functional domain, secondary and three dimensional structure of protein was implemented via both bioinformatics software and web tools. On one hand, the results indicated that TTV1-ORF1-XJ was translated into 638 amino acids, and the phosphorylation sites mainly located in serine, threonine and tyrosine. According to prediction, the N-terminal was arginine-rich and the encoded protein located both inside and outside the membrane. Meanwhile signal peptide was absent, and one transmembrane domain and thirty-one B cell lineal epitopes were discovered. The stability prediction of amino acids suggested there existed unstable area in the protein.On the other hand, TTV2-ORF1-XJ was translated into 625 amino acids, while the remaining prediction results were similar with that of TTV1-ORF1-XJ.3. The matched gene of TTV1-ORF1-XJ and TTV2-ORF1-XJ were subcloned into expression vectors p EASY-E1, and then transferred into E. coli BL21 competentcells, followed by expression induced by IPTG at different time, temperature and concentration. The results of SDS-PAGE and Western Blot revealed that the protein expression amount was too low to meet the purification standard.