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Y. E. Khudyakov, M. E. Cong, B. Nichols, D. Reed, X. G. Dou, S. O. Viazov, et al., “Sequence Heterogeneity of TT Virus and Closely Related Viruses,” Journal of Virology, Vol. 74, No. 7, 2000, pp. 2990-3000. doi:10.1128/JVI.74.7.2990-3000.2000

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• TITLE: Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera

  AUTHORS: Shiwani Singh, Akanksha Singh, Dhananjay Singh Mankotia, Kalpana Luthra, Mohammad Irshad

  KEYWORDS: TTV; ORF-2; Anti TTV-IgG; Expression; N-22

  JOURNAL NAME: Advances in Infectious Diseases, Vol.3 No.3, September 5, 2013

  ABSTRACT: The present study describes the cloning and expression of ORF-2 region of TTV genome and the use of expressed peptide in developing immunoassay for detection of anti-TTV antibodies in serum. Presence of TTV-DNA in serum was detected by PCR amplifying N-22 region of ORF-1 of TTV genome. This was followed by genotyping of TTV by RFLP using N-22 amplicon. Using genotype-1 positive serum as the source of TTV, the ORF-2 region was amplified by PCR and subsequently cloned and expressed in pET-19b vector. The expressed protein, identified as 20 kDa protein on SDS-PAGE gel, was purified by affinity chromatography and then used as antigen to develop western blot assay for detection of anti-TTV antibodies in serum. Analysis of sera for anti-TTV antibodies and their comparison with presence of TTV-DNA, produced encouraging results. There was a good relation between presence of anti-TTV and TTV-DNA in these sera samples. Anti-TTV antibodies could be detected in all TTV-DNA positive sera irrespective of the presence of TTV-genotype. This investigation demonstrates that ORF-2 peptide may be used in developing immunoassay for identification of TTV infection.