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

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DOI: 10.4236/aid.2013.33033    2,824 Downloads   4,575 Views   Citations

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.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

S. Singh, A. Singh, D. Mankotia, K. Luthra and M. Irshad, "Expression of TTV-ORF2 Protein for Detection of Anti-TTV IgG Antibodies in Human Sera," Advances in Infectious Diseases, Vol. 3 No. 3, 2013, pp. 223-229. doi: 10.4236/aid.2013.33033.

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