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.