Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies

Abstract

BackgroundTorque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose.MethodsFull length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay.ResultsAnalysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed.ConclusionsThis concludes that TTV N22 region may be expressed and safely used as antigen for blot assay to detect anti-TTV antibodies in sera.

Development of an Immunoassay for Detection of Torque Teno Virus (TTV) Antibodies Using the N22 Expression Product from TTV Genotype 2

“…The material and methods, as well as the overall workflow used in this study, are described in our previous report [30]. The complete study plan was approved by the Ethics Committee of the All India Institute of Medical Sciences, New Delhi, India.…”

“…The reaction contained digested vector and digested insert DNA in a 1: 3 ratio along with 3 Weiss units of T4 DNA Ligase (Promega Corporation, USA) and was incubated overnight at 16 ° C. The resulting recombinant plasmids were transformed into E. coli DH5α-competent cells (Invitrogen, USA) via the heat shock method and screened on LB-agar medium plates containing 50 μg/mL kanamycin (Sigma-Aldrich, USA). Insertion was confirmed by colony PCR and nucleotide sequencing, as described in our previous publications [30][31][32]. Database searches were performed using the BLAST program (www.ncbi.nlm.nih.gov/BLAST) of the National Center for Biotechnology Information (NCBI).…”

“…The standard measure of culture growth was optical density at 600 nm (OD 600 ) after dilution in water to concentrations that gave readings below 0.40 in a 1-cm path-length cuvette in a BioTek PowerWave XS spectrophotometer. To assess the time course of protein production, aliquots corresponding to a 1-OD cell density were drawn at regular intervals and processed as described in our previous study [30]. A parallel setup containing untransformed BL21 (DE3) cells without the expression vector was used as a control experiment and processed under similar conditions.…”

“…The recombinant protein was expressed on a large scale by culturing the selected clone in 500 mL ZYM-5052 medium for 24 h at 25 ° C. Cells were harvested, lysed by sonication, and analyzed on SDS-PAGE to determine the solubility and localization of the expressed peptide, as described earlier [30]. Protein purification was performed using nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography columns (QIAexpress Kit, Qiagen) under denaturing conditions.…”

“…An immunoassay to detect TTV infection in human sera was reported from our Centre [30]. It was developed using the N22 region from TTV genotype 1 and showed promising results.…”