Sera were collected with informed consent from 88 adult patients with liver and renal diseases attending the Outpatient Department at All India Institute of Medical Sciences, New Delhi. Liver disease group included patients with acute viral hepatitis (AVH), chronic viral hepatitis (CVH) and fulminant hepatic failure (FHF), while renal disease group included patients with chronic renal failure (CRF). These patients were diagnosed on the basis of accepted clinical, and biochemical criteria. Twenty five age and sex matched healthy subjects were used as controls. 6–10 mL of venous blood was drawn and aliqouted in plain tubes without any anticoagulant. Sera were separated from whole blood after centrifugation and stored at -70°C until further analysis. Repeated freezing and thawing of serum was avoided as far as possible.
Detection of DNA and genotyping of TTV
Sera were analysed for the presence of TTV-DNA and subsequent genotyping of all TTV-positive sera were carried out using RFLP as detailed in our previous publication
. Total DNA was purified from 200 μl of sera using High Pure Viral Nucleic Acid kit (Roche Applied Science, Germany) following manufacturer’s instruction. The extracted DNA was used as template for PCR amplification. All steps were performed under laminar air flow hoods using disposable racks and aerosol-resistant tips to avoid sample to sample cross-contamination.
Amplification of full length N22 region
Full length N22 region of TTV genotype-1 was amplified from sera using Taq PCR Core kit (Qiagen, Germany). Primers used were: 5′- ATGTCCTACTTTGAAA -3′ (forward) and 5′- TTACCAGATCCACTTA -3′ (reverse), and were designed using JustBio (http://www.justbio.com) and Primer3 software (http://primer3.ut.ee/). The PCR reactions (25 μL) contained 1X Q-buffer, 1X PCR buffer, 10 mM of each dNTP, 25 pMole of each primer, 2 mM MgCl2 and 0.75U of Taq Polymerase. The PCR cycles consisted of : 94°C for 5 min; 35 cycles of 94°C for 30 sec, 42°C for 45 sec, 72°C for 45 sec; followed by final extension at 72°C for 5 min. Second round comprising 25 cycles was performed under the same conditions using 5 μL of the first round product. The amplified DNA was electrophoresed on 1.2% agarose gel stained with ethidium bromide (EtBr) and identified as ~500 bp amplicon after exposure to UV light.
TA cloning and sequence analysis
The ~500 bp amplicon corresponding to N22 region was excised and extracted with the QIAquick Gel Extraction kit (Qiagen, Germany). The purified PCR product was cloned using commercial vector kit (Promega Corporation, WI, USA). Reaction mixture contained 50 ng pGEM®-T Easy vector, purified amplicon, 1X ligation buffer and 3 Weiss units of T4 DNA Ligase and resulting plasmids were transformed into Escherichia coli DH5α cells (Invitrogen, USA) by heat shock method. The transformed cells were grown on LB-agar plates containing 100 μg/mL ampicillin (Sigma-Aldrich, USA). Insertion was confirmed by colony PCR and sequencing using T7 and target specific primers, as described in our previous studies
[29, 31]. Database searches were performed using NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Phylogenetic analysis was performed using neighbour joining algorithm of PHYLIP program package (http://evolution.gs.washington.edu/phylip.html). Data set was bootstrap re-sampled 1000 times to ascertain support for major branches of the tree.
Cloning in expression vector
To facilitate cloning into expression vector, the N22 region cloned in pGEM®-T vector was re-amplified using sense 5′-TACTACGGATCCATGTCCTACTTTGAA-3′ and anti-sense primer 5′- AACTATGAGCTCTTACCAGATCCACTTA-3′ containing BamHI and SacI restriction sites (shown in italics) at the 5′ and 3′ ends respectively. The amplification conditions were same as used earlier, except for annealing step that was performed at 55°C for 45 sec. The PCR product was digested sequentially using restriction enzymes BamHI and SacI (NEB, USA) and ligated into pET-28a(+) expression vector (Novagen, Germany). The recombinant vector was transformed in E. coli DH5α cells and screened using LB-agar media plates containing 50 μg/mL kanamycin (Sigma-Aldrich, USA). Positive clones were confirmed by colony PCR and bi-directional sequencing. Plasmid from selected recombinant clones was extracted using Qiagen Plasmid Mini kit (Qiagen, Germany).
Expression of TTV protein
N22 region was subsequently expressed as a fusion protein containing hexa-histidine tag (His6-G1N22-pET-28a) in Escherichia coli BL21 cells (Invitrogen, USA) using LB growth medium. Isopropyl-ß-D-thiogalactoside (IPTG) at 0.5– 2.0 mM concentrations was used for 2, 4, 6, 8 and 20 hour inductions, both at 25°C and 37°C. Parallel cultures, without addition of IPTG, were used as controls. Cells from induced and control setups were harvested by centrifugation at 10,000 g for 5 min at 4°C. Whole-cell lysates were studied by 12% SDS-PAGE
 and protein bands were visualized with Coomassie stain (SRL, India).
Expression in ZYP-5052 auto-induction medium
Attempts were made to conduct expression in auto-induction medium also. Principle of this medium is based on presence of carbon sources in the medium that are metabolized differentially to promote high density cell growth and automatically induce protein expression from lac promoters. The medium contains both glucose and lactose as the carbon source. Lactose, often present in undefined components of complex media such as Tryptone, has been shown to cause unintended, sporadic induction of expression in LB media. This can lead to basal expression of target protein, even without addition of IPTG
Use of auto-induction medium was envisaged for a better growth/yield of expression product. For this, freshly screened colonies were grown to saturation in a non-inducing defined medium (PG) at 37°C and this saturated culture was used to inoculate ZYP-5052, a complex auto-inducing growth media containing 100 mM phosphates and 150 μg/ml kanamycin and incubated at 25°C with shaking at 200 rpm. Aliquots were drawn at regular intervals and culture was grown to saturation. To check the time course production of recombinant protein, cells corresponding to 1 OD cell density were harvested, re-suspended in 4X SDS-PAGE sample buffer and analysed on SDS-PAGE. Cells grown to saturation in PG growth medium were used as control. Final culture was harvested by centrifugation at 10,000 g for 5 min at 4°C. The pellet was retained and stored at -80°C till further analysis.
Proteins from SDS-PAGE were transferred electrophoretically to nitrocellulose membrane (Genetix, India) using mini-transblot (Biorad, USA) at 30 V overnight at 4°C
 and the membrane was blocked with 3% BSA (Amresco, USA) in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). For immunodetection of the His fusion proteins, the primary antibody used was His probe (H-3) mouse monoclonal IgG (1 : 500; Santa Cruz Biotechnology, USA) and the secondary antibody was HRP-conjugated anti-mouse IgG antibody (1 : 2000; Santa Cruz Biotechnology, USA). Bound antibodies were detected using di-aminobenzidine (DAB) (Amresco, USA) as substrate.
Purification of expressed N22 protein
After confirmation on western blot, protein was expressed on large scale. 500 mL culture of recombinant E. coli BL21 was grown in ZYP-5052 medium till saturation. The culture was harvested 45 hours post-induction by centrifugation at 10,000 g for 5 min at 4°C and pellet was re-suspended in ice-cold lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, pH 8.0). Sonication was used to lyse the cells (six 10 sec bursts/ 10 sec cooling cycles at 200 W in a Branson Sonicator, USA). Cleared lysate obtained after centrifugation (10,000 g for 20 min at 4°C) was separated from cellular debris (pellet). Both, cleared lysate (soluble protein fraction) and pellet (insoluble protein fraction), were analysed on SDS-PAGE to determine whether recombinant protein is expressed in soluble form or as aggregate in inclusion bodies. To solubilize recombinant protein present in inclusion bodies, 8 M Urea in 100 mM NaH2PO4, 10 mM Tris-Cl, pH 8.0 was used. Purification was performed using Ni-NTA agarose columns (Qiagen, Germany) under denaturing conditions using manufacturer’s instructions. The lysate obtained after treatment with 8 M urea was incubated with Ni-NTA agarose for 30 min on a rotary shaker at room temperature and loaded into an empty column. Flow-through obtained was collected and the column was washed twice with 2 bed volumes of wash buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M Urea, pH 6.3) to remove non-specific proteins. The bound His-tagged recombinant protein was eluted in two steps from column using elution buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M Urea, pH 4.5). Protein concentration in all fractions obtained were determined using Bradford’s reagent
 and subsequently analysed on 12% SDS PAGE.
Development of blot assay
The purified protein was dialyzed to remove urea and subsequently coated on nitrocellulose membrane (50 mm × 5 mm). The control strip was coated with PBS only. After drying, the strips were blocked with 3% BSA in PBS followed by incubation overnight at 4°C. Human sera serially diluted in PBS (1: 500, 1: 1000, 1: 2000, 1: 3000 and 1: 5000) were used as primary antibody for detection of anti-TTV antibodies. A serum without TTV-DNA (1: 500 dilution) was used in negative control. Diluted sera were added to the NC strips and incubated at room temperature for 2 hours with constant shaking. Secondary antibody (goat anti-human IgG-HRP conjugated antibody; Santa Cruz Biotechnology, USA) was added in 1: 4000 dilution in PBS and incubated for 2 hours at room temperature. The strip treated with anti-His (His probe H-3) primary antibody was used as positive control. The blot was developed using HRP-conjugated anti-mouse IgG antibody as detailed earlier. Finally, the substrate (DAB) was used for detection of antigen-antibody complex in all strips. The development of colour in the coated region of strips confirms the binding of anti-TTV antibodies to expressed N22 translational product.
Detection of anti-TTV antibodies in human sera
Panel of sera from liver and renal disease patients as well as healthy individuals were analysed using this blot assay. All sera from patients were used at a dilution of 1: 1000 simultaneously using control sera at a dilution 1: 500. The presence of dot blot on nitrocellulose membrane was used as an indication of anti-TTV antibodies in serum. The results of anti-TTV immunoassay were compared with presence of TTV-DNA in same panel of sera.
The study was approved by the Ethics Committee of All India Institute of Medical Sciences, New Delhi.