Synthesis of cDNA and amplification of NS3 and NS5A genes of HCV3a
After the identification of chronically infected HCV genotype 3a patient from the Division of Molecular Virology and Molecular Diagnostics, the cDNA was synthesized of the extracted RNA as described [15]. The synthesized cDNA was used as a template for the full-length amplification of the NS3 and NS5A genes of HCV 3a genotype of the local isolate using gene-specific primers [15]. The amplified products of NS3 and NS5A were submitted to GeneBank, and accession numbers were obtained.
Identification of antigenic sites within the NS3 and NS5A genes
From the confirmed sequences of NS3 and NS5A genes of HCV 3a genotype, the antigenic sites were selected by using the software Antigenicity Plot ((http://www.bioinformatics.org/JaMBW/3/1/7/). The selected antigenic regions of NS3 and NS5A genes were designated as NS3.1 (nt ~ 328 bp) and NS5A.1 (nt ~ 747 bp) and were used as a template.
Amplification of the NS3.1 and NS5A.1 antigenic sites
To amplify the antigenic sites NS3.1- and NS5A.1-specific primers containing the restriction enzyme, sites were designed. For NS3.1 primers, HindIII and XhoI restriction enzyme sites were added to the primers while NS5A.1 primer contained EcoR1 and HindIII restriction enzyme sites.
Construction of expression vector containing NS3.1 and NS5A.1 antigenic sites and bacterial transformation
To construct an expression vector containing the NS3.1 and NS5A.1 antigenic regions, both the amplified sites and vector were treated with restriction enzymes. For the construction of the NS3.1 expression vector, the gene and vector were treated with HindIII and XhoI restriction enzymes. In order to construct the NS5A.1 vector, the amplified antigenic site and vector were treated with EcoR1 and HindIII enzymes. The digested genes were ligated into the digested plasmid to construct the vector expressing the NS3.1 and NS5A.1 antigenic regions of HCV 3a. After the confirmation of correct ligation by sequencing 2 μl of plasmids harboring the antigenic regions (NS3.1 and NS5A.1) of NS3 and NS5A, genes of HCV 3a genotype of the local isolate were transformed into competent BL21 DE3 pLysS bacterial cells by heat shocking for 90 s at 42 °C. LB medium of 500 ml free of antibiotics was added and incubated for 1 h at 37 °C. Selection of transformants of NS3.1 and NS5A.1 was done on LB-agar plates containing kanamycin (25 mg/ml) and chloramphenicol (34 mg/ml) and incubated overnight at 37 °C.
Expression studies of NS3.1 and NS5A.1 antigenic sites
The expression of the antigenic sites of the NS3 and NS5A genes of the HCV 3a genotype was determined by western blotting. Individual pET28 clones NS3.1-28 and NS5A.1-28 was transformed into the bacterial strain by using the heat shock method as mentioned previously; 2 μl of plasmid was transformed into the BL21 (DE3) pLysS, and selection was done on L-agar plates supplemented with chloramphenicol (34 mg/ml) and kanamycin (25 mg/ml). Isolated colonies were selected and inoculated into L-broth supplemented with chloramphenicol and kanamycin and incubated at 37 °C overnight on a shaker.
Western blot analysis
The confirmed clones (colony PCR) were then used for expression analysis. Individual colonies were given induction of 0.5 M IPTG for 4 h. For protein, isolation cells were harvested, and for 1 g of pellet, 10 ml of lysis buffer was added. Thaw the pellet at room temperature. Prepare a protein sample by mixing 65 μl of the sample with 35 μl of 6X protein loading dye. This is the sample of the total extract. Now, add 0.004 g of lysozyme to the remaining extract and mix well. Sonicate (Mixsonic, USA) the lysed cells for 15 s × 4 pulses. Transfer the pellet into a separate tube and wash the pellet with three times with lysis buffer. Now resuspend the pellet 400 μl of 1X PBS. Prepare the protein sample by mixing 65 μl of pellet and 35 μl of 6Xprotein loading dye. Heat shock for 7 minutes in boiling water bath and snap cool on ice for 5 min. Run-on 15% SDS-PAGE gel at 60 V for 90 min. Western blot was performed following [15] using the gene-specific and vector-specific His tag antibodies.
