Protein was extracted after induction, and samples were identified through western blot by using gene-specific as well as vector-specific antibodies. 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 and restriction enzymes. In order to construct the NS5A.1 vector, the amplified antigenic site and vector were treated with and 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?l 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 LIPG 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?l 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?l of the sample with 35?l 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?l of 1X PBS. CNQX disodium salt Prepare the protein sample by mixing 65?l of pellet and 35?l 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  using the gene-specific and vector-specific His tag antibodies. Results Selected antigenic site of NS3 and NS5A genes To determine the antigenic regions of the NS3 and NS5A genes, antigenicity plot was used. The full sequence of NS3 and NS5A genes was analyzed for the analysis of the antigenic CNQX disodium salt sites. Figure ?Figure11 illustrates the antigenic sites NS3.1 determined in the NS3 gene of the virus. In Fig. ?Fig.2,2, the determined antigenic site NS5A.1 of NS5A gene of the virus is shown. Open in a separate window Fig. 1 Full gene CNQX disodium salt sequence of NS3 gene indicating (highlighted) specific antigenic site NS3.1 Open in a separate window Fig. 2 Full gene sequence of NS5A gene indicating (highlighted) specific antigenic site NS5A.1 PCR amplification of antigenic site To clone the antigenic site regions into the pET-28a vector the determined antigenic regions NS3.1 and NS5A.1 were PCR CNQX disodium salt amplified by utilizing the antigenic site-specific restriction primers. Designed primers for NS3.1- and NS5A.1-specific antigenic sites having a start codon ATG and specific restriction sites were synthesized. Figure ?Figure44 revealed the amplified regions of the specific antigenic site NS3.1 of the NS3 gene. The expected band size of NS3.1 was 328?bp which was observed on the gel. In Fig. ?Fig.3,3, 747?bp gene band of the NS5A.1 antigenic region was observed. This indicates that the determined antigenic sites were successfully amplified. The PCR amplified products were observed onto 2% agarose gel which is stained with ethidium bromide. Both the amplified.