バイオテクノロジーとバイオマテリアル

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Cloning and Expression of Vaccine Peptide Containing NS3, E2, NS5A Genes of Hepatitis C Virus in Pichia Pastoris

Saeed Amel Jamehdar, Reza Karimi, Arezoo Esmaili, Samira Tabaei, Baratali Mashkani

Introduction: Hepatitis C is one of the most important causes of chronic hepatitis in developed countries. So far, no useful studies have been performed to design a cost-effective, high-immunity immunization vaccine for hepatitis C virus (HCV) infection. The aim of this study was to clone and express the vaccine peptide containing the NS3, E2, NS5A genes of hepatitis C virus in the yeast of Pichia pastoris.

Materials and methods: In this study, we used the methylotrophic yeast Pichia pastoris as our host to make the recombinant protein. In the next step, the dominant immuno-peptide genes NS3, NS5A and E2 were selected. The mouse IgG2a was selected as desired protein fraction. Linker peptide -GGGGS- was used to make the fusion peptide. The peptides were optimized using Geneious and Gen Script software before synthesis. Then, these genes were cloned into the expression vector pPICZαA. Pastoris strain GS115 strain was transferred to P. pastoris host cells.

Results: Spot blotting and SDS-PAGE techniques confirmed the expression of high levels of NS3-NS5A-E2 -Fc fusion peptide. The results of the other part of the study showed that the possibility of high protein expression is associated with the Codon Compatibility Index (CAI). The CAI of FC-NS3 -E2-NS5A components for Pichia pastoris expression host was 0.68. The other part of the study also showed that after optimizing the GC content became more uniform during the coding sequence. The results also showed that 39% of FC-NS3 -E2-NS5A codons were in the range of 91-100 codons (higher frequency codons) before optimization and this percentage reached 82% after gene optimization.

Conclusion: As a result, this study showed that fusion peptide is expressed in GS115 strains, as confirmed by dot blot technique.