Transfection of Sf9 insect cells by recombinant Baculovirus containing Core+1 gene of HCV JFH1 genotype 2a in order to express Core+1 protein

Subject Areas : Microbial Biotechnology

Farideh Sadat Sajadian Fard ¹ , Pooneh Rahimi ²

1 - M.Sc., Department of Microbiology, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences branch, Islamic Azad University, Tehran, Iran.
2 - Assistant Professor, Department of Hepatitis and AIDS, Pasteur Institute of Iran, Medical Virology Group, Tehran, Iran.

Received: 2015-10-03 Accepted : 2015-11-26 Published : 2016-08-01

Keywords: Hepatitis C Virus, JFH1 strain, Insect cells (Sf9), Baculovirus expression system, Protein Core +1,

Abstract :

Background & Objectives: Recently, a new protein, named Core+1, has been reported to be expressed through a +1 ribosomal frame shift in the Core protein coding region of Hepatitis C virus. The purpose of this study was to design a recombinant Baculovirus vector containing HCV-2a (JFH1) Core+1 sequence, and also, to verify the production of this recombinant Core+1 protein in Baculovirus expression system. Materials & Methods: Core+1 gene of HCV-2a (JFH1) was synthesized into pUC57 plasmid. The synthesized target gene was sub-cloned into the plasmid pFastBac-HTA. The recombinant vector was used to transform the competent E. coli DH10Bac containing the donor clone. The recombinant Baculovirus bacmid was produced following transposition. Recombinant bacmid was verified by PCR and then was transfected into Sf9 insect cells to package a new recombinant Bbaculovirus. SDS-PAGE and Western blotting was used to confirm the expression of Core+1 protein in insect cells. Results: Sequence analysis and white-blue colony selection confirmed a successful cloning of the Core+1 sequence of HCV (JFH1) into the pFastBac-HTA vector and transformation of E. coli DH10Bac. Production of recombinant Baculovirus. The HCV-2a (JFH1) Core+1 protein was successfully expressed and confirmed in SDS-PAGE and Western blot. Conclusions: Baculovirus expression system provides a high yield post-translational modification tool for HCV Core+1 protein expression, which is similar to Core+1 protein produced during natural infection with HCV.

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