Cloning, optimization of expression condition, purification and immunological characterization of hydrophilic section of HCV core Ag, expressed in E-coli by T7-ara BAD promoter

The capsid or core Ag of Hepatitis C virus is a multifunctional protein which has the principal pathogenesis and diagnostic role in HCV related infections and most of these properties are attributed to the hydrophilic section [amino acids 2-122] of this protein. For different research and diagnostic applications, high amounts of this protein in pure and original form are required. So, the aim of this study was to clone the gene, optimize the expression condition, purify it in the original form, and immunologically characterize hydrophilic section of HCV Core Ag, expressed by T7-araBAD promoter system in E.coli. The PCR amplified region corresponding to 2-122 section of this Ag from genotype lb was cloned in pIVEX 2.3, a T7 promoter derived vector. The proper construct after digestional analysis and sequencing confirmations was transformed into BL2 1 -Al E. coli, and protein expression under control of araBAD promoter by addition of 0.2% Arabinose was induced. After optimization of expression condition, purification of protein by NI-NTA agarose gel chromatography in native condition by immidazole yielded about 3.5mg/L of HCV core Ag. Immunological studies by western blotting through application of core specific mAbs and results of ELISA tests indicated that the protein is with desired immunological properties. AraBAD promoter can be perfectly utilized to produce the hydrophilic section of HCV core in high yields, and purification through NI-NTA in native condition may provide the antigen for different research and diagnostic applications