Cloning and periplasmic soluble expression of hepatitis B surface antigen gene in Escherichia coli.

ABSTRACT

OBJECTIVE: The objective of this study was to clone and express the hepatitis B surface antigen gene (HBsAg) in Escherichia coli (E. coli), thereby aiming to develop potential local therapeutics for combating Hepatitis B virus (HBV) infection in the Pakistani community by producing HBsAg in E. coli. MATERIALS AND METHODS: Blood serum samples were collected from hepatitis B-infected patients, and their genomic DNA was extracted. Real-time and nested polymerase chain reaction (PCR) was performed to amplify the HBsAg gene. The gene of interest was cloned into the pET20b expression vector and transformed into E. coli BL21 (DE3) using Isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction. The gene's precise size was confirmed with gene-specific external and internal primers (681 bp and 400 bp, respectively). RESULTS: The HBsAg gene was successfully sequenced and submitted to GenBank, exhibiting 98% homology with targeted HBV sequences worldwide. The expression of HBsAg protein was confirmed through silver staining, Coomassie staining, western blot, and dot blot analysis. CONCLUSIONS: The expressed protein clones are now available for further development as a local recombinant DNA vaccine to prevent hepatitis B viral infection in the local community.