The Expression of Codon Optimised Hepatitis B Core Antigen (HBcAg) of Subgenotype B3 Open Reading Frame in Lactococcus lactis

Abstract

Hepatitis B virus (HBV) is currently causing around million people worldwide with chronic HBV infection, while approximately another one million died each year on disease related to liver failure [1]. The HBV is classified into eight genotypes (A-H) based on nucleotide array and each genotype is classified into several subgenotypes. There are three dominant HBV genotypes in Indonesia, namely, HBV/B (70.9%), HBV/C (27.5%), and HBV/D (1.6%). HBV/B is the most dominant HBV genotype in Western Indonesia while HBV/B3 is the most HBV subgenotype in Java region [2, 3]. In other studies, analysis of 214 blood donors (age 18−64 years) and 171 patients with HBV-associated liver diseases samples from Makassar have been shown that HBV/B and HBV/ C are dominant in which subgenotype B3 and C1 reach to 95% and 82.2%, respectively [4, 5]. The HBV is subjected as second carcinogen after tobacco, considering about 80% hepatocellular carcinoma related to HBV infection [6]. Milestone of chronic HBV treatments were adequately improved since 1990’s. Early on, most HBV’s Hepatitis B treatments using immune therapy are gaining interest because of the improvements in dendritic cell performance for antigen presentation, which induces an appropriate immune response and raises patient survival rates. This research aims to produce a significant amount of the HBcAg antigen, which can induce an immune response and have a curative effect on HBV infection. In this study, the HBV subgenotype B3 of the HBcAg gene was used, which is dominant in Indonesia. Further, Lactococcus lactis bacteria was used as the host because of its safety and tightly regulated protein expression. The codon usage for the HBcAg gene was optimized to improve protein expression in L. lactis, which is important because a codon is not random between species. The HBcAg gene is attached to a pNZ8148 plasmid and transformed into the L. lactis NZ3900 expression host. The results confirm that a positive protein band (21 kDa) in two fractions of purified HBcAg was recognized by both western blotting and dot blot hybridization, even if the HBcAg optimized codon has higher GC contents than that suggested for L. lactis expression. Overall, this research strengthens the broad use of L. lactis bacteria for any protein expression, including higher protein expression of codon optimized HBcAg gene compared to non-optimized genes. Furthermore, the improvement in the codon optimization of the HBcAg gene significantly increases the total protein expression by 10–20%, and the expression level of the codon optimized HBcAg increases 1.5 to 3.2-times that of the native HBcAg.