High level virion production and surface antigen expression with 1.5 copies of hepatitis B viral genome

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• 作者：Qingming Dong ; Zheng Liu ; Yangchao Chen ; Chu-Yan Chan ; Marie C. Lin ; Hsiang-fu Kung ; Henry L.Y. Chan ; Joseph J.Y. Sung ; Ming-Liang He
• 关键词：Hepatitis B virus ; Basal core promoter ; HBsAg ; HBeAg ; Pregenomic RNA
• 刊名：Journal of Virological Methods
• 出版年：2009
• 出版时间：August 2009
• 年：2009
• 卷：159
• 期：2
• 页码：135-140
• 全文大小：695 K

文摘

The present study aimed to construct a 1.5X hepatitis B virus (HBV) replication system in vitro that could generate high level of HBV viruses. This system would help compare the replication capacity among the virus strains associated with high and low risk of hepatocellular carcinoma (HCC). Four strains of HBV were isolated from two HCC patients and two HBV carriers. After molecular cloning, four corresponding constructs named as HBV-1.5Xs were generated. Each of them has one and a half copies of HBV 3.2 kb genome, a 5′-end redundant sequence of 1.1 kb to nt715 and a 3′-end redundant sequence of 500 bp to nt2325 that situated after the poly (A) sequence. The HepG2 cells were transfected with the HBV-1.5Xs, and the levels of HBsAg, HBeAg and viral DNA were then detected in both the supernatant and the cells. After 24 h and 48 h of transfection, a high OD value of HBsAg of 3.5 was observed in the supernatant and also in some of the diluted cell lysate samples. The HBeAg level was relatively low in all strain samples of HBV. The log₁₀ values of viral loads were also determined with the cell lysate having a higher value (10–11 per ml) than that of the supernatant (6–7 per ml). The results showed that the novel HBV-1.5X system was capable to generate high level of HBV in a consistent manner. However, no significant difference was found among the replication capacities among these strains in vitro. The HBV-1.5X system may be a useful platform that assists the establishment of stable cell lines and transgenic mice for the investigation of viral pathogenesis, particularly for the various strains of HBV.