Establishment of a cell line supporting the gene expression and replication of hepatitis B virus-genotype C

Abstract

Abstract: Objective To establish a novel cell line supporting the gene expression and replication of hepatitis B virus (HBV)-genotype C, and provide a new cell model for antiviral research. Methods HBV DNA was extracted from the serum of hepatitis B patients. Full-length of HBV genome was amplified by polymerase chain reaction (PCR). The circularized HBV full-length genome was generated by Sap I restriction enzyme cleavage and T4 ligase ligation, and was used as a template to amplify 0.2 copies of HBV genome (nt1402-nt1989) and one copy of HBV (nt1402-nt3215-nt1407), respectively. The two HBV DNA PCR products were cloned into pcDNA3(-)-EGFP-△CMV vector to obtain a p1.2×HBV-EGFP plasmid, followed by sequencing verification. The recombinant plasmid was transfected into HepG2 cells with lipofectamine 2000. Positive clones were selected by culturing the cells in the presence of G418 (700μg/ml) for 2-3 weeks. The levels of HBsAg, HBeAg, and HBV DNA in the cell culture supernatants were detected by ELISA and real-time PCR, respectively; The intracellular HBV replication intermediates were detected by Southern blotting, and the intracellular HBsAg and HBcAg were detected by immunofluorescence. Results A novel cell line, namely HepG2X15, was established for supporting the replication of HBV DNA -genotype C that derived from clinical isolates. It was evidenced that high levels of HBsAg, HBeAg, and HBV DNA were detectable in the culture supernatant, and intracellular HBV replication intermediates, HBsAg, and HBcAg were detectable in the cells. Compared with HepG2.2.15 cell line, HepG2X15 cells secrete higher levels of HBsAg and HBV virions into the culture supernatant. Conclusion A novel cell line HepG2X15 supporting the replication of HBV- genotype C was successfully established, with high production of HBsAg, HBeAg, and HBV virions, and intracellular HBV replication intermediates. It can be used for screening of anti-HBV drug and for investigating the mechanisms of anti-HBV drug resistance.

Key words: Hepatitis B virus, Clinical isolates, DNA replication, Cell line

LI Rui-ming, YANG Yan, ZENG Xian-huang, MENG Zhong-ji. Establishment of a cell line supporting the gene expression and replication of hepatitis B virus-genotype C[J]. Chinese Hepatolgy, 2021, 26(9): 998-1002.

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