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目的构建乙型肝炎病毒(HBV)复制表达载体,验证其在HepG2细胞系中的复制和表达。方法通过引物设计合成,以实验室构建的HBV C1亚型质粒通过PCR、酶切、克隆等分子生物学技术,构建HBV复制表达载体。利用转染试剂(Fugene HD Transfection Reagent)将载体质粒导入HepG2细胞,分别检测乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)和HBV DNA在不同时间点的表达。结果酶切和测序结果均证实载体构建成功,通过瞬时转染细胞证实培养上清液中可检测到HBsAg、HBeAg和HBV DNA。结论利用分子克隆技术成功构建了HBV复制表达载体,可以在HepG2细胞系中复制表达。通过进一步改造,可以为HBV表型耐药以及复制活性研究提供支持。
Objective To construct hepatitis B virus (HBV) replication expression vector and verify its replication and expression in HepG2 cell line. Methods The HBV replicative expression vector was constructed by primer design and synthesis, and molecular biological techniques such as PCR, restriction enzyme digestion and cloning were used to construct HBV C1 subtype plasmids. The vector was transfected into HepG2 cells with Fugene HD Transfection Reagent to detect the expression of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV DNA at different time points. Results The results of enzyme digestion and sequencing proved that the vector was constructed successfully. HBsAg, HBeAg and HBV DNA were detected in the culture supernatant by transient transfection. Conclusion The HBV replication expression vector was successfully constructed by molecular cloning technology and could be replicated in HepG2 cell line. Through further transformation, it can provide support for HBV phenotype drug resistance and replication activity research.