HePG2.2.15细胞株中乙型肝炎病毒基因异质性的研究

目的以乙型肝炎病毒(HBV)前C/C基因异质性来探讨在HepG2.2.15细胞体内是否存在HBV准种。方法依据Genebank中HBVayw亚型全基因序列,选择保守区域设计出特异性多聚酶链反应引物,自HepG2.2.15细胞培养上清中扩增HBV前C/C基因序列,克隆入pGEM-TEasy载体,挑选15个克隆测序以比较病毒的变异程度。结果测序结果发现HepG2.2.15细胞HBV前C/C基因存在异质性,碱基序列之间的同源性大于93%,突变类型全部为碱基替换。结论结果提示,长期培养HepG2.2.15细胞内有HBV准种共存,这种细胞模型内HBV异质性现象可能影响其在基因治疗等方面的应用。

Experiment study on the HBV gene heterogeneity in HepG2.2.15 cell

Objective To investigate the HBV quasispecees groups in the long-t erm cultured HepG2.2.15 cell. Methods A set of specific primers was synthesized according to the HBV DNA sequence of the subtype ayw in the GenBank, and the w hole pre-core/core region was amplified by the PCR method from the supernatant of HepG2.2.15 cell. Then the PCR products were subcloned into pGEM-T Easy vectors. Clones to be sequenced were selected, and then the sequence comparison was done to find the difference. Results One sequence of selected clones was found to be different; The homology of the nucleic acid sequence from the same patient was above 93.0%. The mutation type in this region was all substitution. Conc lusion There are HBV quasispecies groups in the HepG2.2.15 c ell. The HBV heterogeneity in the HepG2.2.15 cell may influence the application of the cell on hepatitis B gene therapy.