乙型肝炎病毒C基因逆转录病毒载体的构建及在永生化人外周血B细胞中的表达

Construction of hepatitis B virus C gene retroviral vector and its expression in immortalized human peripheral blood B cell lines

目的 体外研究乙型肝炎病毒Ｃ（ＨＢＶ／Ｃ） 基因变异的生物学意义。方法 应用逆转录病毒载体ｐＸＴ１ 构建ＨＢＶ／Ｃ 基因表达载体，并转染永生化人外周血Ｂ 细胞使之稳定表达ＨＢｃＡｇ。结果 重组质粒ｐＸＴ１ＨＢＶ／Ｃ 经聚合酶链反应（ＰＣＲ） 和ＢｇｌⅡ及ＸｈｏⅠ双酶切鉴定均阳性，转染后的永生化人外周血Ｂ 细胞ＰＣＲ 鉴定含目的ＤＮＡ，流式细胞仪分析显示，约４７ ．４ ％ 的细胞膜上表达了ＨＢｃＡｇ。结论 ＨＢＶ／Ｃ基因逆转录病毒表达载体有较高的转染效率，目的基因能在宿主细胞中稳定表达，有利于系列研究ＨＢＶ／Ｃ基因变异的生物学意义。

Objective In order to study the biological significance of HBV/C gene variant in vitro. Methods Retroviral vector pXT1 was used to construct the HBV/C gene expression vector and the recombinant plasmid pXT1 HBV/C was used to transfect immortalized human peripheral blood B cell lines to express HBcAg steadily in the host cells.Results plasmid pXT1 HBV/C was detected positive by PCR as well as enzyme digestion with BglⅡ and XhoⅠ. Meanwhile, transfected cells was detected to contain HBV/C gene by PCR and HBcAg was expressed in 47.4% of the cells by means of flow cytometry. Conclusion Retroviral expression vector with HBV/C gene can transfect into eucaryotic cells effectively and express the goal gene steadily. The recombinant cells may be used in the systematic studies on the biological significance of HBV/C gene variant.