乙型肝炎病毒DNA转染细胞的一种内参标准:真核细胞报告基因SEAP  

An internal control for transfection of hepatitis B virus gene: eukaryotic cell reporter gene SEAP

作　　者：姚忻[1] 何建文[1] 袁正宏 何丽芳 

机构地区：[1]上海医科大学医学分子病毒学卫生部重点实验室

出　　处：《上海医学》1999年第1期38-41,共4页Shanghai Medical Journal

基　　金：美国中华医学基金

摘　　要：目的建立用真核细胞报告基因ＳＥＡＰ转染人肝癌细胞系（Ｈｕｈ７、ＨｅｐＧ２）及鸡肝癌细胞系（ＬＭＨ）的表达系统，供研究ＨＢＶ基因功能应用。方法分别将带有分泌性碱性磷酸酶（ＳＥＡＰ）报告基因的ｐＢＣ１２／ＰＬ／ＳＥＡＰ及ｐＢＣ１２／ＣＭＶ／ＳＥＡＰ质粒ＤＮＡ转染Ｈｕｈ７、ＨｅｐＧ２及ＬＭＨ细胞，并用ｐＢＣ１２／ＣＭＶ／ＳＥＡＰ质粒ＤＮＡ与Ｓ基因区变异体的克隆ＨＢＶＤＮＡ共转染ＨｅｐＧ２细胞。培养后收取细胞培养液，用ＥＬＩＳＡ法测定ＳＥＡＰ活性及ＨＢｓＡｇ。结果在三种细胞中ｐＢＣ１２／ＣＭＶ／ＳＥＡＰ均可表达，以７２小时表达的酶活性测定最为适宜；而ｐＢＣ１２／ＰＬ／ＳＥＡＰ表达仅处于低水平。以ＳＥＡＰ表达量作为内参对ＨＢｓＡｇ的表达量进行比较，发现Ｓ基因１２９Ｌ变异体表达ＨＢｓＡｇ量显著高于野毒株。结论ＳＥＡＰ作为内参具有快速简便，不需同位素及特殊仪器的优点，有较高的实用价值。Objective To develop transfection and assay system of eukaryotic reporter gene (SEAP) in human hepatoma cell lines and chicken hepatoma cell line, as internal standard control for studies of hepatitis B virus gene function. Methods DNA from two plasmid consturcts (pBC 12 /PL/SEAP,pBC 12 /CMV/SEAP), were separately used to transfect Huh 7, HepG 2 and LMH cell lines and supernatant samples were collected from cultures and assayed for SEAP activity. DNA from cloned HBV contructs with mutation in the S region were used to cotransfect with the reporter gene. Results pBC 12 /CMV/SEAP could be exprexxed in hepatoma cell lines and reached a plateau 72 hours after transfection, whereas, pBC 12 /PL/SEAP was expressed at very low level. Cotransfection of DNA from cloned HBV constructs with mutation in the S region together with the DNA of reporter gene, showed that the expression level of SEAP could be sued as the internal control for the efficiency of transfection. Compared to the wild tpye virus, it was found that mutations in codons 145(Arginine) and 129 (Leucine) resulted in lower and higher level of HBsAg expression respevtively. Conclusion As an internal control, SEAP has the advantages of rapid, simple, and does not need isotopes and specical equiment. Therefore, this internal control can be used to study the structure and function of hepatitis B genome.

关 键 词：肝肿瘤 乙型肝炎病毒 基因转染 SEAP 

分 类 号：R735.702[医药卫生—肿瘤] R373.21[医药卫生—临床医学]