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

目的 建立用真核细胞报告基因ＳＥＡＰ转染人肝癌细胞系及鸡肝癌细胞系（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ＨＢＶ ＤＮＡ共转染ＨｅｐＧ２细胞。培养后收取细胞培养液，用ＥＬ

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

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.