人FⅧ基因高效表达质粒的构建及其在Cos-7细胞中的表达

目的：构建人ＦⅧ真核表达质粒——ｐＡｄＣＭＶＬｉｎｋＦⅧＤＢ并观察其在Ｃｏｓ－７细胞中的表达活性。方法：采用缺失大部分Ｂ结构域的人凝血因子ⅧｃＤＮＡ（ＦⅧＤＢ），长度为４．６ｋｂ，将其插入含腺病毒序列表达质粒——ｐＡｄＣＭＶＬｉｎｋ１，构建了ＦⅧ真核表达质粒——ｐＡｄＣＭＶＬｉｎｋＦⅧＤＢ，用脂质体介导法将其转染Ｃｏｓ－７细胞，转染后２４，４８，７２小时分别用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）、ＥＬＩＳＡ法及一期法测定培养细胞中ＦⅧＤＢｍＲＮＡ及ＦⅧ的含量与活性。结果：ＲＴ－ＰＣＲ法可检测到ＦⅧＤＢｃＤＮＡ转录的ｍＲＮＡ，ＥＬＩＳＡ法测得７２小时后ＦⅧ的含量为每２４小时１８ｎｇ／１０６细胞，一期法测得活性为每２４小时０．６Ｕ／１０６细胞，相当于正常人血浆中１００μｇ／ＬＦⅧ所产生的凝血活性的６０％。结论：所构建的ＦⅧ真核表达质粒在Ｃｏｓ－７细胞中具有一定的表达活性。

Construction of a human factor VIII gene-containing plasmid and its expression in Cos-7 cells]

OBJECTIVE: To construct an eukaryotic expressing plasmid--pAd CMV Link F VIII DB and express it in Cos-7 cells. METHODS: An eukaryotic expressing plasmid--pAd CMV Link F VIII DB was constructed by inserting human factor VIII cDNA (F VIII DB, 4.6 kb), in which most part of B domain was deleted, into an adenovirus sequence-containing plasmid, and then Cos-7 cells were transfected with the constructed plasmid by liposome-mediated gene transfer method. F VIII DB mRNA, F VIII: Ag and F VIII: C in the transfected Cos-7 cells were assayed by RT-PCR, ELISA and one-stage method, respectively, at 24, 48 and 72 hours (hrs) after transfection. RESULTS: F VIII DB mRNA was positive and, F VIII: Ag and F VIII: C were 18 ng/10(6) cells/24 hrs and 0.6 U/10(6) cells/24 hrs, respectively at 72 hrs after transfection, which was comparable to 60% of the activity produced by 100 micrograms/L F VIII in normal human plasma. CONCLUSION: The constructed plasmid is proved to be expressed in Cos-7 cells.