不同启动子引导的人凝血因子Ⅷ基因载体在HC11细胞和小鼠乳腺中表达的研究  

A Study on the Expression of hFⅧ Gene Vectors Directed by Differ- ent Promoters in HCll Cells and Mouse Mammary Gland

作　　者：王晴[1] 任晓叶[1] 蔡勤[1,2] 龚秀丽[1,2] 黄英[1,2] 曾溢滔[1,2] 

机构地区：[1]上海市儿童医院,上海交通大学附属儿童医院,上海医学遗传研究所,上海市200040 [2]卫生部医学胚胎分子生物学重点实验室,上海市胚胎与生殖工程重点实验室,上海市200040

出　　处：《医学分子生物学杂志》2012年第5期313-319,共7页Journal of Medical Molecular Biology

基　　金：国家高技术研究发展计划（863计划）（No.2011AA100602）（致谢感谢黄淑帧、任兆瑞教授对本文工作提出的宝贵建议.）

摘　　要：目的应用山羊β-酪蛋白基因启动子（P1A3），构建真核细胞表达人凝血因子Ⅷ（humancoagu—lationfactorⅧ，hFⅧ）全长cDNA载体（pcDNA3．1．P1A3-hFⅧ）和hFⅧB区缺失（humancoagulationfactorⅧB—domain．deleted，hFVⅧBD）的cDNA载体（pcDNA3．1-P1A3-hFⅧBD），研究它们在乳腺细胞中的表达情况，同时与巨细胞病毒（cytomegalovirus，CMV）启动子引导的相应的hFⅧ载体进行比较。方法采用常规DNA分子克隆技术构建人凝血因子Ⅷ（hFⅧ）基因载体，转染小鼠乳腺上皮细胞（HC-11），应用RT—PCR以及real—timePCR技术检测hFⅧ基因转录本；对NSL期母鼠乳腺注射DNA载体后，采集试验母鼠乳汁，应用ELISA检测乳腺细胞分泌hFⅧ蛋白的瞬时表达水平。结果应用构建的CMV启动子和P1A3启动子指导的hFⅧ基因载体转染HC-11细胞后，在mRNA水平均有表达；瞬时转染哺乳期母鼠的乳汁检测表明，转染CMV和P1A3启动子的hFⅧ载体的母鼠，乳汁中的hFⅧ蛋白表达量分别为2．81μg／ml（CMV—hFⅧBD），2．01μg/ml（CMV—hFⅧ），1．82μg／ml（P1A3-hFⅧBD），1．20μg／ml（P1A3．hFⅧ）。结论构建的乳腺特异性表达hFⅧ基因的载体，转染HC-11细胞和乳腺组织后的mRNA及蛋白表达水平均证实了该载体的有效性：CMV启动子引导的hFⅧ载体表达虽稍高于P1A3启动子载体，但由于后者具有乳腺特异表达的特点，更适用于乳腺生物反应器。我们构建hFⅧ基因的载体的经验，为乳腺生物反应器的研制提供了有价值的科学依据。Objective To construct mammary specific expression vectors containing human co- agulation factorⅧ （hFⅧ） full-length eDNA or B-domain-deleted eDNA driven by goat B-casein promoter （ P1 A3 ） , and to investigate their expression in mammary gland cells. The expression levels in mouse mammary epithelial （HCI1） cells and mouse mammary glands transfected with vectors di- rected by CMV or P1 A3 promoters were compared. Methods The construction of the vectors was performed according to the conventional molecular cloning methods. The vectors were transfected into HC-11 cells using Lipofectamine 2000. hFⅧ transcripts were detected by RT-PCR and real time PCR. The hFⅧ vectors were injected into mouse mammary glands during lactating period, and the milk was collected for the transient expression detection of hFⅧ protein by ELISA. Results RT- PCR and real-time PCR analysis showed the specific hFⅧ transcripts in the transfected HCll cells. ELISA results demonstrated that hFⅧ protein in the milk can be expressed at a variety of a- mount after transient injection of the vectors to mouse mammary glands, with the hFⅧ contents of 2.811μg/ml （CMV-hFⅧBD）, 2.011μg/ml （CMV-hFⅧ）, 1.82μg/ml （P1A3-hFⅧBD）, and 1. 20μg,/ml （P1 A3-hFⅧ） , respectively. Conclusion hFⅧ vectors can be effectively expressed in HC11 cells as well as mouse mammary glands. Our results indicate that, although the hFⅧ expres- sion level of CMV-direeted vectors was slightly higher than that of P1A3-directed vectors, the mam- mary gland-specific hFⅧ vectors provide valuable scientific information for the generation of mam- mary gland bioreactor.

关 键 词：人凝血因子Ⅷ 哺乳动物乳腺 启动子 特异表达 

分 类 号：R349.6[医药卫生—基础医学]