非全长型人卵透明带-3蛋白慢病毒表达载体的构建及其在中国仓鼠卵巢细胞中表达的研究  

Expression of Nonfull-long Human Zona Pellucida-3 in CHO Cell with Lentiviral Vector

作　　者：陈小佳[1] 李丽玲[1] 朱伟杰[2] 谢秋玲[1] 洪岸[1] 李菁[3] 徐万祥[4] 

机构地区：[1]暨南大学生物工程研究所,广州510632 [2]暨南大学生殖免疫研究中心,广州510632 [3]暨南大学医学院,广州510632 [4]上海市计划生育科学研究所,上海200032

出　　处：《生殖与避孕》2007年第3期161-165,177,共6页Reproduction and Contraception

基　　金：广东省科技计划项目(2001C12001)

摘　　要：目的:构建含非全长型人卵透明带-3蛋白(hZP3)的慢病毒载体,转染293FT细胞获得重组慢病毒颗粒,感染中国仓鼠卵巢细胞(CHO)使其表达hZP3。方法:将hZP3基因序列插入到慢病毒系统的入门载体pENTR-11中构建为pENTR-hZP3,采用LR重组酶将pENTR-hZP3和pLenti6/V5-DEST进行重组反应,形成新重组载体pLenti6/V5-DEST-hZP3。将该重组载体和其它viruspower 3个质粒载体充分混合,用阳离子脂质体转染293FT细胞,培养和待细胞完全裂解后收集富含hZP3基因的病毒颗粒上清液,取适量上清液感染CHO-K1细胞,采用杀稻瘟Blastcidin筛选,挑单克隆对其扩增培养,进行基因水平和蛋白水平的鉴定。结果:获得的含hZP3基因的病毒颗粒上清液滴度为1×105TU/ml,随机挑取的16株单克隆细胞中,有9株表达hZP3。结论:成功构建含hZP3的慢病毒表达载体,获得表达hZP3的CHO株,为后续研究与开发奠定了基础。Objective： To construct lentiviral expression vector with the nonfull-long human zona pellucida- 3 （hZP3） DNA, and to express the hZP3 protein in Chinese hamster ovary cell（CHO）. Methods： pENTR-hZP3 was constructed by cloning hZP3 DNA into pENTR-11 vector. Then, pLenti6/VS-DEST-hZP3 was obtained by reacting between pENTR-hZP3 and pLenti6/V5-DEST with LR enzyme. The pseudoviral particles containing the expressed construct were generated by lentiviral packaging system in 293FT cells, which were infected CHO-K1 to select monoelonal cells by using blastcidin added. The expression of hZP3 was determined by both mRNA and protein level. Results： The pseudoviral particles with hZP3 DNA were obtained, and the titer was up to 1 × 10^5 TU/ml. Among 16 monoclonal ceils randomly selected, 9 cell lines were expressed hZP3. Conclusions： The lentiviral vector with hZP3 DNA was successfully constructed. The CHO with expressing hZP3 was successfully obtained. These results could benefit to subsequent hZP3 research and its application.

关 键 词：人卵透明带 慢病毒载体 中国仓鼠卵巢细胞 表达 

分 类 号：Q78[生物学—分子生物学]