【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。更多还原

【Abstract】 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/V5-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 monoclonal 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 × 105 TU/ml. Among 16 monoclonal cells 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.更多还原