【作者】 刘祖龙； 刘起展； 李爱萍； 周建伟；

【Author】 LIU Zulong, LIU Qizhan, LI Aiping, ZHOU Jianwei.Department of Molecular Toxicology, Public Health School, Nanjing Medical University, NanJing 210029, China

【机构】 南京医科大学公共卫生学院分子毒理实验室；

【摘要】 [目的] 构建JWA反义RNA真核表达载体pEGFP-C1-asJWA,建立并鉴定JWA缺陷细胞株,并观察该缺陷细胞的某些生物学效应,用于JWA功能及有关生物学意义的研究。[方法] 利用RT-PCR方法扩增出JWA基因中一段长约339bp的目标片断,经由pGEM-T载体反向插入绿色荧光蛋白表达载体pEGFP-C1中,从而构建JWA基因反义RNA真核表达载体pEGFP-C1-asJWA。并用该载体转染Hela细胞,用蛋白免疫印迹法鉴定转染细胞中JWA的表达水平。同时观察转染细胞生长形态,绘制生长曲线,分析细胞周期。[结果] pEGFP-C1-asJWA载体在转染细胞内可较稳定表达,JWA蛋白缺陷细胞株JWA的蛋白表达水平下降了42%,转染后JWA缺陷细胞生长形态、生长速度及细胞周期无明显变化。[结论] JWA蛋白缺陷细胞株成功建立和鉴定,且该缺陷不足以单独引起可观察的某些生物学效应,为JWA功能研究及有关生物学意义的研究提供了一种有效细胞株。更多还原

【Abstract】 [Objective] To construct the pEGFP-C1-asJWA vector, an eukaryotic expression plasmid of JWA antisense RNA and to construct JWA protein deficient cell strain which can be used in studying the functions and biological effects of JWA gene and observe the some biological characters induced by JWA protein deficiency. [Methods] A 339bp fragment of JWA gene was amplified by RT-PCR and then was inserted and subcloned into pEGFP-C1 vector reversedly after had been cloned into pGEM-T vector. The pEGFP-C1-asJWA vector was constructed.Then, Hela was transfected with pEGFP-C1-asJWA to construct JWA protein deficient cells(named as "asJWA-Hela"). The protein expression levels of JWA gene in Hela and asJWA-Hela were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected asJWA-Hela were observed. [Results] pEGFP-C1-asJWA vector was successfully expressed in Hela. The protein expression level of JWA gene in asJWA-Hela was decreased 42% as compared with that in C1-Hela. No obvious changes of the biologic characters were observed in asJWA-Hela. [Conclusion] The JWA protein deficient cell strain was successfully constructed which offered an effective cell strain for studying the function and biological effects of JWA gene. Only the JWA protein deficient didn’t induced obvious changes of the biological characters in asJWA-Hela.更多还原