摘要
[目的]构建人死亡结构域相关蛋白(hDaxx)的干扰载体,为研究hDaxx在HPV致宫颈癌发生与发展过程中的作用提供实验基础。[方法]以hDaxx全基因序列为模板,设计hDaxx RNA干扰引物并扩增干扰片段,将其连接到pGPU6/GFP/Neo真核表达载体,构建pGPU6/GFP/Neo-SiDaxx干扰载体,转染至HeLa细胞,利用荧光显微镜观察转染效率以及通过RT-PCR、Western Blot检测siRNA对HeLa细胞中hDaxx表达的影响。[结果]在转染了pGPU6/GFP/Neo-Si Daxx载体的HeLa细胞中,其hDaxx mRNA和蛋白的表达水平与空载体对照组比较明显降低。[结论]成功构建了hDaxx的干扰载体pGPU6/GFP/Neo-Si Daxx,该干扰载体能抑制HeLa细胞中hDaxx mRNA基因和蛋白的表达。
[ Objective] To construct the interference vectors for human Death domain associated protein, and provide the experimental foundation about the function of hDaxx on the development of cervical cancer. [ Methods ] Using the hDaxx genome as template, the interference primers are designed, and the interference fragments for hDaxx RNA are amplified, and connected with pGPU6/GFP/Neo eukaryotic expression vectors for constructing the pGPU6/GFP/Neo - SiDaxx interference vectors. The vectors are digested by endonucleases, and therefore identified for sequencing. The interference vectors are transfected into HeLa cells, and the ratios of transfection are detected by dfluorescence microscopy, and the effect of siRNA on the expression of hDaxx in the HeLa cells is tested by RT- PCR, Western Blot. [ Results ] Compared with the blank vectors,, the mRNA espression and protein expression of hDaxx were significantly decreased in the Hela cells with transfeeted pGPU6/GFP/Neo - SiDaxx. [ Conclusion] The interference vectors for hDaxx pGPU6/GFP/Neo -SiDaxx are successfully constructed, and the interference vectors could inhibit the mRNA expression and protein expression of hDaxx in HeLa cells
作者
余斓
唐双阳
申海艳
伍虹蓉
朱翠明
万艳平
Lan Yu;Shuangyang Tang;Haiyan Shen;Hongrong Wu;Cuiming Zhu;Yanping Wan(Institute of Pathogenic Biology,University of South Chin;Hunau Provincial Key Laboratory for Special Pathogens Prevention and ControL,Hengyang 421001,China)
出处
《生物技术》
CAS
2018年第4期341-344,371,共5页
Biotechnology
基金
国家自然科学基金项目("人乳头瘤病毒16型E2蛋白对Daxx调控细胞凋亡的影响"
No.81402169)
湖南省自然科学基金项目("Daxx对人乳头瘤病毒16型E2蛋白转录调控E6和E7的影响"
No.2018JJ3452)
特殊病原体防控湖南省重点实验室资助项目(湘科计字[2014]5号)
湖南省分子靶标新药研究协同创新中心资助项目(湘教通[2015]351号)
关键词
死亡结构域相关蛋白
干扰
表达
抑制
death domain associated protein
interference
express
inhibit
