摘要
目的构建Bag-1短发卡RNA（shRNA）干扰载体并通过内源性筛靶实验检测Bag-1基因mRNA和蛋白的表达，筛选出最佳的干扰靶序列。方法应用干扰技术构建Bag-1shRNA干扰载体，在细胞转染预实验中将构建好的带有绿色荧光蛋白的质粒转染Lovo细胞，通过观察绿色荧光蛋白来判断转染的效率，分别应用逆转录一聚合酶链反应（RT—PCR）和Westernblot法检测Bag-1在LoVo细胞中的表达。在shRNA干扰序列筛选实验中，通过定量PCR和Westernblot法来检测构建的干扰载体在LoVo细胞中的表达。结果重组质粒分别经双酶切分析，琼脂糖凝胶电泳鉴定可见清晰地切出与Bag-1及pGPHl／GFP／Neo载体大小相符的片段并与DNAMarker相符。用带绿色荧光基因的pGPHl／GFP／Neo-shNC质粒转染LoVo细胞，在转染后的第24、48及72小时分别观察到逐渐增强的绿色荧光蛋白的表达。分别应用RT—PCR和Westernblot法检测Bag-1在LoVo细胞中的表达，可见Bag-1在LoVo细胞中的mRNA和蛋白表达水平均较高。在Bag-1有效shRNA干扰序列筛选实验中，综合定量PCR及Westernblot内源筛靶结果，筛选出Bag-1-homo-825作为最佳干扰靶序列。结论成功构建针对小鼠Bag-1基因的特异性shRNA质粒，获得稳定转染的结肠癌Lovo细胞株，结果证明所构建的pGPHl／GFP／Neo—Bag-1-homo-825作为最佳干扰靶序列，显著抑制Lovo细胞Bag-1mRNA和蛋白的表达。
Objective To construct the short hairpin RNA （shRNA） plasmids carrying Bag-1 gene of human and test the expression of Bag-1 mRNA and protein. Methods Recombinant plasmids con- taining green fluorescent protein reporter genes were constructed using gene cloning methods. The shRNA plasmids for Bag-1 gene were constructed by RNA interference technology. Fluorescent plasmid-transfected target cells were used in the cell transfection experiments, and the transfection efficacy of plasmids was an- alyzed by observing the fluorescence amount. Three synthetized shRNAs were transfected into target screen- ing cells, and reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting were used to identify the difference of target gene transfection and translation level in cells. Results Tl^e specific shRNA plasmid for Bag-1 gene was successfully recombinanted and stabty transfected colon cancer LoVo cell lines were obtained. The constructed shRNA plasmids significantly inhibited the expression of Bag-1 mRNA and protein of Lovo cells, and could maintain the effect for a long term. Conclusion pGPH1/GFP/ Neo-Bag-l-homo-825 was screened as the optimum sequence of interference, and can significantly inhibit the expression of Bag-1 mRNA and protein.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第12期2503-2506,共4页
Chinese Journal of Experimental Surgery
基金
山东省自然科学基金资助项目（ZR2012HM002）
中国博士后科学基金资助项目（20100481271）
山东大学自主创新（交叉）基金资助项目（2011JC016）
