筛选喉癌相关基因LCRG1的有效沉寂序列  

Screening the effective target sequences of laryngeal carcinoma related gene LCRG1

作　　者：段朝军[1] 蒋铁斌[2] 李萃[1] 章晓鹏[1] 李茂玉[1] 肖志强[1] 汤参娥[1] 易红[1] 陈主初[1] 

机构地区：[1]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410008 [2]中南大学湘雅三医院血液科,长沙410013

出　　处：《中南大学学报（医学版）》2008年第6期468-475,共8页Journal of Central South University ：Medical Science

基　　金：国家“973”计划(2001CB510207);国家自然科学基金项目(30500558,30670990);教育部新世纪优秀人才培养计划基金(教育部科技函[2007]70)~~

摘　　要：目的:拟采用RNA干扰技术筛选有效的针对LCRG1基因的打靶序列。方法:首先采用PCR定点突变技术改造pSuper载体,而后利用该改造后的载体构建针对LCRG1基因的5对打靶序列的真核表达载体。将构建的重组pSuper362,398,432,789,903表达载体和pSuper空白载体分别转染He-la细胞,经抗性药物筛选获得抗性细胞克隆和池克隆;通过RT-PCR及荧光定量PCR鉴定阳性克隆,进行平板克隆集落形成试验,以检测打靶序列沉寂LCRG1基因mRNA表达水平的效果。结果:应用PCR定点突变技术改造的pSuper载体,可被BglⅡ酶切;利用RT-PCR和荧光定量PCR检测各重组载体转染细胞池克隆LCRG1mRNA的表达发现,362组,398组,432组的基因均能封闭内源性LCRG1基因表达,尤以362组为显著;鉴定362组筛选的各个抗性克隆,发现A2和A5克隆的LCRG1mRNA表达水平明显降低。平板克隆实验结果提示362组的A2,A5和池克隆的细胞增殖能力明显强于载体和空白对照组(P<0.05)。结论:成功改建了pSuper真核表达载体;362siRNA相对其他siRNA具有较好的打靶效果,这对于应用RNAi方法研究LCRG1基因的功能和其作用分子机制具有重要的指导意义。Objective To screen the effective target sequences of laryngeal carcinoma related gene LCRG1 using RNAi.Methods PCR site mutation method was used to reconstruct pSuper vector.Five pairs of siRNA sequences designed by siRNA software were annealed and inserted into the reconstructed pSuper vector.The reconstructed pSuper 362,398,432,789,903,and pSuper vectors were transfected into Hela cell lines and selected with the appropriate drugs to get resistant and pool cells,respectively.The colonies were identified by RT-PCR or real-time RT-PCR analysis.The silence effects were observed by cloning formation analysis.Results pSuper vector was reconstructed to restore BglⅡ restriction enzyme sites using PCR mutation.The RT-PCR or real-time RT-PCR results of pool clones showed 362,398,and 432 pool clones all had better effects of LCRG1 gene-silence,especially 362 pool clones.The expression level of LCRG1 mRNA of selected 362 group anti-puromycin clones A2 and A5 was decreased.The results of clone forming efficiency revealed that the cellular proliferation in A2 of 362 group was significantly higher than that of the vector and control Hela cells（P〈0.05）.Conclusion The reconstructed pSuper vector is successfully constructed.The 362 group has better gene silence and has 2 effective 362 group anti-clones,suggesting that methodology has important values in studing the function and molecular mechanism of LCRG1.

关 键 词：LCRG1基因 PCR定点突变 RNAI 

分 类 号：R739.65[医药卫生—肿瘤]