部分替换法构建SCN 1A基因突变载体  

The construction of SCN1A mutational vector by partial replacement method

作　　者：刘超 段现来 肖乐 王爱民 曾乐平[3] 

机构地区：[1]长沙市第一医院,湖南长沙410005 [2]长沙市第三医院,湖南长沙410015 [3]中南大学湘雅医学院,湖南长沙410013

出　　处：《湘南学院学报（医学版）》2015年第2期1-4,共4页Journal of Xiangnan University（Medical Sciences）

基　　金：国家自然科学基金资助(81100663);教育部博士点新教师基金资助(20100162120072)

摘　　要：目的构建SCN1A基因突变载体。方法首先将要含有需要突变位点的SCN1A基因片段克隆到p GEM-11Zf(+)载体,定点诱变试剂盒对其进行定点诱变,经测序筛选阳性克隆。然后再将重组质粒上的突变片段替换PCMV-SCN1A表达质粒上的对应片段,经测序筛选阳性克隆。结果成功构建了SCN1A基因突变载体,SCN1A基因第377位精氨酸密码子(CGA)突变为谷酰胺密码子(CAA)。结论该方法先将目的基因部分片段在小质粒上进行定点诱变,然后再替换到大质粒,减少了意外突变的可能性,适合于较大载体的定点诱变。Objective To construct the SCN1A mutational vector. Methods The SCN1A fragment which contained the mutation site was subeloned into the pGEM-11Zf（＋） vector. Its Mutational vector was per- formed by site-directed mutagenesis kit. The correct clones were screened by sequencing analyses. The frag- ment from SCN1A expressing construct was replaced by mutant fragment by a series of enzyme digesting and ligation action. The correct clones were screened by sequencing analyses. Results The SCN1A mutational vector was successfully constructed and mutation was identified at the directed site as SCN1A R377Q. Con- clusion The site-directed mutagenesis of partial gene fragment on small plasmid was first performed, and then the mutant fragment was replaced into the large plasmid. Partial replacement method is suitable for site directed mutagenesis of unstable and large plasmids, because short fragment amplified by PCR will reduce efficiency of unexpected mutations.

关 键 词：突变 SCN1A 癫痫 

分 类 号：R742.1[医药卫生—神经病学与精神病学]