摘要
目的通过构建具有HA标签的DJ-1 L166P突变体(DJ-1 L166P-HA),建立一种快速构建具有标签的突变体的方法——重叠法联合长引物法。方法 PCR分别扩增DJ-1的N端和C端。扩增C端的反向引物DJ-1-HA-R包含HA标签的编码序列,是具有60个核苷酸的长引物。然后,DJ-1的N端和C端重叠延伸获得DJ-1 L166P-HA全长。最后,克隆DJ-1 L166P-HA至质粒pcDNA3.1(+)。结果只需一次连接反应,成功地构建了具有HA标签的DJ-1 L166P突变体的重组质粒pcDNA3.1(+)-DJ-1 L166P-HA。将该重组质粒转染HEK293细胞后,DJ-1 L166P-HA融合蛋白质成功表达。结论重叠法联合长引物法具有简便、快捷,易掌握等特点,具有良好的应用前景。
[ Objective ] To establisha rapid method for constructing mutant with tag, overlap method combined with long-primer method, by constructing DJ-1 L166P mutant with HA tag（DJ-1 L166P-HA）. [Methods] DJ-1 N terminus and C terminus were amplified by PCR. The reverse primer for amplifying DJ-1 C terminus, DJ-1-HA-R, was a 60 nucleotides primer which included the coding sequence of HA tag. Then, the DJ-1 L166P-HA full-length sequence was obtained by overlap extension of DJ-I N terminus and C terminus. At last, the DJ-1 L166P-HA frag- ment was cloned to plasmid pcDNA3.1 （＋）. [Results] The recombinant plasmid, pcDNA3.1 （＋）-DJ-1 L166P-HA, which has DJ-1 L166P mutant with HA epitope, was successfully constructed by one time ligation. After the recombinant plasmid transfected HEK293 cell, the DJ-1 L166P-HA fusion protein was expressed successfully. [Conclusions ] The overlap method combined with long-primer methodis characterized by concise, fast and easy to be mastered.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2014年第19期6-9,共4页
China Journal of Modern Medicine
基金
国家自然科学基金(No:31100764)
湖南省自然科学基金(No:14JJ4024)
中南大学中央高校基本科研业务费-青年助推项目(No:2011QNZT131)
湖南省科技计划(No:2013FJ6067)
中南大学大学生创新训练项目(No:CY12373)
