利用反式剪接核酶修复突变绿色荧光蛋白mRNA

为构建修复突变绿色荧七蛋白（GFP）基因的反式剪接核酶，分别构建包含突变的GFP基因的XYQ5／10-pGEM重组质粒、XYQ5／10—pEGFP—C2重组质粒及用于修复该突变基因的反式剪接核酶载体trans—rib—CMV2。通过对体外共转录XYQ5／10—pGEM和trans—rib—CMV2重组质粒的RNA产物进行RT—PCR检测核酶细胞外剪接效果；通过XYQ5／10-pEGFP-C2和trans—rib—CMV2重组质粒共转染HeLa细胞检测核酶细胞内的剪接效果。结果显示，XYQ5／10—pGEM、XYQ5／10-pEGFP-C2及trans—rib—CMV2重组质粒构建成功，反式剪接核酶在细胞外及细胞内都可以修复突变基因。虽然效率不高，但为今后更大规模地研究设计反式剪接核酶打下了基础。

Functional Repair of a Mutant GFP Gene Using a Trans-Splicing Ribozyme

To construct a trans-splicing ribozyme used to repair mutant green fluorescence protein(GFP)gene,the mutant GFP gene express recombinant plasmids XYQ5/10-pGEM,XYQ5/10-pEGFP-C2 and the trans-splicing ribozyme recombinant plasmid trans-rib-CMV2 were constructed.The in vitro trans-splicing function of the trans-ribozyme was detected by RT-PCR with the co-transcribed RNA production of the XYQ5/10-pGEM and trans-rib-CMV2 recombinant plasmid,and the in vivo trans-splicing function of the trans-ribozyme was detected by co-transfection of the HeLa cell with the XYQ5/10-pEGFP-C2 and trans-rib-CMV2 recombinant plasmid.The results appeared that XYQ5/10-pGEM,XYQ5/10-pEGFP-C2 and trans-rib-CMV2 recombinant plasmid were successfully constructed;the mutant GFP gene was repaired by trans-splicing ribozyme,although the efficiency was low.This work will be a good foundation for the farther research of the trans-splicing ribozyme.