利用反式剪接核酶修复突变绿色荧光蛋白mRNA

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

为构建修复突变绿色荧光蛋白(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。

To construct a trans-splicing ribozyme used to repair mutant green fluorescence protein （GFP） gene, the mutant GFP gene express recombinant plasmids XYQS/10-pGEM, XYQS/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 XYQS/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 XYQS/10-pEGFP-C2 and trans-rib-CMV2 recombinant plasmid. The results appeared that XYQS/10-pGEM, XYQS/10-pEGFP-C2 and trans-rib-CMV2 recombinant plasrnid 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.