线粒体DNA缺失质粒pMDD-Z的构建及鉴定  

Construction and identification of mitochondrial DNA deficient plasmid pMDD-Z

作　　者：张朝明[1] 赵文霞[1] 汤树生[1] 靳溪[2] 张婷[3] 刘凤英[1] 肖希龙[1] 

机构地区：[1]中国农业大学动物医学院国家兽药安全评价中心,北京海淀100193 [2]北京师范大学生命科学学院,北京西城100875 [3]中国科学院北京基因组研究所,北京朝阳100029

出　　处：《中国兽医杂志》2014年第1期14-17,共4页Chinese Journal of Veterinary Medicine

摘　　要：分别以pEGFP-Mito和pAN4质粒为模板,扩增出MTS-EGFP和EcoRⅠ基因片段,利用快速体外基因重组技术-重组PCR,将两者连接得到MTS-EGFP-EcoRⅠ片段,构建克隆载体pTRE2hyg-EE,经AgeⅠ和NotⅠ双酶切得到MTS-EGFP-EcoRⅠ融合基因产物,连接克隆于真核表达质粒pEGFP-Mito后获得pMDD-Z质粒。琼脂糖凝胶电泳结果显示,MTSEGFP和EcoRⅠ基因片段大小分别是831 bp和860 bp。测序结果显示,插入pTRE2hyg--EE克隆载体中的MTS-EGFPEcoRⅠ片段大小1 672 bp,且基因序列正确,没发生点突变。琼脂糖凝胶电泳结果显示,pMDD-Z质粒的双酶切产物分别为1 587 bp的插入片段和4 024 bp的载体片段。测序结果显示,pMDD-Z质粒中含1 659 bp的开放阅读框,编码553个氨基酸,从N端至C端,分别为线粒体信号导肽、绿色荧光蛋白和限制性内切酶EcoRⅠ,且未发生移码突变。To obtain the mitochondrial DNA deficient plasmid pMDD-Z, the MTS-EGFP and EcoR I gene fragments were amplified through a rapid in vitro recombinant DNA technology-the recombinant PCR, utilizing pEGFP- Mito and pAN4 plasmids as templates, followed by subcloned into a cloning vector pTRE2hyg to obtain the plasmid pTRE2hyg- EE , which was then digested with Age I and Not I restriction enzyme to acquire MTS-EGFP-EcoR I fusion gene product. Subsequently, the fusion gene product was cloned into the eukaryotic expression plasmid pEGFP- Mito to derive pMDD- Z plasmid. Agarose gel electrophoresis showed that the size of MTS- EGFP and EcoR I gene fragments were 831 bp and 860 bp , respectively. Sequence analysis showed that the size of MTS- EGFP- EcoR I fragment which inserted into the vector pTRE2hyg was 1 672 bp , and the gene sequence was also correct. Agarose gel electrophoresis showed that the double digestion products of pMDD-Z plasmid included inserted fragments of 1 587 bp and vector fragments of 4 024 bp. The sequencing results showed that pMDD-Z plasmid contained an open reading frame of 1 659 bp , encoding 553 amino acids which were comprised of the mitochondrial signal leader peptide, green fluorescent protein and restriction endonuclease EcoR I,from the N-terminal to C-terminus.

关 键 词：线粒体DNA Rho^0细胞 克隆 重组PCR 质粒构建 

分 类 号：S852.23[农业科学—基础兽医学]