大鼠寡霉素敏感相关蛋白(OSCP)基因的体外扩增及克隆

Amplification and Cloning of Rat OSCP Gene

目的 构建大鼠寡霉素敏感相关蛋白 (OSCP)基因重组质粒。方法 从雌性SD大鼠脑组织中提取总RNA ,根据OSCP基因已知序列 ,设计合成 1对引物 ,用PCR方法扩增编码OSCP的基因片段 ,插入pGEM Teasy质粒 ,转化大肠杆菌JM1 0 9感受态细胞 ,经酶切鉴定 ,而后进行测序。结果 OSCP基因体外扩增产物大小为 70 0bp ,重组质粒经酶切鉴定表明获得正确重组子 ,测序结果与已知序列基本吻合。结论 在国内初次克隆了大鼠OSCP基因 。

Objective To construct a recombinant plasmid containing oligomycin sensitivity conferring protein (OSCP) gene of rat. Methods Total RNA was extracted from the rat brain tissue and used as templates for RT PCR. Primers were designed based on the published sequence of the OSCP gene and used to amplify the rat OSCP cDNA using RT PCR method. The OSCP gene obtained by PCR technique was directionally cloned into plasmid pGEM T easy vector. The constructed recombinant plasmid pGEM T easy OSCP was transferred into E.coli JM109. The transformants were screened and identified by restriction analysis. The DNA sequence of the gene was determined by Sanger′s method. Result The size of amplified OSCP gene was 700bp. The correct recombinant plasmid pGEM T easy OSCP was isolated and confirmed by restriction analysis. The DNA sequence of cloned gene was the same as the published sequence. Conclusion The OSCP gene successfully amplified and cloned into plasmid pGEM T easy vector. The cloned OSCP gene could be used to produce recombinant protein and for the study of its function.