摘要
【目的】克隆人类DNA聚合酶 (pol β)基因全长cDNA ,构建该基因的真核高效表达载体。【方法】抽提人胚肺纤维细胞HLF总RNA进行RT PCR扩增 ,用pGEM T载体经T/A克隆、DNA测序确定后 ,用双酶切将 pol β全长cDNA定向克隆到绿色荧光蛋白表达载体 pEGFP C1,转染大肠杆菌DH5α ,筛选阳性克隆 ,双酶切鉴定重组质粒。【结果】经RT PCR获得 1 0 3kb的产物 ,经DNA测序 ,确定所扩增片段为人类 pol β基因全长cDNA ,进而成功筛选出真核表达载体 pEGFP C1 β。【结论】成功构建人 pol β基因全长cDNA真核表达载体 。
To construct an efficient Eukaryotic expression vector of human DNA polymerase β(pol β). Following total RNA being extracted from human embryo lung fibroblast HLF and RT PCR being carried out, the PCR product was cloned into pGEM T vector. The recombinant plasmid certified by sequencing. The pol β entire cDNA was cloned into expression vector pEGFP C1,and then transferred into E.coli DH5α. The recombinant plasmids were identified by restriction endonuclease enzyme analysis 1 03 kb fragment containing restriction sites on 5′ stream was obstained through RT PCR. The object fragment is homology with pol β. The recombimant plasmid pEGFP C1 β was successfully selected [Conclusion] The efficient expression vector of human pol β has been constructed, it can be used in establishing the human cell line of overexpression of pol β.
出处
《中山医科大学学报》
CSCD
北大核心
2002年第3期187-189,共3页
Academic Journal of Sun Yat-sen University of Medical Sciences
基金
国家自然科学基金资助项目 (3 0 10 0 15 2 )
