现代医药卫生2016，Vol.32Issue(20)：3106-3108,3112,4.DOI:10.3969/j.issn.1009-5519.2016.20.004
人LDH-B基因克隆及其真核表达载体的构建
Cloning of human lactate dehydrogenase B gene and construction of its eukaryotic expression vector
摘要
Abstract
Objective To clone human lactate dehydrogenase B(LDH-B) gene and to construct its eukaryotic expression vector. Methods Total RNA was extracted from human breast cancer cell line MDA-MB-231,cDNA was generated by reverse transcription,cDNA was used as the template and the sequence segment of LDH-B gene coding region was amplified by poly merase chain reaction(PCR),both LDH-B gene segment and pUC19 plasmid were digested with two restriction endonucleases of BamHⅠand KpnⅠ,pUC19-LDH-B cloning vector was constructed by ligation reaction and then transformed into E. coli DH5αcompetence cells,the single white bacterial colony was selected in the blue-white selection plates,positive recombinant plasmid was identified by double restriction endonucleases digestion and DNA sequencing. Eukaryotic expression vector of pcDNA3.1 (-)-LDH-B was constructed by subcloning LDH-B gene segment with the right sequence into pcDNA3.1 (-) vector. Results A spe-cific DNA band about 1.0 kb was found by PCR amplification , which agreed with the expected fragment length of LDH-B;two DNA bands about 2.6 kb and 1.0 kb were seen after digestion of pUC19-LDH-B with BamHⅠand KpnⅠ,which agreed with the fragment length of pUC19 and LDH-B respectively,the LDH-B sequencing result was consistent with its reference sequence in Genebank;two DNA bands about 5.5 kb and 1.0 kb were seen after digestion of pcDNA3.1 (-)-LDH-B with BamHⅠand KpnⅠ, which agreed with the fragment length of pcDNA3.1 (-) and LDH-B respectively. Conclusion Human LDH-B gene is successfully cloned in this experimental study and its eukaryotic expression vector is successfully constructed ,which lays an essential founda-tion for further exploring LDH-B biological functions.关键词
L鄄乳酸脱氢酶/基因/克隆,分子/遗传载体/真核细胞Key words
L-lactate dehydrogenase/Genes/Cloning,molecular/Genetic vectors/Eukaryotic cells引用本文复制引用
胡湘麟,卿鑫,曾凡才..人LDH-B基因克隆及其真核表达载体的构建[J].现代医药卫生,2016,32(20):3106-3108,3112,4.基金项目
国家级大学生创新创业训练计划项目（201410632010）。 （）
