小鼠乳酸脱氢酶C亚基在大肠埃希菌中的表达与鉴定  

Expression and Characterization of Subunit C of Mouse Lactate Dehydrogenase in Escherichia coli.

作　　者：熊永忠[1] 郑德柱[1] 谢飞[1] 涂向东[1] 兰风华[1] 

机构地区：[1]南京军区福州总医院实验科,福建福州350025

出　　处：《中华男科学杂志》2004年第1期9-11,共3页National Journal of Andrology

基　　金：福建省重点科技攻关项目 ( 2 0 0 2Y0 3 2 );全军医药卫生"十五"基金项目 ( 0 1MA0 3 2 )资助

摘　　要：目的 :构建小鼠特异性乳酸脱氢酶 (LDH)C亚基的原核重组载体并进行表达和鉴定。　方法 :提取小鼠睾丸总RNA ,逆转录合成cDNA ,自行设计引物 ,PCR扩增小鼠LDH C亚基编码序列 ,PCR产物经BamH Ⅰ/EcoR Ⅰ 双酶切后 ,克隆至谷胱甘肽S转移酶 (GST)融合表达载体pGEX 2T中 ,在E .coliBL2 1中诱导GST融合蛋白的表达。　结果 :在异丙基 β 硫代半乳糖苷 (IPTG)的诱导下 ,重组菌高效表达出一个相对分子质量约为6 0 0 0 0的产物。对重组质粒的序列分析表明 ,插入片段的序列与GenBank登录的编码序列完全一致。　结论 :小鼠LDH C亚基的全长编码序列已被克隆至GST融合表达载体pGEX 2T ,并在E .coliBL2Objectives: To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21. Methods: The coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers, and cloned into pGEX-2T after the restriction digestion with BamH I and EcoRⅠ. GST fusion protein was expressed after induction with IPTG. Results: Sequencing and restriction digestion of the recombinant plasmid revealed the existence of coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60 000 could be induced by IPTG in the recombinant plasmid. Conclusions: The coding sequence of mouse lactate dehydrogenase subunit C was introduced into the pGEX-2T plasmid and a GST-fused protein could be induced at a high level.

关 键 词：乳酸脱氢酶C亚基 精子 克隆 基因表达 小鼠 PCR LDH同工酶 避孕疫苗 

分 类 号：R346[医药卫生—基础医学]