乳酸脱氢酶A(ldha)基因的克隆与原核表达

Cloning and Prokaryotic Expression of Idha Gene

乳酸脱氢酶是一种糖酵解酶,在厌氧条件下能催化丙酮酸生成乳酸;当动物体内缺乏葡萄糖时,还可氧化乳酸生成丙酮酸并经葡萄糖异生途径转变为葡萄糖。通过反转录PCR获得了Hela细胞中的ldha基因,并将它克隆到pET28a(+)载体上,获得重组表达载体pET28a(+)-ldha。重组质粒通过转化法转入宿主E.coli BL21(DE3)溶源菌中。SDS-PAGE分析表明,在1.0 mmol·L-1异丙基硫代-β-D半乳糖苷(IPTG)的诱导下,重组子成功表达了一条分子质量约为36 kDa的目标蛋白。

The lactic dehydrogenase is one kind of enzymes in glycolysis.It can catalyze the pyruvic acid into lactic acid under the anaerobic condition.When animal body lacks the glucose,it may also oxidize the lactic acid to pyruvic acid and transform it into glucose by gluconeogenesis in vivo.In this study,ldha gene was obtained from Hela cell by using the reverse transcription polymerase chain reaction（RT-PCR） method.And the protein expression system of Escherichia coli BL21（DE3） for ldha gene was constructed with vector pET28a（＋）.SDS-PAGE analysis showed that the transformant with recombinant expression plasmid pET28a（＋）-ldha produced a protein with corresponding molecular weight of 36 kDa when induced by 1.0 mmol·L-1 IPTG.