重庆理工大学学报：自然科学
重慶理工大學學報：自然科學
중경리공대학학보：자연과학
Journal of Chongqing Institute of Technology
2012年
11期
23-26,44
,共5页
Hela细胞%ldha基因%克隆%原核表达
Hela細胞%ldha基因%剋隆%原覈錶達
Hela세포%ldha기인%극륭%원핵표체
Hela cell%ldha gene%cloning%prokaryotic expression
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。
乳痠脫氫酶是一種糖酵解酶,在厭氧條件下能催化丙酮痠生成乳痠;噹動物體內缺乏葡萄糖時,還可氧化乳痠生成丙酮痠併經葡萄糖異生途徑轉變為葡萄糖。通過反轉錄PCR穫得瞭Hela細胞中的ldha基因,併將它剋隆到pET28a（＋）載體上,穫得重組錶達載體pET28a（＋）-ldha。重組質粒通過轉化法轉入宿主E.coli BL21（DE3）溶源菌中。SDS-PAGE分析錶明,在1.0 mmol·L-1異丙基硫代-β-D半乳糖苷（IPTG）的誘導下,重組子成功錶達瞭一條分子質量約為36 kDa的目標蛋白。
유산탈경매시일충당효해매,재염양조건하능최화병동산생성유산;당동물체내결핍포도당시,환가양화유산생성병동산병경포도당이생도경전변위포도당。통과반전록PCR획득료Hela세포중적ldha기인,병장타극륭도pET28a（＋）재체상,획득중조표체재체pET28a（＋）-ldha。중조질립통과전화법전입숙주E.coli BL21（DE3）용원균중。SDS-PAGE분석표명,재1.0 mmol·L-1이병기류대-β-D반유당감（IPTG）적유도하,중조자성공표체료일조분자질량약위36 kDa적목표단백。
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.
