乳酸脱氢酶B基因的克隆及其活性分析

Cloning and activity analysis of lactate dehydrogenase B gene

目的构建带Flag标签的乳酸脱氢酶B(lactate dehydrogenase B,LDHB)基因的真核表达载体,鉴定其表达产物的生物学活性。方法采用PCR技术从人乳腺文库扩增LDHB基因,将其克隆到p CDH-EF1-MCS-T2A-puro载体中,酶切和测序验证后,转染人胚肾293T细胞,通过蛋白质免疫印迹鉴定其表达;转染人肝癌Hep G2细胞,采用酶和底物反应的方法检测乳酸含量,并通过生长曲线研究LDHB对肝癌细胞生长的影响。结果将人乳腺文库扩增得到约1000 bp的c DNA片段克隆到p CDH-EF1-MCS-T2A-puro载体上,测序结果与目的序列完全一致;转染人胚肾293T细胞后,LDHB基因表达成功;乳酸含量测定结果显示,LDHB促进了肝癌细胞乳酸的生成;还可促进肝癌细胞的生长。结论成功构建了p CDH-Flag-LDHB真核表达载体,表达的LDHB具有生物学活性,为进一步研究LDHB在低氧和肿瘤发生发展中的功能奠定了良好基础。

Objective To construct a eukaryotic expression vector of lactate dehydrogenase B( LDHB) gene with Flag tag and identify the biological activity of the LDHB expression product. Methods LDHB gene was amplified from a human mammary gland library by PCR and cloned into the p CDH-EF1-MCS-T2 A-puro vector,which was confirmed by enzyme digestion and sequencing. The expression vector was transfected into human embryonic kidney 293 T cells and the LDHB expression was detected by Western blotting. The expression vector was transfected into Hep G2 cells,the lactic acid content was detected by enzyme and substrate reaction and a growth curve was observed to study the effects of LDHB on the growth of Hep G2 cells and 7721 cells. Results A c DNA fragment about 1000 bp from a human mammary gland library was cloned into the p CDH-EF1-MCS-T2 A-puro vector and the cloned sequence was identical with the target sequence according to DNA sequencing. LDHB gene was expressed in human embryonic kidney 293 T cells transfected with the expression vector. Analysis of lactic acid content showed that LDHB expression promoted lactic acid production in liver cancer cells.High expressions of LDHB could markedly enhance the proliferation of Hep G2 cells and 7721 cells. Conclusion p CDHFlag-LDHB eukaryotic expression vector has been successfully constructed,and the expressed LDHB has biological activity,which can facilitate further study on the function of LDHB in hypoxia as well as tumor development and progression.