CAJ | 학술논문

目的：构建人乙醛脱氢酶1A1（ ALDH1A1）真核表达载体，并鉴定其生物学功能。方法通过基因克隆技术获得人ALDH1A1基因cDNA序列；测序正确后，将其连接到真核细胞表达载体pEGFP?N1，构建pEGFP?N1?ALDH1A1表达载体，经酶切鉴定、测序正确后，用脂质体转染技术将pEGFP?N1?ALDH1A1表达载体转染人胃癌细胞株MKN?28细胞，利用免疫印迹法鉴定表达产物，紫外?可见分光光度法测定ALDH1A1活性的改变。结果成功克隆到人ALDH1A1基因的全长cDNA，经测序与GeneBank序列完全一致；成功构建pEGFP?N1?ALDH1A1表达载体，经Western blotting鉴定显示ALDH1A1在MKN?28细胞中过表达，活性检测证实转染组的ALDH1A1活性较对照组明显升高。结论成功构建并鉴定了ALDH1A1基因过表达的胃癌细胞模型，为下一步研究ALDH1A1在胃癌中作用机制打下了基础。
목적：구건인을철탈경매1A1（ ALDH1A1）진핵표체재체，병감정기생물학공능。방법통과기인극륭기술획득인ALDH1A1기인cDNA서렬；측서정학후，장기련접도진핵세포표체재체pEGFP?N1，구건pEGFP?N1?ALDH1A1표체재체，경매절감정、측서정학후，용지질체전염기술장pEGFP?N1?ALDH1A1표체재체전염인위암세포주MKN?28세포，이용면역인적법감정표체산물，자외?가견분광광도법측정ALDH1A1활성적개변。결과성공극륭도인ALDH1A1기인적전장cDNA，경측서여GeneBank서렬완전일치；성공구건pEGFP?N1?ALDH1A1표체재체，경Western blotting감정현시ALDH1A1재MKN?28세포중과표체，활성검측증실전염조적ALDH1A1활성교대조조명현승고。결론성공구건병감정료ALDH1A1기인과표체적위암세포모형，위하일보연구ALDH1A1재위암중작용궤제타하료기출。
Objective To construct the pEGFP?N1?ALDH1A1 eukaryotic expression vector and evaluate its biological function in human gastric cancer cell line MKN?28. Methods A cDNA encoding ALDH1A1 was cloned by gene cloning techniques;The pEGFP?N1?ALDH1A1 was constructed by use of recombinant DNA technique and was demonstrated by restriction endonuclease mapping and sequencing.The pEGFP?N1?ALDH1A1 was transfected into human gastric cell line MKN?28 by using lipofectamine 2000. The protein expression of ALDH1A1 was detected by the Western blotting assay. The activity of ALDH was detected with ultraviolet?vis?ible light detector. Results A cDNA encoding ALDH1A1 was successfully cloned;Western blotting showed the level of protein expres?sion of ALDH1A1 in gastric cancer cell line MKN?28 transfected with pEGFP?N1?ALDH1A1 were significantly increased,and the AL?DH1A1 activity in MKN?28 transfected with pEGFP?N1?ALDH1A1 were significantly increased. Conclusion The recombinant plasmid of pEGFP?N1?ALDH1A1 was constructed corrected and the protein of ALDH1A1 overexpressed in human gastric cancer cell line MKN28. It provided solid experiment foundation for the further studies on the function of ALDH1A1 in gastric cancer.