醛缩酶A基因真核表达载体的构建及其生物学活性分析

Construction and biological activity analysis of aldolase A gene eukaryotic expression vector

目的构建带FLAG标签的醛缩酶A(ALDOA)基因的真核表达载体,对其表达产物进行功能检测。方法利用PCR技术从人乳腺文库中扩增ALDOA基因,将其克隆至真核载体pcDNA3.0-FLAG中。经过酶切与测序验证,转染人胚肾293T细胞系,利用蛋白质免疫印迹实验完成其表达鉴定。转染人乳腺癌细胞MCF7和ZR75-1,使用葡萄糖摄取试剂盒检测细胞糖摄取情况,乳酸试剂盒检测细胞外乳酸产量。通过细胞生长曲线及克隆形成实验探究pcDNA3.0-FLAG-ALDOA对乳腺癌细胞生长的影响,通过划痕实验探究ALDOA对乳腺癌细胞迁移能力的影响。结果通过PCR技术从人乳腺文库中扩增得到约1100 bp cDNA片段,克隆到pcDNA3.0-FLAG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,pcDNA3.0-FLAG-ALDOA基因表达成功。糖摄取及乳酸含量鉴定结果表明,pcDNA3.0-FLAG-ALDOA对乳腺癌MCF7和ZR75-1细胞糖摄取及乳酸的生成有促进作用;生长曲线及克隆形成实验结果表明,pcDNA3.0-FLAG-ALDOA可促进乳腺癌MCF7和ZR75-1细胞生长;迁移实验结果表明,pcDNA3.0-FLAG-ALDOA可促进乳腺癌MCF7和ZR75-1细胞迁移。结论FLAG-ALDOA真核表达载体构建成功,表达产物ALDOA具有生物学活性,可促进乳腺癌细胞的葡萄糖摄取、乳酸生成、细胞生长及迁移,为进一步研究ALDOA在肿瘤发展中的意义奠定了理论基础。

Objective To construct a eukaryotic expression vector of the aldolase A gene with FLAG tag,and to perform a functional test of its expression products.Methods Fructose bisphosphatealdolase A(ALDOA)gene was amplified from the human breast library by PCR technology and cloned into the eukaryotic vector pcDNA3.0-FLAG.After restriction enzyme digestion and sequencing,FLAG-ALDOA was transfected into human embryonic kidney 293 T cell line,and the expression was identified by Western blotting.The glucose uptake and extracellular lactate production in human MCF7 and ZR75-1 breast cancer cells with transfected FLAG-ALDOA were examined by corresponding kits.Cell proliferation was examined by cell growth curves and colony formation assays.Cell migration was determined by wound healing assays.Results A cDNA fragment of about 1100 bp was amplified from the human breast library by PCR and cloned into the pcDNA3.0-FLAG vector.The sequencing result was completely consistent with the target sequence.After transfection of HEK-293 T cells,ALDOA gene expression was successful.Glucose uptake and lactate assays showed that FLAGALDOA promoted glucose uptake and lactate production in breast cancer cells.Cell growth curves and colony formation assays showed that FLAG-ALDOA promoted cell proliferation of MCF7 and ZR75-1 cells.Wound healing assays showed that FLAG-ALDOA promoted cell migration of MCF7 and ZR75-1 cells cells.Conclusion An FLAG-ALDOA eukaryotic expression vector has been constructed.The expression product FLAG-ALDOA has biological activity and can promote glucose uptake,lactate production,cell growth and migration of breast cancer cells,which can contribute to further research on the significance of ALDOA in tumor development.