TMEPAI蛋白表达对溶酶体稳定性的影响

摘要

The purpose of the research is to generate stable cell lines overexpressing TMEPAI and then study the effect of TMEPAI expression on lysosome stability.The recombined expression vector,pEF-IRES-TMEPAI-Flag plasmid,was constructed with genetic engineering technology.The expression plasmid was positively identified with restriction enzyme digestion,PCR detection and DNA sequencing.A549 lung cancer cells were transfected with pEF-IRES-TMEPAI-Flag plasmid.Stable cell lines were generated after puromycin screening,and verified by Western blot and immunofluorescence.Furthermore,MTT assay,flow cytometry and AO staining assays indicated that the expression of TMEPAI increased the stability of lysosome.%本研究旨在构建稳定表达TMEPAI细胞株,研究TMEPAI表达对溶酶体稳定性的影响.采用基因工程技术构建重组表达载体pEF-IRES-TMEPAI-Flag,经酶切、PCR检测及测序鉴定,证实表达质粒构建正确.将表达质粒转染A549肺癌细胞,经抗生素puromycin筛选单克隆阳性细胞株,采用免疫印迹实验(Western blot)和免疫荧光技术证实稳定表达TMEPAI细胞株构建成功.通过MTT法、流式细胞技术和吖啶橙染色实验表明,TMEPAI蛋白能够增强溶酶体的稳定性.