摘要
目的:构建ABCE1基因的pEGFP重组表达载体pEGFP-C1-ABCE1,并将其转染至小细胞肺癌细胞(NCI-H446),观察其表达,并检测转染前后细胞增殖情况。方法:RT-PCR扩增ABCE1基因cDNA,酶切后和携带绿色荧光蛋白报告基因的真核表达载体pEGFP-C1重组获得重组表达载体pEGFP-C1-ABCE1。经酶切,电泳,DNA测序鉴定后应用Fugene-HD将重组表达载体pEGFP-C1-ABCE1及空载体pEGFP-C1分别转染NCI-H446细胞,观察pEGFP-C1-ABCE1和pEGFP-C1在小细胞肺癌细胞的表达,MTT法检测转染ABCE1基因对小细胞肺癌细胞增殖的影响。结果:ABCE1基因cDNA片段成功重组到pEGFP-C1载体中。经酶切和DNA测序验证,插入载体的cDNA片段为ABCE1基因。荧光显微镜观察转染pEGFP-C1-ABCE1的NCI-H446细胞绿色荧光蛋白主要在胞浆表达而转染pEGFP-C1的NCI-H446细胞绿色荧光蛋白在胞浆及胞核均有表达。MTT实验提示转染pEGFP-C1-ABCE1的细胞与转染pEGFP-C1和未转染的同类细胞相比增殖能力明显增加。结论:成功构建了pEGFP-C1-ABCE1真核细胞重组表达载体,并将该载体转染入小细胞肺癌细胞,该载体的绿色荧光主要在NCI-H446细胞胞质表达,提示ABCE1基因可能主要在胞质表达。该基因与小细胞肺癌的增殖活性有关。
Objective:Construct eukaryotic expression vector of ABCE1 with green fluorescent protein and transfect small cell lung carcinoma cells ( NCI - H446 ), then investigate the ABCE1 expression in small cell lung carcino- ma cells( NCI- H446), detect the change of proliferation. Methods:ABCE1 cDNA fragment was amplified with reverse transcription polymerase chain reaction ( RT - PCR) from NCI - H446 cells. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein. The ABCE1 cDNA fragment was inserted into the multiclone sites of the vector then construct a new plasmid pEGFP - C1 - ABCE1 . Plasmid DNA was identifed by cutting with Hind Ⅲ ,BamH Ⅰ nuclease and by DNA sequencing, pEGFP -C1 -ABCE1 or pEGFP-C1 was transfected into NCI - H446 cells and protein expression was identified by fluorescent microscopy. After transfected successfully,the proliferation of small cell lung cancer cells was assayed by MTT. Results:ABCE1 cDNA fragment was inserted into the vector pEGFP - C1 correctly. The fragment was defined to be ABCE1 gene by nuclease digestion and DNA sequencing. NCI - H446 cells were transfected with the new recombinant plasmid DNA. The green fluorescent protein was mainly expressed in the cytoplasm in cells transfected with pEGFP - C1 -ABCE1 group,while the green fluorescent protein was mainly in the nucleus and cytoplasm in cells transfected with pEGFP - C1 group. MTT test showed that proliferation of the cells which transfected with pEGFP - C1 - ABCE1 was much higher compared with the control parental vector pEGFP-C1 and normal group. Conclusion:The pEGFP- C1 - ABCE1 expression vector were successfully constructed,the vector were transfected into small cell lung cancer cells which mainly expressed in kytoplasm of NCI - H446cells.ABCE1 gene may related with the the proliferation of small cell lung cancer.
出处
《现代肿瘤医学》
CAS
2009年第3期393-397,共5页
Journal of Modern Oncology
基金
国家自然科学基金资助项目(编号:30170914)
辽宁省教育厅资助项目(编号:2004D162
20060966)