ABCE1基因pEGFP重组表达载体构建及转染小细胞肺癌细胞

Construct pEGFP vector of ABCE1 gene and transfect it into small cell lung cancer cell

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.