PEA-15真核表达载体的构建及表达  

Construction and expression of eukaryotic expression vector of PEA-15

作　　者：张弘广[1] 庄晓飞[1] 王春利[1] 郭石平[1] 马炎炎[1] 

机构地区：[1]山西省肿瘤医院胸外二科,太原030013

出　　处：《肿瘤研究与临床》2014年第5期298-301,共4页Cancer Research and Clinic

基　　金：基金项目：山西省回国留学人员科研资助项目（115）

摘　　要：目的构建PEA-15真核表达载体pcDNA3．1-PEA-15，并在人类食管癌细胞（EC-109）中表达。探讨PEA-15对EC．109细胞的影响。方法采用反转录聚合酶链反应（RT—PCR）技术检测EC-109细胞中的PEA-15基因表达。构建重组真核表达载体pcDNA3．1-PEA-15。酶切及测序鉴定重组质粒正确后，脂质体介导转染至EC-109细胞中。采用RT—PCR、Westernblot法检测基因及蛋白表达。四甲基偶氮唑蓝（MTT）法检测细胞生长抑制率。结果RT—PCR显示PEA-15在EC-109细胞中表达。PCR扩增片段与预期片段大小相符，测序结果表明真核表达载体pcDNA3．1-PEA-15构建成功。转染后的细胞中PEA-15表达均增加（t值分别为4．078、5．269，均P〈0．05）。转染质粒72h后，细胞的生长抑制率较转染空质粒组降低[（7．12±0．86）％、（12．55±1．78）％，t=6．163，P〈0．05]。结论成功构建重组真核表达载体pcDNA3．I-PEA-15，并在EC-109细胞中进行了表达；转染后对食管癌细胞生长有促进作用，其表达可能促进食管癌的发生。Objective To construct the eukaryotic expression vector of pcDNA3.1-PEA-15 and express it in the human esophageal cancer （EC-109） cells, and to explore the effect of PEA-15 on EC-109 cells. Methods The PEA-15 gene was amplified from EC-109 by reverse transcriptase polymerase chain reaction （RT-PCR） and ligated to eukaryotic expression vector pcDNA3.1. After confirmation of recombinant plasmid was correctly by endonucleases digestion and DNA sequencing, the construct was transfected it into EC-109 through liposome inducing. The expression of PEA-15 in transfected EC-109 was detected by RT-PCR and Western blot. The cell growth inhibition ratio was evaluated by MTT assay. Results RT-PCR indicated that PEA-15 was highly expressed in EC-109 cells. The amplified fragment by RT-PCR was coincident with hypothesis enzyme digestion analysis and DNA sequencing confirmed that the pcDNA3.1-PEA-15 was constructed successfully. The expression of PEA-15 gene was increased obviously in the transfected EC-109 detected by RT-PCR and Western blot respectively （t = 4.078, 5.269, P 〈 0.05）. The cell growth inhibition ratio in the group which transfected pcDNA3.1-PEA-15 was significantly lower compared with the pcDNA3.1 transfect group after 72 hours （t = 6.163, P 〈 0.05）. Conclusions The recombinant eukaryotie expression vector peDNA3.1-PEA-15 is constructed successfully, and it can be expressed in EC-109. It also shows that PEA-15 has the function on cell growth which suggests that PEA-15 can inhibit the apoptosis of EC-109 cells and its expression involved in esophageal cancer development.

关 键 词：食管肿瘤 PEA-15 真核表达载体 

分 类 号：R735.1[医药卫生—肿瘤]