PRKACB基因真核表达载体的构建及稳转细胞株的筛选  

Construction of EGFP-PRKACB vector and screening of its stable transfection cells

作　　者：郭剑波[1] 陈拥[2] 田大力[2] 高英[3] 

机构地区：[1]中国医科大学附属第四医院普外科,辽宁沈阳110032 [2]中国医科大学附属第四医院胸外科,辽宁沈阳110032 [3]中国医科大学附属第四医院病理科,辽宁沈阳110032

出　　处：《现代肿瘤医学》2016年第22期3543-3547,共5页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:30973502);辽宁省科技厅科学技术计划项目(编号:2012225072)

摘　　要：目的:克隆人c AMP依赖性蛋白激酶催化亚单位β基因(PRKACB),构建重组真核表达载体p EGFP-C1-PRKACB,并筛选其稳定转染肺癌细胞株。方法:应用逆转录聚合酶链反应(RT-PCR)技术,从真核细胞中扩增得到人PRKACB的全长序列,克隆至表达增强型绿色荧光蛋白载体中,经酶切、PCR鉴定后测序证实克隆成功。将重组载体转至真核细胞,采用RT-PCR、Western blot技术检测外源基因PRKACB的表达。应用新霉素(G418)筛选出稳定转染p EGFP-C1-PRKACB的非小细胞肺癌细胞株。结果:测序结果证实成功将PRKACB基因克隆至真核表达载体中,酶切片段与预期大小一致(1 200bp)。RT-PCR及Western blot结果显示:转染后的细胞中PRKACB在转录和翻译水平的表达均有明显提高。稳定转染p EGFP-C1-PRKACB的细胞株经Western blot证实其PRKACB蛋白上调最显著。结论:成功构建了真核表达载体p EGFP-C1-PRKACB,并筛选其稳定转染后上调最显著的细胞株,为进一步研究PRKACB的生物学功能奠定了基础。Objective： To construct the eukaryotic expression vector p EGFP- C1- PRKACB and screen its stable transfection cells. Methods： The full- length PRKACB gene sequence was amplified from LTEP- a- 2 cells by reverse transcription polymerase chain reaction（ RT- PCR）. The fragment was inserted into Green Fluorescent protein（ GFP） expression vector digested by Bam HI and Hind III enzymes,the same with PCR product digestion. The recombinant was transfected into LTEP- a- 2 cells and the exogenous expression of PRKACB gene was detected by RT-PCR and Western blot. The p EGFP- C1- PRKACB stable transfection cells were screened using G418. Results： Sequencing results verified that the p EGFP- C1- PRKACB eukaryotic expression vector was successfully constructed and the inserted sequence was 1 200 bp in accordance with our expectation. RT- PCR and Western blot showed the predominant elevation of PRKACB expression in transfected cells at both transcription and translation levels. The PRKACB protein level of p EGFP- C1- PRKACB stable transfection cells was up- regulated. Conclusion： The p EGFP- C1- PRKACB eukaryotic expression vector was successfully constructed and PRKACB was significantly expressed in stable transfection cells,which provides the foundation for the biologic function study of PRKACB.

关 键 词：PRKACB 克隆 表达载体 

分 类 号：R734.2[医药卫生—肿瘤]