摘要
目的克隆细胞凋亡抑制蛋白Survivin异构体之一Survivin-1(SVV-1)基因的编码序列,构建含SVV-1基因的真核细胞表达载体并在肺腺癌细胞系A549中高表达,为进一步研究该基因在肺腺癌发病中的作用奠定基础。方法根据已发表的SVV-1基因的核苷酸序列自行设计一对分别含有BamHⅠ和Xho l双酶切位点的survivin-1基因上下游引物,以A549细胞系抽提的RNA为模板进行逆转录聚合酶链反应(RT-PCR),扩增产物用Bam HI和XhoI双酶酶切后定向克隆到真核细胞表达载体pcDNA3.0中,用限制性内切酶酶切重组质粒pcDNA30-SVV-1和DNA序列测定进行鉴定。用脂质体法将pcDNA3。0-SVV-1导入肺腺癌细胞系A549中,嘌呤霉素选择培养,经免疫组化法和western blot鉴定其表达。结果RT-PCR扩增出长448bp的特异性片段,经克隆至pcDNA3.0后酶切鉴定证实,并测序表明序列与Gen-Bank报道完全一致。pcDNA3.0-SVV-1在A549细胞中有稳定表达。结论成功克隆了SVV-1的编码序列,构建了其真核细胞表达载体pcDNA3.0-SVV-1,有助于对SVV-1基因在腺癌发生中的致瘤机制做进一步研究。
[Objective] To construct the recombinant plasmid vector containing human survivin-1, and transfect it into A549 cells. [Methods] Full length survivin-1 cDNA was obtained from A549 by RT-PCR. The PCR product was double-digested with restriction endonucleases BamH Ⅰ and Xho Ⅰ, and inserted orientationally into pcDNA3.0. The plasmid of pcDNA-svv-1 was reproduced, and the fragment containing survivin-1 was reclaimed and transfected into E. cob JM109, After having been screened, the extracted plasmid containing surviving-lwas transfected into A549 cells with liposome, and the expression of surviving-1 was proved by Western blot analysis and immunohistochemistry. [Results] The recombined plasmid pcDNA-survivin-1 was constructed successfully and expressed in adenocarcinoma A549 cells was observed by Western boltting. [ Conclusion] A recombinant plasmid vector containing human surviving-1 was constructed successfully, and the survivin-1 protein was expressed highly in A549 cells.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2007年第6期683-685,共3页
China Journal of Modern Medicine
