| 摘要: |
| 目的 构建人survivin真核表达载体,稳定转染入小鼠黑素瘤B16细胞,建立稳定表达人survivin的小鼠黑素瘤细胞系。方法 采用PCR方法扩增出人survivin全长基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×his标签,得到重组表达质粒pIRES-neo-SUR-(his)6。利用阳离子脂质体介导法将其稳定转染入小鼠黑素瘤B16细胞,经G418加压筛选出阳性克隆。利用RT-PCR、蛋白免疫印迹及免疫荧光等检测方法验证人survivin基因在稳定转染B16细胞株中的表达。结果 经限制性内切酶鉴定及序列分析,pIRES-neo-SUR-(his)6重组体构建正确。RT-PCR结果表明:从稳定转染pIRES-neo-SUR-(his)6的B16细胞所抽提的总RNA中能够扩增出survivin基因(约530 bp)；蛋白免疫印迹结果表明:利用特异抗体能够从稳定转染pIRES-neo-SUR-(his)6的B16细胞中检测到survivin蛋白条带,而对照空质粒转染后的细胞则没有相应的条带；流式细胞仪和激光共聚焦显微镜检测结果显示,稳定转染pIRES-neo-SUR-(his)6的B16细胞用特异性抗体检测到高效的荧光表达,其中5号单克隆的荧光表达率为91.38%,而对照空质粒转染后的细胞则没有相应的荧光表达。结论 成功构建了真核表达载体pIRES-neo-SUR-(his)6,建立了稳定转染高效表达人survivin的小鼠黑素瘤细胞系。 |
| 关键词: survivin 稳定转染 实验性黑素瘤 基因疫苗 肿瘤免疫治疗 |
| DOI：10.3724/SP.J.1008.2010.0359 |
| 投稿时间：2009-12-14修订日期：2010-02-03 |
| 基金项目:国家自然科学基金(30772002),国家高技术研究发展计划(“863”计划,2007AA02Z451). |
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| Establishment and identification of B16 cell line stably expressing human survivin gene |
| ZHANG Liang1, LI Xiao-xiao2, YAN Jin-qi 1, WANG Yue1, XIAO Yi1, GAO Kun1, YU Ji-yun1* |
| (1. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China;2. Department of Clinical Laboratory, Beijing Jishuitan Hosptal, Beijing 100035, China) |
| Abstract: |
| Objective To construct the eukaryotic expression vector of human survivin gene and stably transfect it into mouse melanoma B16 cell line. Methods The full length human survivin cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo; the restriction enzyme position and 6 his tag were added to obtain recombinant plasmid pIRES-neo-SUR-(his)6 , which was then transfected into B16 cells by Lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, and the transcription and expression of the human survivin gene were identified by RT-PCR, Western blotting analysis and immunofluorescence assay. Results The result of restriction enzyme digestion and the sequence analysis showed that the recombinant of pIRES-neo-SUR-(his)6 was successfully constructed. RT-PCR results showed survivin gene (about 530 bp) was amplified from the total RNA in the group stably transfected with pIRES-neo-SUR-(his)6. Western blotting analysis showed the expression of survivin protein in pIRES-neo-SUR-(his)6 transfection group, but not in the control group. FACS and immunofluorescence assay showed high fluorescence signal in pIRES-neo-SUR-(his)6 transfection group, with the fluorescence positive rate being 91.38% when No. 5 monoclonal antibody was used; no fluorescence signal was found in the control group(transfected with pIRES-neo). Conclusion We have successfully constructed the eukaryotic expression vector of human survivin pIRES-neo-SUR-(his)6, and established a B16 cell line stably and highly expressing human survivin gene. |
| Key words: survivin stably transfection experimental melanoma gene vaccine tumor immunotherapy |
