摘要
目的构建人端粒酶反转录酶(hTERT)和生存素(survivin)基因表达的RNA共干扰载体,并检测其对人结肠癌SW480细胞中hTERT基因和survivin基因的干扰作用。方法根据Genbank提供的hTERT和survivin cDNA序列,设计、筛选出高效、特异性强的干扰序列,构建特异性干扰hTERT基因和survivin基因的重组体pGenesil-1.1-survivin、pGenesil-1.2-hTERT,酶切、测序鉴定正确后,两个重组体均用HindⅢ和BamHⅠ双酶切,将pGenesil-1.1-survivin回收大片段,pGenesil-1.2-hTERT回收小片段,将回收大、小片段用T4 DNA连接酶定向连接,即构建成重组体pGenesil1.1-survivin-hTERT。将3个重组体分别转染SW480细胞,RT-PCR检测细胞中hTERT和survivin基因表达水平,MTT检测对SW480细胞增殖的影响。结果构建的各质粒经限制性酶切及DNA测序均证明其质粒构建正确,RT-PCR检测hTERT和survivin基因表达水平明显降低(P<0.05),各干扰质粒重组体对细胞增殖有明显的抑制作用(P<0.05)。结论靶向survivin和hTERT基因的双干扰载体构建成功,为后续的结肠癌基因治疗研究奠定理论基础。
Objective To construct the double interference vector targeting both survivin and hTERT gene and de- tect its silencing effects on survivin and hTERT gene in human colon carcinoma SW480 cell line. Methods Two effective targeting sequences were chosen according to hTERT and survivin cDNA sequences in Gen Bank, Then they were inserted into pGenesil-1.1 and pGenesil-1.2, pGenesil-1.1-survivin and pGenesil-1.2-hTERT were con- structed. The recombinant plasmids were identified by restriction endonuclease and DNA sequencing, pGenesil- 1. 1-survivin and pGenesil-1.2-hTERT were digested by Hind 111 and BamH I se sil-1.1-survivin and small fragment of pGenesil-1.2-hTERT were collected. Then parately, long fragment of pGene- long fragment and small fragment were connected with T4 DNA ligase orientation, the double interference vector pGenesill. 1-survivin-hTERT wasconstructed. These siRNA recombinant expression vector separately were transfected into SW480 cell line, The effect of siRNA recombinant expression vector was detected by RT-PCR and MTT. Results By restriction endonuclease and DNA sequencing, specific RNAi expression vector targeting survivin and hTERT gene was correctly constructed. The growth rate of SW480 cells transfected with siRNA recombinant expression vector was significantly decreased. Conclu- sions The multiple siRNA recombinant expression vector targeting hTERT and survivin gene simultaneously has been constructed successfully. This result could provide theoretical information for the further research.
出处
《基础医学与临床》
CSCD
北大核心
2013年第11期1440-1445,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(30901779)
潍坊市科技局资助项目(20121227)
山东省自然科学基金(ZR2010HM065
ZR2010HM006)
