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目的:构建含酵母菌自杀基因CD的质粒表达载体pcDNA3.1(-)CMV.CD,并利用该载体进行喉癌Hep2细胞株转染研究,体外实验观察前体药物5FC对稳定表达CD基因的喉癌Hep2细胞株的杀伤作用。方法:通过RTPCR从酵母菌RNA内扩增出CD基因全长CDS序列,将其定向克隆到质粒表达载体pcDNA3.1(-)CMV中,重组体质粒经XhoI/HindⅢ双酶切鉴定,并对重组体中的CD基因片段进行序列分析,将鉴定好的阳性重组质粒pcDNA3.1(-)CMV.CD运用电穿孔法转入喉癌Hep2细胞中。经300～600mg/LG418正筛选14d和10mg/L前体药物5FC负筛选,获得稳定表达CD基因的喉癌Hep2细胞株,提取该细胞株细胞的总RNA,经RTPCR鉴定CD基因的表达。四甲基偶氮唑盐(MTT)法观察不同浓度5FC对稳定表达CD基因的喉癌Hep2细胞及转染空白载体pcDNA3.1(-)CMV的对照组喉癌Hep2细胞的杀伤作用。结果:阳性重组质粒pcDNA3.1(-)CMV.CD经XhoI/HindⅢ双酶切后获得5353bp的片段及496bp的插入片段,DNA自动序列分析证明重组体质粒含完整的477bp长的CD基因CDS序列。RTPCR从转染细胞总RNA中扩出453bp的预期片段。当添加不同浓度的5FC时,表达CD基因的Hep2细胞不同程度地被杀死,而对照组的细胞几乎未受到影响,两组细胞的相对生存率差异具有统计学意义(P<0.05)。结论:成功构建了质粒表达载体pcDNA3.1(-)CMV.CD,建立了稳定表达酵母菌CD基因的喉癌Hep2细胞株,表达CD基因的Hep2细胞可以被5FC杀死。
OBJECTIVE: To construct a plasmid expression vector pcDNA3.1 (-) CMV.CD containing yeast suicide gene CD and to study the transfection of laryngeal carcinoma Hep2 cell line by using this vector. The effect of priming drug 5FC on the expression of CD gene Killing effect of laryngeal carcinoma Hep2 cell line. Methods: The CDS sequence of CD gene was amplified from yeast DNA by RTPCR and cloned into the plasmid vector pcDNA3.1 (-) CMV. The recombinant plasmids were identified by double digestion with XhoI / Hind Ⅲ Recombinant CD fragment was sequenced, and the positive recombinant plasmid pcDNA3.1 (-) CMV.CD was transfected into Hep2 laryngeal carcinoma cells by electroporation. The positive clones were screened by 5FC with 300-600mg / L LG418 positive screening for 14d and 10mg / L prodrug 5FC. The Hep2 cell line stably expressing CD gene was obtained. The total RNA was extracted from the cell line and the expression of CD gene was identified by RTPCR. The cytotoxicity of 5FC on Hep2 cells stably expressing CD gene and Hep2 cells transfected with blank vector pcDNA3.1 (-) CMV were observed by MTT assay. Results: The positive recombinant plasmid pcDNA3.1 (-) CMV.CD was digested with XhoI / Hind Ⅲ to obtain a 5353bp fragment and a 496bp insert. The DNA sequence analysis showed that the recombinant plasmid contained a complete 477bp CDS CDS sequence . RTPCR extended 453 bp of the desired fragment from the total RNA of transfected cells. When different concentrations of 5FC were added, the Hep2 cells expressing CD gene were killed to some extent, but the cells in the control group were hardly affected. The relative survival rates of the two groups were statistically different (P <0.05). CONCLUSION: Plasmid expression vector pcDNA3.1 (-) CMV.CD was successfully constructed and Hep2 cell line stably expressing yeast CD gene was established. Hep2 cells expressing CD gene could be killed by 5FC.