自杀基因质粒表达载体pcDNA3.1(-)CMV.CD的构建及进行喉癌Hep-2细胞株转染研究

A study on construction of plasmid pcDNA3.1(-) CMV.CD and transfection into laryngeal cancer cell Hep-2

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 recombinant plasmid containing the yeast germ suicide gene CD and explore the killing effect of CD gene on Hep-2 cells in combination with 5-FC in vitro. Method： A fragment containing full-lengh coding region of CD was subcloned into expression plasmid pcDNA3.1 （-）CMV to construct recombinant plasmid pcDNA3.1 （-）CMV. CD identified by enzyme digestion of XhoⅠ/Hind Ⅲ, then the recombinant plasmid was conducted into the Hep-2 cell line by electroporation. Hep-2 cells stably expressing CD was obtained by 14-day positive select with 300-600 mg/L G418 and negative select with 10 mg/L 5-FC. Total RNA was extracted and the expression of the CD gene in transfected Hep-2 cells was identified by RT-PCR. MTT assay was used to observe the killing effect of 5-FC of different concentration on Hep-2 cells stably expressing CD and the con- trolled group was Hep-2 cells transfected by pcDNA3.1 （-）CMV. Result： A fragment of 5353bp and inserted frag- ment of 496 bp were got by cutting positive recombinant plasmid of pcDNA3.1 （-）CMV. CD with XhoⅠ/Hind Ⅲ. Automatic DNA sequence analysis demonstrated that the recombinant plasmid pcDNA3.1 （-）CMV. CD contained integral coding region of CD of 477bp that was totally the same with that published in GenBank. The expression of CD gene was detected by RT-PCR.. The relative survival rate of Hep-2 cells stably expressing CD were separately lower than those in the control group（ P〈0.05）. Conclusion： pcDNA3.1 （ - ）CMV. CD was successfully constructed and CD-expressing Hep-2 ceils can be killed by 5-FC, so the recombinant plasmid may be a candidate vector for laryngeal cancer therapy.