中文摘要：

目的：构建含自杀基因胞嘧啶脱氨酶（CD）的真核表达载体pcDNA3．1／HA—myc-His（-）Z—CD，并进行哺乳动物细胞HEK293转染研究。方法：以本实验室保存的含CD基因全长的质粒为模版，用PcR方法扩增CD基因阅读框序列，并定向克隆到带有HAtag的pcDNA3．1／HA—myc-His（-）Z载体上，使目的基因与HAtag在同一阅读框。重组体质粒经EcoRI和BamHI双酶切鉴定，并对插入的CD基因片段进行测序，将鉴定好的阳性重组质粒pcDNA3．1／HA—myc-His（-）Z—CD用脂质体介导转染HEK293，提取细胞蛋白，western blot检测CD基因的表达情况.结果：阳性重组质粒pcDNA3．1／HA—myc-His（-）ZCD经Eco砌和BanHI双酶切后，获得约为5．5kb片段和1．3kb插入片段，序列分析表明插入的片段与GenBank发布的序列一致．western blot检测到CD基因的表达。结论：成功构建了含自杀基因CD的真核表达质粒。

英文摘要：

Objective： To construct a recombinant plasmid containing the suicide gene CD and study the expression of CD gene in HEK293 cell. Methods： A fragment containing full - lengh coding region of CD was subcloned into HAtag- tagged vector pcDNA3.1/HA - myc -His（ - ）Z to construct recombinant plasmid pcDNA3.1/HA - mye - His（ - ）Z - CD. CD was identified by enzyme digestion of EcoRl/ BamHI and sequence, then tlie positive recombinant plasmid was transfected into HEK293 cells using a routine lipofectamine method. After 48h, total protein was extracted and the expression of the CD gene in transfected HEK293 cells was identified by western blot. Results： A fragment of 5.5kb and inserted fragment of 1.3kb were got by cutting positive recombinant plasmid of pcDNA3.1/HA- myc- His（ - ）Z- CD with EcoRI/ BamHI. Automatic DNA sequence analysis demonstrated that sequence of the recombinant plasmid pcDNA3.1/HA- myc- His（ - ）Z- CD was totally the same with that published in GenBank. The expression of CD gene was detected by western blot. Conclusion： pcDNA3.1/HA - myc - His（ - ）Z- CD was successfully constructed.