CD真核表达载C 体的构建及在 2KHE39 细胞中的表达

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。

A Study on Construction of Plasmid pcDNA3.1/HA - myc - His( - ) Z- CD and Transfection into HEK293 Cell

Objective:To construct a recombinant plasmid containing the suicide gene CD and study the expression of CD gene in HEK293 cert.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-myc-His(-)Z-CD.CD was identified by enzyme digestion of EcoRI/ BamHI and sequence,then rite 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.