诱发口腔鳞癌细胞凋亡嵌合基因表达载体的构建

目的：获取含凋亡基因fas表达调控元 件cdc25A片段和fas开放阅读框架的cdc25A -fas嵌合基因，构建和鉴定真核表达载体pAdTrack-CMV-cdc25A -fas和pAdTrack-cdc25A-fas，为将口腔鳞癌细胞固有的 促细胞增殖信号转变为促细胞凋亡信号的研究奠定基础。方法:根据fa s基因、cdc25A已知序列设计合成引物，采用PCR技术从pBLF58-1质 粒中扩增得到嵌合基因cdc25A-fas；然后定向克隆至真核表达载 体pAdTrack-CMV和pAdTrack，分别转化大肠杆菌E.coli DH5α感受态细胞；卡那霉素筛 选阳性克隆；进行PCR、酶切鉴定和序列分析。结果：PCR扩增得到特异 的 1 603 bp的cdc25A-fas片段；筛选并鉴定得到含pAdTrac k-CMV-cdc25A-fas和pAdTrack-cdc25A- fas的大肠杆菌E.coli DH5α阳性克隆。结论:成功构建了真核表 达载体pAdTrack-CMV-cdc25A-fas和pAdTrack-cdc25 A-fas,为下一步研究其在肿瘤基因治疗中的作用奠定基础。

Construction of chimeric gene expression vector inducing apoptosis of oral squamous carcinoma cells

AIM:To gain cdc25A-f as chimeric gene bearing the regulative fragment cdc25A an d opening read frame of fas in order to construct and identify eukaryotic ex pre ssion vectors,pAdTrack-CMV-cdc25A-fas and pAdTrack-cd c25A-fas,which have the potential to transfer the tumor p roliferative signal to promoting-apoptosis signal through up-regulate the fas expression by c-myc.METHODS:A pair of primers were designed according to the known sequences of cdc25A and fas.The cdc25 A-fas chimeric gene was obtained by PCR reaction and inserted into the two plasmids pAdTrack-CMV and pAdTrack,respectively.The two recombinant plasmids w ere transferred into E.coli DH5α.The positive clones were screened in LB media with 50 mg/L kanamycin and identified by agarose gel electrophoresis,endo nuclease digestion and PCR.The nucleotide sequence of inserted cdc25 A-fas was determined by dideoxy chain termination method.Using so ftware,BLAST was conducted to analyze the structure and sequence of the target fragments and compared with GenBank.RESULTS:The chimeric target gene,cdc25A- fas,of 1 603 bp was obtained.The positive host bacteria E.coli DH5 α of recombinant plasmids were screened and amplified.The double-enzyme digest ion showed the pAdTrack-CMV-cdc25A-fas and pAdTrack-cd c25A-fas presenting 9.2 kb,1.6 kb bands,and 8.3 kb,1 .6 kb bands respectively.The sequence analysis confirmed that the two shuttle plasmids containing 1 597 bp cdc25A-fas with the ORF of fas.CONCLUSION:The eukaryotic expression plasmids pAd-Track-CMV-c dc25A-fas and pAdTrack-cdc25A-fas were successfully constructed.