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目的构建反义MBD1基因片段真核表达载体,为研究MBD1基因功能提供工具。方法根据MBD1基因cDNA序列中编码序列,设计PCR引物,在引物5′端分别添加Xba I和Kpn I 酶切位点,将PCR片段反向插入真核表达载体pcDNA3．1(+)的多克隆位点上,构建反义MBD1基因片段真核表达载体,并用PCR、酶切法和DNA测序鉴定。结果PCR鉴定得到322 bp特异性条带, 双酶切得到327 bp目的基因片段和5．4 kb载体片段,DNA测序说明插入片段序列是正确的。结论采用基因克隆技术,成功构建了反义MBD1基因片段真核表达载体,为研究MBD1基因在DNA甲基化和肿瘤发生中的功能提供了实验工具。
Objective To construct the eukaryotic expression vector of antisense MBD1 gene fragment and provide a tool for studying the function of MBD1 gene. Methods According to the coding sequence of MBD1 cDNA sequence, PCR primers were designed. Xba I and Kpn I restriction sites were added to the 5 ’end of the primer. The PCR fragment was inserted into the pcDNA3.1 (+) polyclonal Site, the antisense MBD1 gene fragment eukaryotic expression vector was constructed and identified by PCR, restriction enzyme digestion and DNA sequencing. Results A 322 bp specific band was obtained by PCR. The 327 bp gene fragment and the 5.4 kb vector fragment were double digested. DNA sequencing showed that the insert sequence was correct. Conclusion The eukaryotic expression vector of antisense MBD1 gene fragment was successfully constructed by gene cloning technology, which provided an experimental tool for studying the function of MBD1 gene in DNA methylation and tumorigenesis.