【摘要】 目的构建6种人DNA甲基转移酶(DNMT)3B典型的异构体的真核表达载体并对其进行过表达,初步探讨其在人胚肾细胞系293A中的生物学作用。方法以人HCT116 c DNA为模板,采用高保真PCR获得DNMT3B1和DNMT3B2的全长,并以DNMT3B2为模板,利用特异的引物扩增出DNMT3B3、DNMT3B4、DNMT3B5和DNMT3B7,将PCR产物连接到真核表达载体p CMV-2B上,并对上述载体进行Bam HⅠ、EcoRⅠ双酶切和测序鉴定,鉴定正确后,将重组质粒转染293A细胞并利用G418筛选出稳定表达的克隆株,MTT法检测细胞增殖活力。结果双酶切和测序结果表明克隆了6种人DNMT3B异构体CDS序列,经蛋白印迹检测,DNMT3B异构体在293A细胞中获得稳定表达;稳定过表达DNMT3B3、DNMT3B4和DNMT3B7可以抑制细胞增殖。结论构建了人DNMT3B异构体真核表达载体及获得了其稳定表达细胞株,DNMT3B异构体蛋白对胚肾细胞系293A的增殖有不同的作用。更多还原

【Abstract】 Objective To construct six kinds eukaryotic expression vectors of human DNA methyltransferase 3 B isofroms and overexpress these recombinant DNMT3 B isoforms,study its biological effects on human embryonic kidney( HEK) cell line 293 A. Method Human DNMT3 B1 and DNMT3 B2 were amplified from HCT116 using high fidelity Taq PCR. Other isoforms were amplified from DNMT3 B1 or DNMT3 B2. Then the PCR product waslinked into eukaryotic expression vector p CMV-2B. These recombination plasmids were identified by double enzyme digestion with Bam HⅠand EcoRⅠand by sequence. These recombination plasmids were transfected into 293 A cells when the sequencing results of recombination plasmids were correct. The G418 was used to get the stable cell line which express DNMT3 B isofoms respectively. MTT assay was used to detect the proliferation of cells which stable express the DNMT3 B isoform. Results Six kinds of Human DNMT3 B CDS sequence were cloned. The stable293 A cells overexpressing human DNMT3 B isoforms were established. Overexpression DNMT3B3、DNMT3B4 and DNMT3B7 can inhibit the growth of 293 A cells. Conclusions Eukaryotic expression vectors encoding human DNMT3 B isoforms and corresponding stable overexpression cells are constructed. Different human DNMT3 B isoforms protein may have different effects the growth of on 293 A cell.更多还原