靶向性甲基化酶真核表达载体pcDNA3.1-myc-3a的构建

Construction of Targeting Methyltransferase pcDNA3.1-myc-3a in pcDNA3.1 Expression Vector

目的:在pcDNA3.1真核表达载体中构建表达融合myc-6his标签的靶向性甲基化酶pcDNA3.1-myc-3a并进行鉴定。方法:以含有myc全长基因的pcDNA3.1-myc-Luc质粒为模板,通过PCR方法扩增获得myc(DBD),再以含有靶向性甲基化酶的pcDNA4.0-GBD-3a-myc-6his质粒为模板,通过PCR的方法扩增获得融合有myc-6his标签序列的目的区段3a,然后将两者克隆入表达载体pcDNA3.1;以双酶切和PCR方法对构建的载体进行鉴定。结果:通过PCR方法获得了靶向性甲基化载体pcDNA3.1-myc-3a,并通过免疫印记方法验证了抗myc标签抗体可以特异性识别目的蛋白。结论:正确构建了靶向性甲基化酶pcDNA3.1-myc-3a的真核表达载体。

Objective：To clone and express the targeting methyltransferase myc-3a gene fused with myc-6his tag in eukaryotic expression system and to identify the protein of fused expression. Methods：The myc DNA binding domain was amplified from pcDNA3. 1-c-myc-Luc, and the methyltransferase catalysis domain 3a was amplified from pcDNA4.0-GBD-3a-myc-6his plasmid with PCR, and both cloned into the expression vector pcDNA3.1. The vector was identified by PCR and double enzymes digestion. The expression product was analyzed with Western blot. Results：The target methylransferase pcDNA3.1-myc-3a gene was amplified with PCR, and the interest protein could be specifically recognized by anti-myc-tag monoclonal antibody. Conclusion：The targeting DNA methyltransferase myc-3a was cloned into the expression vector correctly and expressed successfully in eukaryotic expression system.