野生型MLK3真核表达载体的构建及其表达

Construction and expression of the wild-type MLK3 eukaryotic expression plasmid vector

目的构建野生型MLK3真核表达质粒并转染至COS-7细胞中表达。方法以大鼠海马总RNA为模板,通过RT-PCR分段扩增MLK3的序列,采用酶切、链接的方法先将目的基因克隆至pGEM-T,筛选正确的质粒后,再亚克隆至pcDNA3.1(+)。以脂质体转染法将构建好的重组质粒转染至COS-7细胞,通过免疫印迹鉴定重组质粒的表达。结果限制性内切酶酶切和测序结果表明,扩增出的野生型MLK3的序列正确。免疫印迹显示,转染的重组质粒在相对分子质量92×103处有相应条带出现。结论成功构建了MLK3的真核表达质粒,重组质粒在COS-7细胞中高效表达。

Objective To construct the recombinant of wild-type MLK3 eukaryotic expression plasmid and to express the recombinant in transfected COS-7.Methods According to the cDNA sequence of MLK3（GenBank,BC081952）,the sequence of wild-type MLK3 fragments were expanded by RT-PCR,using rat hippocampal total RNA as the template.Then these fragments were cloned into the plasmid of pcDNA3.1（＋）.The recombinant of pcDNA3.1-MLK3 was transfected into COS-7 cells by the mediation of lipofectamine reagents.Immunoblot method was used to determine the expression of MLK3 proteins.Results The target gene was confirmed by restriction enzyme digestion and sequencing.The immunoblotting analysis indicated that the recombinant of MLK3 was successfully expressed in COS-7 cells.Conclusion The eukaryotic expression plasmids of MLK3 were successfully constructed and the recombinant plasmid were highly expressed in COS-7 cells.