人YAP1基因亚型的质粒构建及表达鉴定

Construction of the recombinant plasmid of human YAP1 gene and the expression of fusion protein

目的:构建人Yes相关蛋白1(YAP1)基因亚型1δ、2δ的真核表达载体并进行瞬时表达鉴定,为研究YAP1亚型间的区别及其在肿瘤中的作用打下基础。方法:在已有pCI_2-Flag-YAP1-2γ质粒的基础上,利用重叠PCR增加碱基的方法得到YAP1-2δ的片段,再同理删减WW基因序列得到YAP1-1δ片段,通过Cla I和Sal I两个酶切位点将片段和pCI_2载体相连接,构建pCI_2-Flag-YAP1-1δ及pCI_2-Flag-YAP1-2δ质粒。利用测序确认完整的DNA序列。在HEK293细胞中瞬时转染质粒,用Western blot方法检测YAP1两个亚型的蛋白表达。结果:重组质粒经双酶切和基因测序比对鉴定正确,HEK293细胞经瞬时转染表达出YAP1-1δ和YAP1-2δ蛋白。结论:成功构建了pCI_2-Flag-YAP1-1δ及pCI_2-Flag-YAP1-2δ真核表达质粒,并在HEK293细胞内过表达成功。为进一步探讨YAP1各亚型对肿瘤发展的影响奠定基础。

Objective： To construct two isoforms of YAP1 for further research into their difference. Meth-ods： YAP1-1δ and YAP1-2δ fragments were obtained by using overlapping PCR. And then the two DNA fragments were respectively ligated to the plasmid contained partial YAP1 gene via ClaI and SalI two restriction enzyme cutting sites. The recombinant vectors named pCI2-Flag-YAP1-1δ and pCI2-Flag-YAP1-2δ were identified by re- striction enzyme digestion and gene sequencing. After transfection of 293 cells with the two vectors for 48 h, the expression of YAP1 protein was detected by Western blot. Results： The two over-expressing plasmids were suc- cessfully constructed and YAP1-1δ and YAP1-2δ proteins were strongly expressed in HEK293 cells. Conclusion： The HEK293 cells could transiently express YAP1-1δ and YAP1-2δ by using the recombinant plasmids pCI2-Flag- YAPI-1δ and pCI2-Flag-YAP1-2δ, which lays a foundation for further study of the different isoforms of YAP1 and its mechanism in the development of tumorigenesis.