应用酵母双杂交技术筛选和验证与DNA-PKcs磷酸化簇区域相互作用的蛋白

目的 通过酵母双杂交技术筛选与DNA依赖蛋白激酶的催化亚单位（DNA dependent protein kinase catalytic subunit，DNA-PKcs）磷酸化簇区域相互作用的蛋白并进行验证。方法 通过酵母双杂交技术，以之前构建的pGBKT7-DPC载体为诱饵，在人肝组织酵母文库中筛选与DNA-PKcs磷酸化簇区域相互作用的蛋白，对筛选出来的阳性酵母细胞克隆进行PCR鉴定、回转验证以及测序分析；构建诱饵蛋白和阳性克隆蛋白的真核表达载体，分别转染至人胚肾293T细胞中，检测诱饵蛋白和阳性克隆蛋白能否正确表达；最后将阳性克隆蛋白与诱饵蛋白共转染至293T细胞中，通过免疫共沉淀实验检测阳性克隆蛋白与诱饵蛋白之间的相互作用。结果 经过两轮酵母双杂交实验筛选得到12个文库质粒克隆，通过PCR鉴定确定7个插入片段长度互不相同的文库克隆，对7个文库克隆进行回转验证得到3个与DNA-PKcs磷酸化簇区域相互作用的蛋白，经测序分析显示分别为MBNL1、SIK2和YY1AP1。成功构建诱饵蛋白和3个阳性克隆蛋白的真核表达载体，且均能够在293T细胞中正确表达。免疫共沉淀实验证实筛选出来的3个阳性克隆蛋白均能够与DNA-PKcs磷酸化簇区域发生相互作用。结论 成功筛选到与DNA-PKcs磷酸化簇区域相互作用的蛋白并进行了验证。

Screening and verifying the proteins interacting with phosphorylation cluster of DNA-PKcs by yeast two-hybrid assay

Objective To screen and verify the proteins interacting with phosphorylation cluster of DNA dependent protein kinase catalytic subunit ((DNA-PKcs) by yeast two-hybrid assay. Methods To know the proteins interacting with DNA-PKcs phosphorylation cluster,yeast two-hybrid assay was applied to screen the cDNA library of human hepatic tissue with a previously constructed plasmid pGBKT7-DPC.The positive clones were further identified by PCR,rotary validation and sequence analysis.Then the eukaryotic expression vectors of the bait protein and screened positive clone proteins were constructed and transfected into human embryonic kidney 293T cells to detect whether the proteins could been expressed correctly.At last,the bait protein and screened positive clone proteins were co-transfected into 293T cells and protein interaction was detected with Co-Immunoprecipitation (Co-IP) assay. Results After two rounds of screening using the yeast two-hybrid assay,12 candidate clones were obtained.Then 7 clones with different insert fragments were identified by PCR,and 3 positive proteins interacted with DNA-PKcs phosphorylation cluster were further verified by rotary validation.Sequencing analysis demonstrated that these 3 proteins were MBNL1,SIK2 and YY1AP1,respectively.Accordingly,the eukaryotic expression vectors of bait protein and 3 positive clone proteins were constructed successfully and expressed correctly in 293T cells.Finally,the Co-IP assay confirmed that these 3 positive clone proteins could interact with DNA-PKcs phosphorylation cluster. Conclusions Proteins interacting with DNA-PKcs phosphorylation cluster are successfully screened and identified.