DOC-2作用蛋白的筛选及与TGFβⅢ受体相互作用的初步验证

Screening of DOC-2 interactive proteins and identification of the interaction between DOC-2 and TGFβⅢ receptor

背景与目的:人卵巢癌差异表达基因DOC-2既是肿瘤抑制基因也是信号分子,参与细胞的生长分化过程。筛选DOC-2氨基端磷酸酪氨酸作用结构域PID(nDOC-2)的相互作用蛋白并进行初步验证,为研究DOC-2的信号通路提供线索。方法:将含有人DOC-2氨基端PID结构域cDNA的片断插入酵母表达载体pGBKT7中构建诱饵质粒,转化酵母菌AH109并在其内表达,然后转化人胎脑cDNA文库,在营养缺陷培养基和X-α-半乳糖苷酶(X-α-gal)上进行阳性克隆的双重筛选,PCR扩增目的片段,测序,并进行生物学分析,寻找DOC-2氨基端PID结构域的相互作用蛋白。将DOC-2cDNA和TGFβⅢ受体cDNA共转染人卵巢癌细胞系HO-8910,利用免疫沉淀和Western Blot,对TGFβⅢ型受体是否能与DOC-2相互作用进行初步分析。结果:经扩增和筛选胎脑cDNA文库及序列分析,21个侯选阳性克隆中3个克隆为:APLP1、TGFβⅢ型受体的部分mRNA和PCDHGC3(protocadherin gammasubfamily C3)。免疫沉淀及Western blot结果显示,DOC-2与TGFβⅢ型受体相互作用后可形成蛋白复合物。结论:获得的3个基因编码的蛋白可能参与了DOC-2的信号转导,DOC-2与TGFβⅢ型受体可能存在互相作用,并通过TGFβⅢ型受体在TGFβ的信号通路中起作用。

Background and purpose： DOC-2 serves as one of the tumor suppressing genes in human ovarian cancer and plays a role in the process of cell growth and differentiation. This study was to investigate the role of DOC-2 in the TGFβ signal pathway and verification of the interaction between DOC-2 and TGFβⅢ receptor. Methods： The bait vector was constructed by inserting the PID domain of DOC-2 （nDOC-2） into yeast express vector pGBKTT, pGBKTT- nDOC2 was transformed into the yeast AH109 and confirmed to be expressed. After the human foetus brain eDNA library had been transformed, the positive cloneswas screened by both nutrition defect medium and X-et-gal. The putative positive clones were sequenced and analyzed to get the DOC-2 interactive proteins. Furthermore, after the DOC-2 eDNA and TGFβⅢ receptor eDNA had been eo-transfeeted into the human ovarian cancer cell line HO-8910 together, the interaction between DOC-2 and TGFβⅢ receptor was investigated by immunopreeipitation and Western blot. Results： 21 putative positive clones were picked after being screened and sequenced. Three of them were identified as Homo sapiens partial mRNA for betaglycan （TBR Ⅲ gene）, homo sapiens protocadherin gamma subfamily C3 （PCDHGC3） and APLP1 （amyloid beta precursor- like protein 1 ）. The analysis by immunopreeipitation and Western blot showed that the interaction between DOC-2 and TGFβⅢ receptor could form protein complex. Conclusions： The three encoding proteins might participate in the DOC-2 signal pathway. DOC-2 might play as an essential role in the TGFβsignal pathway by interacting with TGFβⅢ receptor.