胃癌血管特异性短肽GEBP11结合受体的免疫沉淀法筛选

Screening of the receptor of vascular-endothelial-cell-specific binding peptide GEBP11 in gastric cancer by immunoprecipitation

目的:筛选胃癌血管特异性短肽GEBP11(CTKNSYLMC)的结合受体。方法:采用Western-blot、免疫细胞化学染色法对共培养人脐静脉内皮细胞(co-HUVECs)CD31和KDR的表达进行检测;化学合成生物素标记的GEBP11,应用免疫沉淀、免疫磁珠法分离富集与GEBP11结合的蛋白质;采用Western-blot检测,确定并获取特异性蛋白条带;利用液相色谱-二级质谱(LC-MS/MS)及生物信息学分析,对条带蛋白进行测序和分析。结果:co-HUVECs表达CD31,KDR表达增加。通过免疫沉淀和Western-blot分析,发现一条与GEBP11特异结合的条带,其分子量约为12KD。经LC-MS/MS和生物信息学分析,获得候选蛋白ATP7B。结论:经鉴定co-HUVECs表达内皮细胞标志性分子且KDR表达上调;利用免疫沉淀-质谱法筛选获得了GEBP11受体的候选蛋白,为研究GEBP11的作用机制奠定了基础。

Objectlve：To screen the receptor of GEBP11 （CTKNSYLMC） binding specifically to vascular endothelial cells in gastric cancer. Methods： The expression of KDR and CD31 in co -cuhrued human umbilical vein endothelial cells （ co - HUVECs） was detected by Western - blot and immunocytochemistry. Biotin labeled GEBP11 was obtained by ehemosynthesis. By using immunoprecipitation and Western - blot, the protein band that specifically bound to GEBP11 was screened and then identified and analyzed by Liquid Chromatography -Tandem Mass Spec- trometry （ LC - MS/MS） and bioinformatics. Results： The co - HUVECs expressed CD31 and the expression of KDR was upregulated. A protein band aboutl2KD was gotten and a candidate protein, ATP7B, was obtained. Conclusion： The property of co - HUVECs was consistent with HUVECs and the expression of KDR was upregulated. A candidate protein was obtained by immunoprecipitation and LC - MS/MS, which provided foundation to the mechanism resrcreh of GEBP11.