采用串联亲和纯化质谱分析DJ-1的相互作用蛋白  

DJ-1 interacting proteins analyzed by tandem affinity purification mass spectrometry

作　　者：魏旺丽[1] 谭潭[1] 崔亚娟 汤参娥[3] Wei Wangli;Tan Tan;Cui Yajuan;Tang Can'e(Department of Laboratory Medicine Center,Chenzhou.First People's Hospital,Chenzhou 423000,China;Department of Hematology,The Second Xiangya Hospital,Central South University,Changsha 410011,China;Center of Medical Science Research,Xiangya Hospital,Central South University,Changsha 410008,China)

机构地区：[1]湖南省郴州市第一人民医院检验医学中心,423000 [2]中南大学湘雅二医院血液内科,长沙410011 [3]中南大学湘雅医院医学科学研究中心,长沙410008

出　　处：《中国医师杂志》2021年第10期1472-1476,共5页Journal of Chinese Physician

基　　金：国家自然科学基金(30972970);湖南省科技创新计划项目(S2017SFYLJS0085);郴州市第一人民医院院级科研项目(N2020-7)。

摘　　要：目的采用RNA干扰技术下调DJ-1基因在肺鳞癌细胞HTB-182中的表达,采用串联亲和纯化质谱(TAP-MS)技术寻找DJ-1在HTB-182细胞系中的相互作用蛋白。方法构建靶向DJ-1基因siRNA慢病毒载体,感染HTB-182细胞(DJ-1 siRNA组),并设立慢病毒载体对照组(Control siRNA组)及空白对照组,采用Western blot法检测DJ-1蛋白表达水平,建立内源性的DJ-1蛋白表达沉默的si-DJ-1-HTB-182细胞。设计DJ-1的特异性引物,构建带有链霉素结合肽标签(SBP)、钙调蛋白结合肽标签(CBP)的DJ-1表达质粒pNTAP-DJ-1,用脂质体稳定转染细胞系DJ-1 siRNA HTB-182,G418筛选阳性克隆,Western blot进行验证,TAP-MS技术寻找DJ-1的相互作用蛋白。结果DJ-1 siRNA干扰组中DJ-1的蛋白质表达明显低于空质粒(NC)组和空白对照(BC)组(P<0.05);成功构建了稳定表达pNTAP-DJ-1质粒的HTB-182细胞系;TAP-MS筛选到DJ-1相互作用的三个蛋白质:细胞角蛋白1(Keratin 1)、细胞角蛋白10(Keratin 10)和NADPH氧化酶活化蛋白P47(P47 Px)。结论Keratin 1、Keratin 10和P47 Px可能是DJ-1蛋白的相互作用蛋白。Objective RNA interference technology(siRNA)was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma HTB-182 cells,then,tandem affinity purification mass spectrometry(TAP-MS)was performed to screen the interacting proteins of DJ-1 in lung cancer cell line of HTB-182.Methods The siRNA lentivirus vector targeting DJ-1 gene was constructed to infect HTB-182 cells(DJ-1 siRNA group),and the lentivirus vector control group(control siRNA group)and blank control group were established.The expression level of DJ-1 protein was detected by Western blot,and the endogenous DJ-1 protein silenced si-DJ-1-HTB-182 cells were established.The specific primers of DJ-1 were designed,and the DJ-1 expression plasmid pNTAP-DJ-1 with streptomycin binding peptide label(SBP)and calmodulin binding peptide label(CBP)was constructed.The cell line DJ-1 siRNA HTB-182 was stably transfected with liposome,and the positive clones were screened by G418.The positive clones were verified by Western blot,and the interacting proteins of DJ-1 were found by TAP-MS.Results The protein expression of DJ-1 in DJ-1 siRNA interference group was significantly lower than that in empty plasmid group and blank control group(P<0.05);HTB182 cell line stably expressing pNTAP-DJ-1 plasmid was successfully constructed;Three proteins interacting with DJ-1 were screened by TAP-MS:cytokeratin 1(keratin 1),cytokeratin 10(keratin 10)and NADPH oxidase activating protein P47(P47 Px).Conclusions Keratin 1,Keratin 10 and P47 Px protein may be DJ-1 interactions protein.

关 键 词：HTB-182细胞 基因 DJ-1 相互作用蛋白 

分 类 号：R734.2[医药卫生—肿瘤]