视网膜母细胞瘤结合蛋白4基因酵母双杂交诱饵载体的构建
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摘要
视网膜母细胞瘤结合蛋白4(RBBP4)是WD-40蛋白家族的成员之一,其在组蛋白乙酰化、细胞周期进展及肿瘤干细胞的分化等方面具有重要功能,研究与RBBP4互作的新蛋白质和认识RBBP4在疾病包括肿瘤的发生发展过程中的分子机制有重要意义。通过RT-PCR扩增获得了RBBP4基因的CDS序列,经双酶切后与pGBKT7酵母诱饵载体连接构建了pGBKT7-RBBP4载体,再通过双酶切鉴定和测序验证,结果表明此酵母双杂交诱饵载体被成功构建,为后续利用酵母双杂交技术筛选与RBBP4蛋白相互作用的新蛋白提供了参考。
Being a member of WD-40 protein family, retinoblastoma binding protein 4(RBBP4) plays an important role in histone acetylation, cell cycle progression and differentiation of cancer stem cells. It is important to study new proteins interacting with RBBP4 and to understand its molecular mechanism in the pathogenesis of diseases,including tumors. In this study, the RBBP4 gene CDS sequence was successfully amplified by RT-PCR, and pGBKT7-RBBP4 was constructed by binding with pGBKT7 yeast bait vector. The results of double enzyme digestion and sequencing confirmed that the bait vector of yeast two-hybrid was successfully constructed, which provided a reference for screening new protein interacting with RBBP4 protein by yeast two-hybrid technology.
Being a member of WD-40 protein family, retinoblastoma binding protein 4(RBBP4) plays an important role in histone acetylation, cell cycle progression and differentiation of cancer stem cells. It is important to study new proteins interacting with RBBP4 and to understand its molecular mechanism in the pathogenesis of diseases,including tumors. In this study, the RBBP4 gene CDS sequence was successfully amplified by RT-PCR, and pGBKT7-RBBP4 was constructed by binding with pGBKT7 yeast bait vector. The results of double enzyme digestion and sequencing confirmed that the bait vector of yeast two-hybrid was successfully constructed, which provided a reference for screening new protein interacting with RBBP4 protein by yeast two-hybrid technology.
引文
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[11]刘超,刘乙蒙,栾治东,等.酵母双杂交技术筛选与蛋白激酶Wee1相互作用的蛋白[J].中国生化药物杂志,2016,36(3):23-26.
[12]吕震.RBBP4在肝细胞肝癌中的表达与作用机制研究[D].杭州:浙江大学,2015.
[2]XU C,MIN J R.Structure and function of WD40 domain proteins[J].Protein&Cell,2011,2(3):202-214.
[3]HANAHAN D,WEINBERG R A.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-674.
[4]KRüGER A V,JELIER R,DZYUBACHYK O,et al.Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C.elegans embryogenesis[J].Developmental Biology,2015,398(2):153-162.
[5]SONG H,XIA S L,LIAO C,et al.Genes encoding Pir51,Beclin 1,RbAp48 and Aldolase b are up or down-regulated in human primary hepatocellular carcinoma[J].World J Gastroenterol,2004(4):509-513.
[6]曹嬿,范松龙,张智弘.胃癌中RBBP4和BCL-2的表达及意义[J].临床与实验病理学杂志,2017(10):1070-1073.
[7]钟晶晶,杨旭锐,麦梅清,等.下调RbAp48表达可抑制人宫颈癌MS751细胞株的迁移侵袭能力[J].南方医科大学学报,2015(11):1564-1569.
[8]CREEKMORE A L,WALT K A,SCHULTZ-NORTON J R,et al.The role of retinoblastoma-associated Proteins 46 and48 in estrogen receptorαmediated gene expression[J].Mol&Cell Endocrinol,2008(1-2):79-86.
[9]黄亚强,黄红星,丘少鹏,等.RBBP4对前列腺癌细胞生长和侵袭能力的影响[J].现代泌尿生殖肿瘤杂志,2016(3):162-166.
[10]NAKAMURA T,HAMADA F,ISHIDATE T,et a1.Axin,an inhibitorofthe wntsignalling pathway,interacts withβ-catenin,GSK-3βand APC and reduces theβ-catenin level[J].Genes toCells,1998,3(6):395-403.
[11]刘超,刘乙蒙,栾治东,等.酵母双杂交技术筛选与蛋白激酶Wee1相互作用的蛋白[J].中国生化药物杂志,2016,36(3):23-26.
[12]吕震.RBBP4在肝细胞肝癌中的表达与作用机制研究[D].杭州:浙江大学,2015.