酵母双杂交技术筛选宫颈癌 HeLa 细胞cDNA 文库中 FAM92 A1-289关联蛋白

目的：应用酵母双杂交技术从宫颈癌HeLa细胞cDNA文库中筛选FAM92A1-289关联蛋白。方法构建酵母双杂交pGBKT7-FAM92A1-289诱饵载体，转化至酵母AH109感受态细胞中。利用Clontech GAL4酵母双杂交系统筛选HeLa细胞cDNA文库中与FAM92A1-289相互作用的蛋白质，用营养缺陷型培养基和X-a-Gal双重筛选实验进行筛选，对筛选结果进行生物信息学分析，对克隆重复率较高的蛋白进行回转验证，将β-半乳糖苷酶活性阳性的克隆进行测序并分析。结果成功构建酵母双杂交pGBKT7-FAM92A1-289诱饵载体，可在酵母细胞中正常表达FAM92A1-289，且对酵母细...

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Bibliographic Details
Published in山东医药 Vol. 56; no. 19; pp. 1 - 4
Main Author 沈君豪 方娟 郭兴荣 桂卉 涂汉军 阮绪芝
Format Magazine Article
LanguageChinese
Published 湖北医药学院基础医学院,湖北十堰,442000 2016
Subjects
1
53
A1-289
athanogene
BCL2-associated
BCL2相关永生基因重组人BCL2相关永生基因1
compartment
endoplasmic
FAM92
FAM92A1-289
galectin-1
HeLa细胞
human
intermediate
marker
recombinant
reticulum-golgi
内质网高尔基体中间室标记物53
半乳糖凝集素1
增殖细胞核抗原
宫颈癌
酵母双杂交技术
半乳糖凝集素1
recombinant human BCL2-associated athanogene 1
yeast two-hybrid technique
宫颈癌
FAM92 A1-289
酵母双杂交技术
增殖细胞核抗原
cervical carcinoma
HeLa细胞
FAM92A1-289
proliferating cell nuclear antigen
BCL2相关永生基因重组人BCL2相关永生基因1
HeLa cell
内质网高尔基体中间室标记物53
endoplasmic reticulum-golgi intermediate compartment marker 53
galectin-1
Online AccessGet full text
ISSN1002-266X
DOI10.3969/j.issn.1002-266X.2016.19.001

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Summary:目的：应用酵母双杂交技术从宫颈癌HeLa细胞cDNA文库中筛选FAM92A1-289关联蛋白。方法构建酵母双杂交pGBKT7-FAM92A1-289诱饵载体，转化至酵母AH109感受态细胞中。利用Clontech GAL4酵母双杂交系统筛选HeLa细胞cDNA文库中与FAM92A1-289相互作用的蛋白质，用营养缺陷型培养基和X-a-Gal双重筛选实验进行筛选，对筛选结果进行生物信息学分析，对克隆重复率较高的蛋白进行回转验证，将β-半乳糖苷酶活性阳性的克隆进行测序并分析。结果成功构建酵母双杂交pGBKT7-FAM92A1-289诱饵载体，可在酵母细胞中正常表达FAM92A1-289，且对酵母细胞无毒性，不存在自激活现象。筛选出在SD／-Ade／-His／-Leu／-Trp四缺培养基及含X-a-Gal的SD／-Ade／-His／-Leu／-Trp四缺培养基上均能生长且β-半乳糖苷酶活性阳性的克隆9个。经生物信息学分析发现4种蛋白重复率较高，即增殖细胞核抗原（PCNA）、半乳糖凝集素1（Galectin-1）、内质网高尔基体中间室标记物53（ERGIC-53）、重组人BCL2相关永生基因1（BAG1），并均与FAM92A1-289存在相互作用。结论利用酵母双杂交技术在Hela细胞cDNA文库中成功筛选出与FAM92A1-289相互作用的蛋白，为进一步研究FAM92A1-289的功能提供了新线索。
Bibliography:37-1156/R
Objective To screen the interacting proteins of FAM 92A1-289 in Hela cell cDNA library by yeast two-hy-brid technique.Methods The bait plasmid pGBKT7-FAM92A1-289 was constructed and then transformed into yeast com-petent cells AH109.Clontech GAL4 yeast two-hybrid assay was performed to screen the interacting proteins of FAM 92A1-289 in the Hela cell cDNA library .SD nutrient medium and X-a-Gal were used for selecting and screening .Moreover , back-cross was performed to confirm the interaction of identified proteins , and bioinformatics was used to analyze the se-quences of clones of positive β-galactosidase.Results The bait plasmid pGBKT7-FAM92A1-289 was successfully con-structed and expressed FAM92A1-289 in yeast cells, showing no toxicity and self-activation.Nine colonies were identified after β-galactosidase test and selection of SD/-Ade/-His/-Leu/-Trp plates containing X-a-Gal.Bioinformatics analysis shows four proteins had high repetition-rate, such as proliferating cell nuclear antigen （ PCNA）
ISSN:1002-266X
DOI:10.3969/j.issn.1002-266X.2016.19.001