| 新基因FAM92A1-289与增殖细胞核抗原PCNA相互作用的验证 |
| 投稿时间：2015-03-03 修订日期：2015-03-13 点此下载全文 |
| 引用本文：桂卉,郭兴荣,方娟,沈君豪,阮绪芝.新基因FAM92A1-289与增殖细胞核抗原PCNA相互作用的验证[J].医学研究杂志,2016,45(3):49-52 |
| DOI：
10.11969/j.issn.1673-548X.2016.03.014 |
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| 基金项目:湖北省自然科学基金资助项目(2012FFB02001) |
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| 中文摘要:目的 利用免疫共沉淀技术验证未知蛋白FAM92A1-289和PCNA(增殖细胞核抗原)之间的相互作用.方法 构建带有嘌呤霉素抗性的载体CMV-SP6--GFP-FAM92A1-289和真核表达载体PCDNA3.1-PCNA,先将CMV-SP6-GFP-FAM92A1-289载体转染入Hela细胞,通过最合适浓度的嘌呤霉素筛选,得到稳定表达FAM92A1-289的Hela/ FAM92A1-289细胞,再将载体PCDNA3.1-PCNA转染入Hela/ FAM92A1-289细胞,利用免疫共沉淀来验证FAM92A1-289和PCNA之间的相互作用.结果 构建的重组表达载体经双酶切、测序鉴定正确,稳定表达株 Hela/ FAM92A1-289细胞构建成功,PCNA单克隆抗体沉淀蛋白复合物后,用GFP抗体进行Western blot法检测,检测出了GFP-FAM92A1-289蛋白.结论 免疫共沉淀证明了FAM92A1-289和PCNA之间存在相互作用. |
| 中文关键词:FAM92A1-289 PCNA 免疫共沉淀 相互作用 |
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| Indentfication of Interaction Between New Gene FAM92A1-289 and Proliferating Cell Nuclear Antigen PCNA. |
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| Abstract:Objective To explore the interactions between the unknown protein FAM92A1-289 and PCNA by co-immunoprecipitation methods. Methods We recombinanted vector CMV-SP6-GFP-FAM92A1-289 and eukaryotic expression vector PCDNA3.1-PCNA. First of all, the vector CMV-SP6 -GFP-FAM92A1-289 was tramsfected into Hela cells. By puromycin screening, the stably-transfected Hela/ FAM92A1-289 cells showed that overexpression of FAM92A1-289 were obtained. Then the vector PCDNA3.1-PCNA was transfected into Hela/ FAM92A1-289 cells. Explore the interactions between FAM92A1-289 and PCNA was explered. Results Double restriction enzyme and gene sequencing showed that the recombinant vector was constructed correctly when PCNA was immunoprecipitated by anti-PCNA antibody. GFP-FAM92A1-289 was identified by western blotting with anti-GFP antibody from immunoprecipitated complex. Conclusion The interactions between FAM92A1-289 and PCNA could be identified by co-immunoprecipitation. |
| keywords:FAM92A1-289 PCNA Co-immunoprecipitation Interaction |
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