| 核糖体蛋白RPS3a腺病毒载体的构建和体外表达 |
| 投稿时间：2012-12-31 修订日期：2013-05-20 点此下载全文 |
| 引用本文：吴红媛,厉建中,张俊平.核糖体蛋白RPS3a腺病毒载体的构建和体外表达[J].药学实践杂志,2013,31(5):347~350 |
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| 基金项目:国家自然科学基金(91013014). |
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| 中文摘要:目的 构建核糖体蛋白RPS3a的腺病毒表达载体,并进一步验证其在细胞中的表达。方法 应用RT-PCR从人胚肾HEK293细胞的总RNA中反转录并扩增出RPS3a基因,并将RPS3a亚克隆到穿梭质粒pAdTrack-CMV中,酶切及测序鉴定后,再将含RPS3a基因的重组穿梭质粒pAdTrack-CMV-RPS3a进行Pme I酶线性化处理,转化到含有骨架质粒的BJ5183感受态菌,进行同源重组。鉴定后,经Pac I线性化转染HEK293细胞,包装重组腺病毒。病毒感染NIH3T3细胞,Western blot 分析RPS3a蛋白的表达。结果 证实pAdTrack-CMV-RPS3a及pAdEas-RPS3a质粒构建正确;Western blot检测病毒感染的NIH3T3细胞总蛋白,与pAd-GFP阴性对照组相比,RPS3a蛋白表达量显著提高。结论 成功构建了能表达RPS3a基因的重组腺病毒载体,为进一步研究其生物学功能奠定基础。 |
| 中文关键词:RPS3a 腺病毒 构建 表达 |
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| Construction and expression of adenovirus expressing ribosomal protein RPS3a |
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| Abstract:Objective To construct the recombinant adenovirus expression vector containing ribosomal protein RPS3a and detect its expression in NIH3T3 cells. Method The RPS3a gene was obtained by RT-PCR.,total RNA was extracted from human HEK293 cell as the template. The cDNA fragments were cloned into the shuttle vector pAdTrack-CMV. After identified by enzyme digested and DNA sequence analysis,the product pAdTrack-RPS3a was lineared by PmeI and transformed into competent E.Coli BJ5183 carrying backbone plasmid pAdeasy-1 for homologous recombination. The identified positive recombinant adenoviral plasmid was then lineared by PacI and transfected into 293 cells for packaging and amplifying. Western blotting was used to detect the expression of RPS3a protein in the NIH3T3 cells infected with adenovirus. Results The pAdTrack-RPS3a and pAd-RPS3a plasmids had been successfully constructed. Protein expression was markedly increased in the NIH3T3 cells infected with Ad-RPS3a compared to that with control Ad-GFP. Conclusion The recombinant adenovirus harboring the RPS3a gene was successfully constructed, which could provide a useful tool for further investigating its biological function. |
| keywords:RPS3a adenovirus construction expression |
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