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核糖体蛋白RPS3a腺病毒载体的构建和体外表达

吴红媛,  厉建中,  张俊平

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文章导航 > 药学实践与服务  > 2013 >  31(5): 347-350
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]. 药学实践与服务, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
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]. 药学实践与服务, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
shu
WU Hong-yuan, LI Jian-zhong, ZHANG Jun-ping. Construction and expression of adenovirus expressing ribosomal protein RPS3a[J]. Journal of Pharmaceutical Practice and Service, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
Citation: WU Hong-yuan, LI Jian-zhong, ZHANG Jun-ping. Construction and expression of adenovirus expressing ribosomal protein RPS3a[J]. Journal of Pharmaceutical Practice and Service, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
shu

核糖体蛋白RPS3a腺病毒载体的构建和体外表达

doi: 10.3969/j.issn.1006-0111.2013.05.007
  • 1.

    福建中医药大学, 福建 福州, 350122;第二军医大学药学院生化药学教研室, 上海 200433

  • 2.

    第二军医大学药学院生化药学教研室, 上海 200433

基金项目: 国家自然科学基金(91013014).

Construction and expression of adenovirus expressing ribosomal protein RPS3a

  • 1.

    Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China;Department of Biochemical Pharmacy, School of Pharmacy, the Second Military Medical University, Shanghai 200433, China

  • 2.

    Department of Biochemical Pharmacy, School of Pharmacy, the Second Military Medical University, Shanghai 200433, China

  • 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。
    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.
  • [1] Lindstrom MS. Emerging functions of ribosomal proteins in gene-specific transcription andtranslation[J].Biochem Biophys Res Commun,2009,379(2):167.
    [2] Lecomte F, Szpirer J, Szpirer C. The S3a ribosomal protein gene is identical to the Fte-1 (v-fos transformation effector) gene and the TNF-α-induced TU-11 gene, and its transcript level is altered in transformed and tumor cells[J].Gene,1997,186(2):271.
    [3] Naora H, Nishida T, Shindo Y, et al. Antisense sequences of the nbl gene induce apoptosis in the human promyelocytic leukemia cell line HL-60[J].Leukemia,1998,12(4):532.
    [4] Song D, Sakamoto S, Taniguchi T. Inhibition of poly(ADP-ribose) polymerase activity by Bcl-2 in association with the ribosomal protein S3a[J].Biochemistry, 2002, 41 (3):929.
    [5] Kashuba E, Yurchenko M, Szirak K,et al. Epstein-Barr virus-encoded EBNA-5 binds to epstein-barr virus-induced Fte1/S3a protein[J].Exp Cell Res,2005,303(1):47.
    [6] Nolte D, Taimor G, Kalff-Suske M,et al. The human S3a ribosomal protein: sequence, location and cell-free transcription of the functional gene[J].Gene1996,169(2):179.
    [7] 孙阿萍,马洪星,郑寒松,等. 细胞凋亡过程中核糖体蛋白S3a水平变化的研究(三)[J].黑龙江医药科学,2002,25(1):1.
    [8] Naora H, Nishida T, Shindo Y,et al. Association of nbl gene expression and glucocorticoid-induced apoptosis in mouse thymus in vivo[J].Immunology,1995,85(1):6.
    [9] Naora H, Nishida T, Shindo Y,et al. Constitutively enhanced nbl expression is associated with the induction of internucleosomal DNA cleavage by actinomycin D[J].Biochem Biophs Res Commun, 1996,224(1):258.
    [10] Russell L, Naora H, Naora H. Down-regulated RPS3a/nbl Expression during retinoid-induced differentiation of HL-60 cells: A close association with diminished susceptibility to actinomycin D-stimulated apoptosis[J].Cell Struct Funct,2000,25(2):103.
    [11] Naora H, Takai L, Adachi M. Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosoma protein S3a gene expression induces apoptosis[J].J Cell Biol,1998,141(3):741.
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  • 收稿日期:  2012-12-31
  • 修回日期:  2013-05-20

核糖体蛋白RPS3a腺病毒载体的构建和体外表达

doi: 10.3969/j.issn.1006-0111.2013.05.007
  • 1. 福建中医药大学, 福建 福州, 350122;第二军医大学药学院生化药学教研室, 上海 200433
  • 2. 第二军医大学药学院生化药学教研室, 上海 200433
    • 基金项目:  国家自然科学基金(91013014).
  • 收稿日期: 2012-12-31
  • 修回日期: 2013-05-20

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。

English Abstract

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]. 药学实践与服务, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
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]. 药学实践与服务, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
shu
WU Hong-yuan, LI Jian-zhong, ZHANG Jun-ping. Construction and expression of adenovirus expressing ribosomal protein RPS3a[J]. Journal of Pharmaceutical Practice and Service, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
Citation: WU Hong-yuan, LI Jian-zhong, ZHANG Jun-ping. Construction and expression of adenovirus expressing ribosomal protein RPS3a[J]. Journal of Pharmaceutical Practice and Service, 2013, 31(5): 347-350. doi: 10.3969/j.issn.1006-0111.2013.05.007
shu

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