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重庆市自然科学基金(2008BB5113)

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相关作者:陈炜徐小峰张俊磊韩俊峰李国利更多>>
相关机构:重庆三峡医药高等专科学校重庆医科大学第三军医大学更多>>
发文基金:重庆市自然科学基金国家自然科学基金重庆市卫生局医学科研项目更多>>
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Antibodies against ribosomal protein S29(RPS29)fused with glutathione's transferase specially react with native RPS29 in mouse and human cells
2011年
The ribosomal protein S29 also known as RPS29, is not only a component of the 40S subunit of ribosome, but also involved in embryonic development, oncogenesis and other pathologic conditions. However, rare commercial antibody against RPS29 restricts the discovery of precise physiological and pathological function of this protein. In this study, the whole RPS29 gene was inserted into plasmid pGEX-6p-1 to express glutathione's transferase (GST) fusion proteins in Escherichia eoli (E. coli) strain BL21. High yields of soluble recombinant proteins were obtained. Mice were immunized with the recombinant RPS29 protein. The serum from the immunized mice could specially react with purified recombinant RPS29 proteins and native RPS29 proteins in CCE cells by western blotting, immunofluorescence staining and flow cytometric analysis. Further more the polyclonal antibodies also reacted specifically with human cell strain ECV304, which showed typical cytoplasmatic fluorescence. The polyclonal antibodies we prepared would be an available tool for studying the roles of RPS29 in embryonic development and human diseases.
Liu JiaHan JunfengLi DongyingJian RuiRao XianCaiChen WeiWang JialiXu XiaofengHu ZhenZhang Junlei
病毒介导的P53低表达HepG2细胞株的建立
2012年
目的构建干扰P53的逆转录病毒载体,建立稳定低表达P53的HepG2细胞株。方法合成干扰P53基因的寡核苷酸序列插入pMSCV-hyg-U6质粒,转化受体菌DH5α,经酶切测序鉴定后瞬时转染HepG2细胞,通过半定量RT-PCR方法检测P53 mRNA表达水平评估干扰效果;选择干扰效果最好的重组质粒转染PT67细胞进行病毒包装,获得逆转录病毒颗粒感染HepG2细胞,通过Hygromycin筛选获得多个细胞克隆,Western blot检测P53蛋白的表达情况;选择P53表达水平最低的细胞克隆。通过5-氟尿嘧啶(5-FU)诱导凋亡实验,检测P53低表达的HepG2细胞p53通路介导的细胞凋亡情况。结果经酶切和测序验证成功构建重组病毒载体;瞬时转染HepG2细胞后P53 mRNA表达下调;病毒颗粒感染HepG2细胞后P53的mRNA、蛋白表达下调;用5-FU处理后P53低表达HepG2细胞凋亡水平明显低于对照。结论成功构建了干扰P53的逆转录病毒载体,建立了P53低表达HepG2细胞株,明显阻断了P53通路依赖的细胞凋亡。
刘佳张俊磊李庆伟韩俊峰徐小峰王嘉丽陈炜饶贤才李国利
关键词:逆转录病毒P53HEPG2
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