Prokaryotic Expression, Purification, and Production of Polyclonal Antibody Against Novel Human Serum Inhibited Related Protein I (SI1)
Prokaryotic Expression, Purification, and Production of Polyclonal Antibody Against Novel Human Serum Inhibited Related Protein I (SI1)
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新型人血清抑制相关蛋白I（SI1）多克隆抗体的原核表达、纯化和生产
DOI：
10.1007/s10930-009-9224-x
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发表时间：
2010-01
期刊：
影响因子：
3
通讯作者：
伍红
中科院分区：
文献类型：
--
作者：
马明星;谭德勇;赵文秀;焦扬;黄惟巍;马洁;石应辉;伍红
本实验室通过对U251细胞在有血清和无血清条件下的mRNA差异显示分析，克隆了一个新的血清抑制相关基因（SI1），在血清饥饿的细胞中加入血清可显著抑制SI1的表达。以往的研究表明，SI1在调节细胞周期中具有潜在的意义。本研究对SI1的质粒构建、蛋白表达和纯化以及抗SI1多克隆抗体的制备进行了研究。将Si1基因的全长cDNA克隆到原核表达质粒pET 28-B（+）中，并在大肠杆菌中得到高效表达。异丙基-b-d-硫代半乳糖苷诱导后的大肠杆菌Rosetta（DE 3）菌株。表达的SI1融合蛋白经Ni+亲和柱纯化后免疫Balb/C小鼠，用Protein A柱纯化抗SI1多克隆抗体。为检测SI1抗体的敏感性和特异性，用抗GFP单克隆抗体和抗SI1多克隆抗体对pEGFP-N2-SI1质粒瞬时转染的Hela细胞裂解液进行鉴定。GFP-SI1融合蛋白和Hela细胞内源性SI1蛋白均能被抗SI1多克隆抗体识别。SI1多克隆抗体的制备为SI1的进一步鉴定提供了有力的工具。
A novel serum inhibited related gene (SI1) has been cloned in our lab by using mRNA differential display analysis of U251 cells in the presence or absence of serum, the expression of SI1 was dramatically inhibited by the addition of serum to serum starved cells. Previous reports suggested the potential significance of SI1 in regulating the cell cycle. In this study, the plasmid construction, protein expression and purification, as well as the generation of anti-SI1 polyclonal antibody are described. A full-length cDNA of Si1 was inserted in a prokaryotic expression plasmid pET28-b(+) and efficiently expressed inE. coliRosetta (DE3) strain after induction by isopropyl-b-d-thiogalactoside. The expressed 6His-tagged SI1 fusion protein was purified by Ni+affinity column and then used to immunize Balb/C mice, and the anti-SI1 polyclonal antibody was purified by protein A column. To determine the sensitivity and specificity of the antibody against SI1, a cell lysate of pEGFP-N2-SI1 plasmid transiently transfected Hela cell was identified by anti-GFP monoclonal antibody and anti-SI1 polyclonal antibody. Both the GFP-SI1 fusion protein and endogenous SI1 protein in Hela cell can be recognized by the anti-SI1 polyclonal antibody. The anti-SI1 polyclonal antibody will provide a useful tool for further characterization of SI1.
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影响因子：
7.7
作者：
Simpson, JC;Wellenreuther, R;Wiemann, S
通讯作者：
Wiemann, S
影响因子：
1.5
作者：
Srsen, V;Kitazawa, H;Hisanaga, S
通讯作者：
Hisanaga, S
在NIH 3T3细胞中，在GO中 - > G1过渡期间，生长阻塞特异性蛋白质GAS2的磷酸化与肌动蛋白重排耦合。
影响因子：
7.8
作者：
Brancolini, C;Schneider, C
通讯作者：
Schneider, C
DOI：
10.1074/jbc.272.19.12699
发表时间：
1997-05
期刊：
The Journal of Biological Chemistry
影响因子：
--
作者：
Swati Gupta;A. Bose;N. Chatterjee;D. Saha;Shiyong Wu;N. Gupta
通讯作者：
Swati Gupta;A. Bose;N. Chatterjee;D. Saha;Shiyong Wu;N. Gupta
影响因子：
4.8
作者：
Ahn, JH;Park, SM;Lee, TH
通讯作者：
Lee, TH