人血脑屏障通透性相关基因Caveolin-1的克隆及其蛋白表达、纯化

摘要
图/表
参考文献(20)
相关文章 (15)

全文: PDF (666 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要目的克隆与人血脑屏障通透性相关基因Caveolin-1，并在大肠埃希菌中表达，最后进行蛋白纯化、鉴定。方法根据基因库中收录的Caveolin-1基因序列，设计其上下游引物，经过PCR反应合成Caveolin-1双链，然后与pET28a（+）载体重组，筛选阳性克隆并对DNA序列分析鉴定。将已构建好的重组质粒pET28a-Caveolin-1转入化大肠埃希菌BL21（DE3），异丙基硫代半乳糖苷诱导融合蛋白表达，表达产物经镍离子亲和层析（Ni2+-NTA）纯化，SDS-PAGE检测和Western blot鉴定。结果 PCR扩增的Caveolin-1 DNA片段经鉴定确认为人Caveolin-1基因。经异丙基硫代半乳糖苷诱导的含有pET28a-Caveolin-1重组质粒的DE3菌表达出重组人Caveolin-1融合蛋白。重组蛋白经Ni2+-NTA亲和层析纯化，获得了较高纯度的融合蛋白。结论本研究成功克隆了人血脑屏障通透性相关基因Caveolin-1，并对其进行蛋白表达、纯化，获得了较高纯度融合蛋白，为进一步的相关临床研究奠定了基础。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	招树涛徐永健陈浩云

关键词 ：血脑屏障, 通透性相关基因, Caveolin-1, 克隆, 表达, 纯化

Abstract：Objective To clone Caveolin-1, a gene related to human blood-brain barrier permeability, and express it in Escherichia coli. and to purify and identify the protein finally. Methods According to the sequence of Caveolin-1 gene contained in the gene bank, the upstream and downstream primers were designed. The Caveolin-1 double strand was synthesized by PCR and then recombined with pET28a (+) vector. Positive clones were screened and identified by DNA sequence analysis. The constructed recombinant plasmid pET28a-Caveolin-1 was transfected into E. coli BL21 (DE3), and the expression of the fusion protein was induced by IPTG. The expressed product was purified by Ni2+-NTA affinity chromatography, identified by SDS-PAGE and Western blot. Results The Caveolin-1 DNA fragment amplified by PCR was identified as human Caveolin-1 gene. The recombinant human Caveolin-1 fusion protein was expressed by IPTG-induced DE3 strain containing the recombinant plasmid pET28a-Caveolin-1. The recombinant protein was purified by Ni2+-NTA affinity chromatography to obtain a fusion protein of higher purity. Conclusion This study successfully cloned the blood-brain barrier permeability-related gene Caveolin-1, and expressed and purified the protein to obtain a higher purity fusion protein, which laid a foundation for further relevant clinical research.

Key words： Blood-brain barrier Permeability related gene Caveolin-1 Cloning Expressing Purifying

基金资助:广东省中医药局科研项目（20161048）。

引用本文:

招树涛徐永健陈浩云. 人血脑屏障通透性相关基因Caveolin-1的克隆及其蛋白表达、纯化[J]. 中国医药导报, 2018, 15(32): 13-15,23.
ZHAO Shutao XU Yongjian CHEN Haoyun . Cloning, expression, purification of human blood-brain barrier permeability related gene Caveolin-1. 中国医药导报, 2018, 15(32): 13-15,23.

链接本文:

http://www.yiyaodaobao.com.cn/CN/ 或 http://www.yiyaodaobao.com.cn/CN/Y2018/V15/I32/13

[1] Jian LK，Rosenberg GA. Matrix metalloproteinases and free radicals in cerebral ischemia [J]. Free Radic Biol Med，2005，39（4）：71-80.
[2] Parton RG，Simons K. The multiple faces of caveolae [J]. Nat Rev Mol Cell Biol，2007，8（6）：185-194．
[3] Song L，Ge S，Pachter JS. Caveolin-1 regulates expression of junctionassociatedproteins in brain microvascular endothelial cells [J]. Blood，2007，109（8）：1515-1523.
[4] élizabeth Beauchesne，Desjardins P，Butterworth RF，et al. Up-regulation of caveolin-1 and blood-brain barrier breakdown are attenuated by N-acetylcysteine in thiamine deficiency [J]. Neurochem Int，2010，57（7）：830-837.
[5] Choi KH，Kim HS，Park MS，et al. Regulation of Caveolin-1 expression determines early brain edema after experimental focal cerebral ischemia [J]. Stroke，2016，5（3）：1336-1343.
[6] 叶棋浓.现代分子生物学技术与实验技巧[M].北京：化学工业出版社，2015.
[7] Janyou A，Wicha P，Jittiwat J，et al. Dihydrocapsaicin attenuates attenuates blood brain barrier and cerebral damage in focal cerebral ischemia/reperfusion via oxidative stress and inflammatory [J]. Sci Rep，2017，7（1）：10 556.
[8] Frieler RA，Chung Y，Ahlers CG，et al. Genetic neutrophil deficiency ameliorates cerebral ischemia-reperfusion injury [J]. Exp Neurol，2017，S0014-4886（17）：30 222-30 224.
[9] Su J，Liu J，Yan XY，et al. Cytoprotective effect of the UCP2-SIRT3 signaling pathway by decreasing mitochondrial oxidative stress on cerebral ischemia-reperfusion injury [J]. Int J Mol Sci，2017，18（7）：E1599.
[10] Jian LK，Rosenberg GA. Matrix metalloproteinases and free radicals in cerebral ischemia [J]. Free Radic Biol Med，2005，39（11）：71-80.
[11] Zhao YL，Song JN，Zhang M. Role of caveolin-1 in the biology of the blood-brain barrier [J]. Rev Neurosci，2014， 25（2）：247-254.
[12] Mandyam CD，Schilling JM，Cui W，et al. Neuron-targeted caveolin-1 improves molecular signaling， plasticity， and behavior dependent on the hippocampus in adult and aged mice [J]. Biol Psychiatry，2017， 81（2）：101-110.
[13] Choi KH，Kim HS，Park MS，et al. Overexpression of caveolin-1 attenuates brain edema by inhibiting tight junction degradation [J]. Oncotarget，2016，7（42）：67 857-67 867.
[14] Nag S，Manias JL，Kapadia A，et al. Molecular changes associated with the protective effects of angiopoietin-1 during blood-brain barrier breakdown postinjury [J]. Mol Neurobiol，2017，54（6）：4232-4242.
[15] 钟义良，张融融，黄思源，等.急性脑梗死患者血清陷窝蛋白1水平与早期神经功能恶化的关系[J].上海交通大学学报：医学版，2017，37（12）：1678-1681.
[16] Han F，Zhu HG. Caveolin-1 regulating the invasion and expression of matrix metalloproteinase （MMPs） in pancreatic carcinoma cells [J]. J Surg Res，2010，159（1）：443-450.
[17] Gu X，Reagan AM，McClellan ME，et al. Caveolins and caveolae in ocular physiology and pathophysiology [J]. Prog Retin Eye Res，2017（56）：84-106.
[18] Kovtun O，Tillu VA，Jung W，et al. Structural insights into the organization of the cavin membrane coat complex [J]. DevCell，2014，31（4）：405-419.
[19] Busija AR，Patel HH，Insel PA，et al. Caveolins and cavins in the trafficking，maturation，and degradation of caveolae：implications for cell physiology [J]. Am J Physiol Cell Physiol，2017，312（4）：C459-C477.
[20] Grzegorz S. Caveolae，caveolins，cavins，and endothelial cell function：new insights [J]. Frontiers in Physiology，2011， 2（2）：120.