摘要
【目的】构建整合素(Integrin)β1和β2亚基胞内区原核表达载体,高效表达整合素β1和β2亚基胞内区蛋白,为进一步研究其在细胞迁移和信号传导中的定位及其多克隆抗体的制备奠定了基础。【方法】采用PCR技术,从cDNA中扩增出整合素β1和β2亚基胞内区基因片断,并将其克隆到pGEX-4T2原核表达载体上,构建了GST-β1和GST-β2质粒,然后将GST-β1和GST-β2质粒分别转化E.coliBL21(DE3)表达菌,用IPTG诱导,成功表达了整合素β1和β2亚基胞内区蛋白,并利用Glutathione SepharoseTM4B对表达产物进行纯化。【结果】利用PCR扩增得到了整合素β1和β2亚基胞内区基因片段,并成功构建了GST-β1和GST-β2质粒;对GST-β1和GST-β2诱导表达发现,这2种融合蛋白均为可溶性蛋白,具有良好的生物学活性。通过纯化得到了高纯度的GST-β1和GST-β2融合蛋白。【结论】整合素β1和β2亚基胞内区基因可以高效表达,并具有良好的生物学活性。
[Objective] High expression of intracellular region of integrin β1 and β2 subunits was presented by constructing the pronucleus expression vectors of intraeellular region of integrin β1 and β2 subunits, which lays a solid foundation for the further study of the location of integrin during cell migration, signal transduction and preparation of polyclonal antibody. [Method] Intracellular regions of integrin β1 and β2 subunits were amplified by PCR and subcloned into pGEX-4T2 vector to constuct GST-β1 and GST-β2 fusion protein express plasmids,then transfered to E. coli BL21 （DE3） and induced with IPTG. Finally the expressed protein was purified by Glutathione SepharoseTM 4B. [Result] Intracellular domains of integrin β1 and β2 protein were highly expressed, finally highly pure and soluble GST-β1 and GST-β2 fusion protein was obtained. [Conclusion] The intracellular domain of integrin β1 and β2 protein can be highly expressed and have good biological activities.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2009年第7期11-15,22,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(30771677)
关键词
整合素
β1和β2亚基
基因克隆
原核表达
蛋白纯化
integrin
β1 and β2 subunit
gene cloning
pronucleus expression
protein purification
