摘要
目的:用体外下拉实验证明人肝再生增强因子(hALR)与其相互作用蛋白Na+-K+-ATPase的体外结合作用。方法:将Na+-K+-ATPaseβ亚基部分基因片段定向克隆到pGEX-4T-1中,转化大肠杆菌DH5α,IPTG诱导,获得Na+-K+-ATPaseβ亚基部分蛋白与谷胱甘肽(GST)的融合表达,经GST偶联的琼脂糖珠纯化,以GST下拉实验检测其与hALR蛋白的体外直接相互作用。结果:还原型SDS-PAGE显示GST-Na+-K+-ATPase融合蛋白泳道有hALR蛋白单体和二聚体条带,Western blotting结果进一步证实为hALR蛋白。结论:Na+-K+-ATPase可在体外与hALR蛋白特异地结合。
AIM ： To check the physical interaction between GST - Na^＋ - K^＋ - ATPase domain and recombinant human augmenter of liver regeneration （rhALR） by GST pull down assay. METHODS： With PCR and genetic recombinant techniques, the coding region of β subunit of Na ^＋ - K ^＋ - ATPase was cloned into expressing plasmid pGEX - 4T and identified by endonuclease digestion and sequencing methods. Under the inducing of 0. 1 mmol/L IPTG, the fusion protein GST - Na^＋ - K^＋ - ATPase domain was highly expressed by E. coli DH - 5α. After hypersound quaasating, the GST - Na^＋ - K ^＋ - ATPase domain was purified by glutathione agarose beads and the physical interaction with rhALR was checked by GST pull down assay. RESULTS： Analysis by SDS - PAGE showed the rhALRs of monomer and dimmer in GST- Na^＋ -K^＋ - ATPase domain lane. The Western blotting of the GST -pull down assay showed the same results as well. CONCLUSION： The Na^＋ -K^＋ -ATPase domain is associated with rhALR specifically in vitro.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第4期734-736,共3页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金重点资助项目(No99B04704G-47-1)
