人肝再生增强因子在体外可与Na+-K+-ATPase 特异性结合

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+-ATPase β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+-ATPase β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。

Interaction between human augmenter of liver regeneration and Na+-K+-ATPase in vitro

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 quassating, 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.