重组人白血病抑制因子受体α亚基细胞外区的分子克隆

Molecular cloning of recombinant extracellular domain of α subunit of human leukemia inhibitory factor receptor

目的：纯化可溶性白血病抑制因子（ＬＩＦ）受体，获得特异性免疫原并利用衰变加速因子（ＤＡＦ）的附膜特性探讨ＬＩＦ受体的生物特性和功能。方法：采用基因重组技术，将ＬＩＦ受体α亚基（ｇｐ１９０）细胞外区（ｇｐ１９０ｓｏｌ）的Ｃ端与ＤＡＦＣ端３７个氨基酸连接，再将该重组基因载入ｐＧＥＸ５Ｔ表达系统。结果：在Ｅ．ｃｏｌｉＸＬ１中可高度表达一种Ｎ端为谷胱甘肽Ｓ转移酶和Ｃ端为１９０ｓｏｌＤＡＦ的融合重组蛋白。此蛋白在ＸＬ１中表达产物为不溶性包涵体，以变性剂８ｍｏｌ／Ｌ尿素溶解并复性后，可与交联在亲和层析柱上的谷胱甘肽（Ｇｌｕｔａｔｈｉｏｎｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ）结合，从而一次性分离ＬＩＦ受体α亚基细胞外区。结论：大分子糖蛋白也能采用ｐＧＥＸ５Ｔ系统在大肠杆菌中表达，并通过谷胱甘肽亲和法纯化而作为免疫原。

Objective: Soluble receptor (gp190 sol, extracellular domain of α subunit of LIF receptor ) with DAF can be used to analyse functions of LIF receptor and to prepare the antibodies. Methods: For the production of recombinant human 190 sol DAF in Escherichia coli, a cDNA coding for the human gp190 sol was separated from gp190 full length cDNA and recomposed with DAF oligo, then cloned into the expression vector pGEX 5T. Results: pGEX 5T 190 sol DAF was highly expressed as a fusion protein with the glutathione S transferase (GST) at its N terminus and human 190 sol DAF at its C terminus in XL 1. The fusion protein was insoluble as the including body in the common solutions, but solubilized in 8 mol/L urea. Afterwards, it was purified to homogeneity by affinity chromatography using a glutathione Sepharose 4B column after the renatured reaction. Conclusion: pGEX 5T can be used to the expression system in Escherichia coli. for large molecular glycoprotein and glutathione affinity column can be used to purify this recombinant protein.