摘要
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ｐＧＥＸ５Ｔ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ｐＧＥＸ５Ｔ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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
1997年第2期105-108,共4页
Academic Journal of Second Military Medical University
基金
国家自然科学基金
