人血小板/T细胞活化抗原1(PTA1)亲和层析柱的制备及PTA1/Ig的纯化  

Purification of PTA1/Ig fusion protein by affinity chromatography for PTA1

作　　者：孙凯[1] 金伯泉[1] 孙忱[1] 朱勇[1] 贾卫[1] 杨琨[1] 刘成刚[1] 

机构地区：[1]第四军医大学免疫学教研室

出　　处：《细胞与分子免疫学杂志》1998年第2期113-116,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金

摘　　要：目的：制备可用于纯化血小板／Ｔ细胞活化抗原１（ｐｌａｔｅｌｅｔａｎｄＴｃｅｌａｃｔｉｖａｔｉｏｎａｎｔｉｇｅｎ１，ＰＴＡ１）融合蛋白的亲和层析柱，纯化真核表达的ＰＴＡ１／Ｉｇ融合蛋白，以进一步研究ＰＴＡ１的功能及其配体。方法：以硫酸铵及阴离子交换色谱法纯化抗ＰＴＡ１ｍＡｂ，并交联Ｓｅｐｈａｒｏｓｅ４Ｂ制备ＰＴＡ１亲和层析柱。通过亲和层析法，从ｐＰＴＡ１／Ｉｇ表达载体转染的ＣＯＳ７细胞培养上清中纯化ＰＴＡ１／Ｉｇ融合蛋白，用夹心ＥＬＩＳＡ及ＳＤＳＰＡＧＥ鉴定纯化结果。结果：硫酸铵及阴离子交换色谱纯化后，获得纯度高和活性好的抗ＰＴＡ１ｍＡｂ，用其交联Ｓｅｐｈａｒｏｓｅ４Ｂ制备ＰＴＡ１亲和层析柱，交联率为９６％。该亲和层析柱可从ｐＰＴＡ１／Ｉｇ转染的ＣＯＳ７细胞上清每１００ｍｌ中，纯化ＰＴＡ１／Ｉｇ融合蛋白约１２６μｇ。结论：制备成功可用于纯化ＰＴＡ１的亲和层析柱，并获得纯化的ＰＴＡ１／Ｉｇ融合蛋白。Aim: Platelet and T cell activation antigen 1 (PTA1) is a novel member of immunoglobulin superfamily. Its ligand and function are still not clear. To investigate these, anti PTA1 affinity chromatography column was prepared and the PTA1 IgG Fc fusion protein from culture supernatant of pPTA1/Ig transfected COS 7 cells was purified. Methods: mAb Leo A1 was purified by ammonium sulfate precipitation and FPLC, and its PTA1 binding activity was assayed by flow cytometry. An affinity chromatography column was prepeared by coupling mAb Leo A1 to CNBr activated Sepharose 4B. After transfection of pPTA1/Ig into COS 7 cells by DEAE dextran, expression of secreting fusion protein was identified by sandwich ELISA with anti PTA1 mAb and HRP anti hIgFc mAb. PTA1/Ig fusion protein was obtained by chromatographic purification from supernatant of PTA1/Ig vector transfected COS 7 cells. Results: mAb Leo A1 remained high binding activity to PTA1 after purification by ammonium sulfate precipitation and FPLC. 126 μg of PTA1/Ig fusion protein was obtained from 100 ml of the COS 7 supernatant after chromatography. Both anti PTA1 and anti hIgFc mAb could recognize this fusion protein with molecular weight M r83 000. Conclusion: PTA1/Ig fusion protein could be purified from with transfected pPTA1/Ig COS 7. This fusion protein could mimic the natural PTA1 protein and could be a potential tool for investigation of ligand and function of PTA1.

关 键 词：PTA1 亲和层析 纯化 抗原 融合蛋白 

分 类 号：R392-33[医药卫生—免疫学]