论文部分内容阅读
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ＣＯＳ７细胞培养上清中纯化ＰＴＡ１／Ｉｇ融合蛋白，用夹心ＥＬＩＳＡ及ＳＤＳＰＡＧＥ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ＣＯＳ７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，为进一步研究ＰＴＡ１的功能及鉴定其配体提供了有力手段
OBJECTIVE: To prepare an affinity chromatography column for the purification of platelet-T cell activation antigen 1 (PTA1) fusion protein and purify the eukaryotic expressed PTA1 / Ig fusion protein to further investigate the function of PTA1 and its ligands. METHODS: Anti-PTA1 mAb was purified by ammonium sulfate and anion exchange chromatography, and PTA1 affinity chromatography was prepared by cross-linking Sepharose4B. Purified PTA1 / Ig fusion protein from the supernatant of COS 7 cells transfected with pPTA1 / Ig expression vector by affinity chromatography, and purified the results by sandwich ELISA and SDS-PAGE. Results: After purification by ammonium sulfate and anion exchange chromatography, the anti-PTA1 mAb with high purity and good activity was obtained. The PTA1 affinity chromatography column was prepared by cross-linking Sepharose 4B, and the cross-linking rate was 96%. The affinity chromatography column can be purified from the supernatant of COS 7 cells transfected with pPTA1 / Ig by about 126 μg of PTA1 / Ig fusion protein per 100 ml. Conclusion: The preparation of PTA1 affinity chromatography column, and the purification of PTA1 / Ig fusion protein were successfully prepared, which provided a powerful tool for further study on the function of PTA1 and its ligand identification