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作 者:张蘋[1] 张利宁[1] 刘军莉[1] 王群[1] 宋静[1] 王晓燕[1]
机构地区:[1]山东大学医学院免疫学研究所,山东济南250012
出 处:《山东大学学报(医学版)》2004年第4期373-376,共4页Journal of Shandong University:Health Sciences
基 金:国家自然科学基金资助项目(NO.30271247);山东省自然科学基金资助项目(NO.413709)
摘 要:目的:在真核细胞中表达CTLA4鄄Ig融合蛋白,获得纯品并测定其生物学活性。方法:用电转染的方法将真核表达载体pREP7鄄CTLA4鄄IgG2c转染SP2/0细胞,经300μg/ml的潮霉素筛选得到阳性克隆;大量培养后收集含有重组CTLA4鄄IgG2c的细胞培养上清,用rProteinA亲和层析柱纯化CTLA4鄄Ig。用还原和非还原性SDS鄄PAGE和蛋白印迹进行鉴定。采用单向混合淋巴细胞培养(3H掺入法)测定CT鄄LA4鄄Ig对T细胞增殖的抑制效应。结果:阳性克隆大量培养的细胞上清经rPrtoteinA亲和层析后得到纯化CTLA4鄄Ig。还原条件下SDS鄄PAGE和Westernblot的结果显示在55KD出现目的蛋白条带;在非还原条件下SDS鄄PAGE和Westernblot在130KD处出现目的蛋白条带。混合淋巴细胞培养5d的结果显示,CTLA4鄄Ig浓度为10μg/ml时可以抑制T细胞的增殖;20μg/ml时抑制达到最大程度(P<0.05);继续增加CTLA4鄄Ig的浓度,其抑制作用不再增强。3、7d时的抑制作用不明显。结论:在SP2/0中表达了小鼠CTLA4鄄Ig融合蛋白,该融合蛋白能抑制T细胞的增殖。Objective: To express and purify mouse CTLA4-Ig fusion protein in eukaryote and to detect the biological activity of CTLA4-Ig. Methods: The pREP7-CTLA4-IgG2c vector was transfected into mouse SP2/0 cells by electrotransport. Positive clones were selected by hygromycin (300靏/ml), and CTLA4-Ig fusion proteins purified from vast culture supernatants of positive SP2/0 cell clones by a rProtein A column. The specificity of purified protein was identified by both reducing and non-reducing SDS-PAGE and Western blot, and the biological activity of CTLA4-Ig detected by mixed lymphocyte reaction (MLR). Results: Two weeks after transfection, the positive cellular clone of SP2/0 was obtained by selection of 300靏/ml hygromycin. A single band at about 55KD was found in reducting SDS-PAGE and Western blot. However, a specific band at about 130KD was detected in non-reducting SDS-PAGE and Western blot. CTLA4-Ig suppressed mouse lymphocyte activation in MLR five days later. MLR assay indicated that 10靏/ml and more than 10靏/ml CTLA4-Ig inhibited the proliferation of T lymphocyte and the highest inhibition was found at the concentration of 20靏/ml. The inhibitory intensity was not enhanced by CTLA4-Ig of more than 20靏/ml. Conclusion: The CTLA4-Ig expressed and purified from SP2/0 transfected with pRER7-CTLA4-IgG2c could suppress T proliferation in MLR.
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