中国免疫学杂志2011，Vol.27Issue(8)：691-695,5.DOI:10.3969/j.issn.1000-484X.2011.08.004
重组hIL-10在毕赤酵母X-33的表达及其生物活性鉴定
Expression of recombinant human interleukin-10 in X-33 Pichia pastoris and identification of its bioactivity
摘要
Abstract
Objective:To explore the appropriate conditions for the expression of IL-10 in Pichia and establish the foundation for the study and development and further clinical application of IL-10 related biological products.Methods:rhIL-10 was expressed in Pichia pastoris X-33 by inducing with methanol 0.5％,the analysis and identification were carried out by SDS-PAGE and Western blot.The expression levels of rhIL-10 were compared under different temperature,pH,with or without PMSF.The content of rhIL-10 in culture solution was determined by ELISA method,and the content of total protein in culture solution was determined by Bradford method,thus the proportion of rhIL-10 in the total protein could be calculated.The biological activites of rhIL-10 were analyzed by lymphocyte transformation test.Results:Protein bands of about 42 kD were observed after SDS-PAGE,and the similar specific protein bands were also observed in Western blot.The expression of rhIL-10 reached the maximum level of 20mg/L when th pH was 6.0,temperature was 28℃,and the inhibitor was used.In the sameperiod,the content of total protein was 50mg/L in the culture solution,and the proportion of rhIL-10 in the total protein was 40％.The lymphocyte transform ationtest showed that rhIL-10 inhibited the proliferation of lymphocytes.Conclusion:hIL-10 could be effectively expressed in Pichia with good biological activities;the fermentation condition have great influence on the expression level of rhIL-10,thus teh expression of rhIL-10 could be increased by improvement of the fermentation condition.关键词
人白细胞介素10/毕赤酵母/细胞增殖/淋巴细胞转化试验Key words
hIL-10/Pichia/Cell proliferation/Lymphocyte transformation test分类
医药卫生引用本文复制引用
陈富超,孙万邦,封建凯,杜联峰,黄俊琼,罗军敏,宋明英..重组hIL-10在毕赤酵母X-33的表达及其生物活性鉴定[J].中国免疫学杂志,2011,27(8):691-695,5.基金项目
本文受贵州省教育厅自然科学基金重点培育项目(黔教科2008032)和贵州省科技厅基金([2009]2176)资助 （黔教科2008032）
