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

作　　者：陈富超[1] 孙万邦[1] 封建凯[1] 杜联峰[1] 黄俊琼[1] 罗军敏[1] 宋明英[1] 

机构地区：[1]遵义医学院免疫学教研室贵州省免疫学研究生教育创新基地,遵义563003

出　　处：《中国免疫学杂志》2011年第8期691-695,共5页Chinese Journal of Immunology

基　　金：贵州省教育厅自然科学基金重点培育项目(黔教科2008032);贵州省科技厅基金([2009]2176)资助

摘　　要：目的:用毕赤酵母表达hIL-10并探索合适的表达条件,为IL-10生物制品研发及临床应用奠定基础。方法:以0.5%甲醇诱导酵母X-33表达rhIL-10,用SDS-PAGE和Western blot分析鉴定rhIL-10,比较不同温度、不同pH值和有无PMSF条件下rhIL-10的表达水平。用ELISA法测定培养液rhIL-10含量,用Bradford法测定培养液总蛋白含量,计算rhIL-10占总蛋白的比例。以淋巴细胞转化试验分析rhIL-10的生物学活性。结果:SDS-PAGE后可见42 kD左右蛋白条带;Western blot也检测到42kD左右特异性蛋白质条带。pH值为6.0、温度在28℃并加入抑制剂时rhIL-10表达量最高值为20 mg/L,同时段培养液中总蛋白质含量为50 mg/L,rhIL-10占总蛋白比例为40%。在外周血淋巴细胞转化试验中,rhIL-10抑制了淋巴细胞的增殖。结论:hIL-10能在毕赤酵母高效表达,表达产物有较好生物活性;发酵条件对rhIL-10的表达水平影响大,改善发酵条件能促进rhIL-10的表达。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 activities 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 20 mg/L when the pH was 6.0,temperature was 28℃,and the inhibitor was used.In the same period,the content of total protein was 50 mg/L in the culture solution,and the proportion of rhIL-10 in the total protein was 40%.The lymphocyte transformation test 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 the 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 

分 类 号：R392.1[医药卫生—免疫学]