IL-10基因克隆化与原核重组IL-10蛋白制备的实验研究  

Cloning of IL-10 gene and preparation of prokaryotic recombinant IL-10 protein

作　　者：濮海平 刘华 刘相萍[2] 隋爱华[2] 杨堃[2] 吕振华[2] 

机构地区：[1]401医院儿科,山东青岛266071 [2]青岛大学医学院附属医院中心实验室,山东青岛266003

出　　处：《实用医药杂志》2004年第12期1092-1095,共4页Practical Journal of Medicine & Pharmacy

摘　　要：目的克隆化人IL-10基因及重组蛋白的制备为IL-10基因表达在小儿炎症性、过敏性疾病中的作用及治疗等提供有力的证据。方法采用过敏症患者发作时期的外周血,提取总RNA。以总RNA为模板,特异性引物进行反转录,RT-PCR进行IL-10基因筛选。PCR产物经酶切分析和DNA序列测定后,构建重组质粒pTrchHis2B-hIL10原核高效表达载体,通过大肠杆菌(工程菌)JM109经IPTG诱导表达重组hIL-10蛋白,并经与Ni-NTA树脂中的6×His配体结合进行亲和层析纯化,表达产物进行SDS-PAGE电泳分析。结果DNA测序显示克隆化人IL-10全长开放读框准确,表达读码框正确。SDS-PAGE分析显示,原核表达系统表达的重组蛋白量和纯度满意。结论采用以总RNA为模板的RT-PCR一步法,能简便、可靠地获得hIL-10基因产物,所构建的原核表达载体pTrcHis2B-hIL10,能够快速、高效制备rhIL-10蛋白,为细胞因子hIL-10功能的进一步研究及临床应用奠定了基础。Objective To provide stable basis for studying effects of IL-10 expression in pediatric inflammatory, allergic disease and related clinical application,by cloning hIL-10 gene and preparing rhIL-10 protein. Methods With peripheral blood from a patient suffered from a acute allergy, total RNA were extracted. Then, with specific primers for reverse transcription, RT-PCR was carried out for IL-10 gene screening using total RNA as a template. Recombinant plasmid pTrcHis2B-hIL10 was constructed. Induced by IPTG, hIL-10 gene was expressed in E. coli JM109. Prokaryotic recombinant IL-10 proteins were extracted and purified by Ni-NTA resin which contained 6×His ligand. SDS-PAGE was used for recombinant products analysis and identification.Results PCR products were verified by DNA sequencing. There were not any error within full-length ORF sequence.And the location of hIL-10 ORF within MCS of vector was in a right direction.SDS-PAGE showed a satisfied amount and purification of recombinant protein.Conclusion Using one-step RT-PCR,hIL-10 gene can be conveniently amplified through total RNA as a template.rhIL-10 can be prepared quickly and effectively with prokaryotic expression vector pTrcHis2B-hIL10. Above results could provide a basis for further research and clinical use of the hIL-10.

关 键 词：IL-10 原核 重组蛋白 基因克隆化 临床应用 治疗 RT-PCR 总RNA 诱导表达 PCR产物 

分 类 号：R512.63[医药卫生—内科学] Q786[医药卫生—临床医学]