小鼠HMGB1 B box基因的原核表达及其活性鉴定  

Prokaryotic expression and activity identification of mouse HMGB1 B box coding sequence

作　　者：侯小强[1] 冯娜[2] 夏咸柱[2] 高玉伟[2] 孙培录[1] 杨松涛[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]解放军军事医学科学院军事兽医研究所,长春130062

出　　处：《中国免疫学杂志》2008年第10期872-875,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金(30471295);“十一五”国家科技支撑计划项目(2006BAD06A01)资助

摘　　要：目的:克隆小鼠HMGB1 B box基因,表达具有明显致炎活性的鼠HMGB1 B box蛋白,为深入研究其生物学功能以及新型抗炎制剂的研发奠定基础,也为进一步探讨其与H5N1禽流感病毒性肺炎的相关性奠定了基础。方法:从小鼠的肺脏组织中提取总RNA,经RT-PCR扩增小鼠HMGB1中的B box的cDNA,构建于载体pMD-18T并进行序列测定,随后构建于原核表达载体pET-28a(+)中。IPTG诱导4小时后可见Mr约13000的蛋白。经Ni2+亲和层析柱纯化获得高纯度的HMGB1 B box蛋白,然后用此蛋白刺激小鼠外周血单核细胞(PBMCs),作用6小时后用ELISA检测PBMCs释放TNF-α、IL-6的含量。结果:经RT-PCR扩增得到了240bp的DNA片段,经序列分析与Genebank中报道的已知序列完全一致;成功构建了含有HMGB1 B box编码序列的原核表达载体;纯化表达的HMGB1 B box蛋白能显著地刺激小鼠PBMCs释放致炎因子TNF-α、IL-6,具有较好的生物活性。结论:原核表达的小鼠HMGB1 B box蛋白具有较好的生物活性,为进一步研究HMGB1 B box的作用机制以及新型抗炎制剂的研究奠定了基础。Objective：To clone the encoding sequence of mouse HMGB1 B box gene and construct the recombinant vector,whereby to study its proinflammatory function.Methods：Total RNA was extracted from mouse lung and the HMGB1 B box cDNA was amplified using RT-PCR.The target DNA fragment was cloned into pET-28a（＋） vector and the target protein was expressed in the presence of IPTG.After purified by Ni2＋-NTA chromatography the target protein was used to stimulate mouse PBMCs for detecting the release of TNF-α and IL-6 by ELISA.Results：Obtained full encoding sequence of HMGB1 B box was identical to that in Genebank.Expressing plasmid pET-28a-H was constructed.Purified 13 kD proteins was obtained.The HMGB1 B box protein could obviously increase the release of TNF-α and IL-6 from mouse PBMCs.Conclusion：The purified prokaryotic expressing mouse HMGB1 B box is obtained and has biological activity to induce production of proinflammatory cytokines.It might provide a foundation for further study on the mechanism of HMGB1 in inflammatory reaction and its role in H5N1 avian influenza viral pheumonia.

关 键 词：高迁移率族蛋白B1 B BOX 原核表达 活性鉴定 

分 类 号：R392.11[医药卫生—免疫学]