摘要
目的 为肿瘤坏死因子 α (TNF α)及其受体 (TNFR)的科学研究提供实验工具。方法 用基因克隆技术构建了人TNF α (rhTNF α)及绿色荧光蛋白 (GFP)与人TNF α的融合蛋白 (GFP TNF)的原核表达质粒pET2 8a/rhTNF及 pET2 8a/GFP TNF ,分别转化大肠杆菌BL2 1;用IPTG诱导重组蛋白的表达 ,超声裂菌 ,包涵体中的rhTNF α和GFP TNF用镍金属螯合层析柱进行纯化 ;用MTT法检测rhTNF α和GFP TNF的生物活性。结果重组表达的rhTNF α及GFP TNF均具有明显的细胞毒效应 ,GFP TNF还具有GFP的绿色荧光特性。结论 rhTNF α和GFP TNF具有生物活性 ,可用于TNF α和TNFR的实验研究。
Objective To supply the experimental tool for the study on the recombinant human TNF-α and GFP-TNF fusion protein expressed in bacterial E.coli. Methods By using gene cloning technique, 2 prokaryotic expression plasmids, pET28a/rhTNF and pET28a/GFP-TNF were constructed to express the recombinant human TNF-α (rhTNF-α) and its fusion protein with green fluorescent protein (GFP) in E.coli BL21, respectively. rhTNF-α and GFP-TNF was tagged with 6 histidine residues at their N terminus and was purified by nickel chelation chromatography; biological activities were assayed with U937 cell line using MTT. Results The recombinant TNF-α and GFP-TNF had cytotoxic effect. GFP-TNF had the character of GFP green fluorescence. Conclusion The prokaryotic expressed and purified GFP-TNF fusion protein has biological function and can be applied to further studies on TNF-α and TNFR.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2004年第5期519-521,534,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目 (No .396 30 32 0 )
