内毒素结合肽的原核表达、纯化及生物学活性鉴定

重组人内毒素结合肽 (endotoxinbindingpeptide ,EBP)融合蛋白在大肠杆菌中表达 ,分离和纯化后对其进行生物学活性观察 .将构建好的PinpointⅩa3 EBP生物素融合表达载体转化大肠杆菌DH5α ,IPTG诱导表达菌株 ,亲和层析法纯化表达产物 ,因子Ⅹa(factorⅩa)切割分离内毒素结合肽 ,采用凝胶过滤和反相液相高效色谱法两步纯化 ,从相对分子质量、N端 1 0个氨基酸的序列分析等方面进行鉴定 ;利用人单核细胞U937对重组内毒素结合肽进行了生物学活性的检测 .结果发现 ,内毒素结合肽以包涵体形式存在 ,因子Ⅹa酶切融合蛋白后得到 3 5kD的内毒素结合肽 ,纯化后内毒素结合肽纯度达 99%以上 ,N端 1 0个氨基酸的分析结果与预期相符 ;初步证实内毒素结合肽具有较好的LPS结合活性 ,能够抑制LPS的作用 .经原核表达及纯化复性 ,获得了具有较好生物学活性的内毒素结合肽 ,为进一步研究其功能奠定了良好的基础

Expression,Purification of Endotoxin Binding Peptide in E.coli and Its Biological Activities

The recombinant endotoxin binding peptide(EBP)fusion protein was expressed in E.coli DH5α.EBP was separated and purified and its biological activities were determined.After the construct of Pinpoint Ⅹa3\| EBP and being transformed into E.coli DH5α,the expression of EBP\|biotinylated fusion protein was induced by IPTG and was captured and purified by Softlink TM Soft\|release avidin resin.After its cleavage by factor Ⅹa, the target EBP was separated and purified by Sephadex G\|25 and reversed phase HPLC.10 amino acids of N\|terminal and relative molecular weight of EBP were detected to evaluate it.The monocyte U937 was used to detect the biological activities of EBP.The fusion protein was expressed in the form of inclusion body.The 3 5 kD target EBP was obtained and its purity was more than 99%.The amino acid assay were matched with expectation.The preliminary results indicated that recombinant human EBP might possess biological activities.These results paved a reasonable way for the study of new endotoxin binding peptides.