新型重组人内毒素结合肽突变体的构建及原核表达纯化

Preparation and Expression of the Recombinant Mutant of Human Endotoxin Binding Peptide in E. Coli

目的构建新型人内毒素结合肽（a new endotoxin binding peptide consisting of 25 amino acid residues，EBP25）及其突变体（mutant of EBP25，mEBP25）的原核表达重组质粒，并在大肠埃希菌中诱导表达。方法采用PCR法，扩增EBP25基因，构建pET-30-EBP25.融合表达载体并转化Ecoli DH5α扩增。重组质粒经酶切和测序鉴定后，应用快速定点突变法将EBP25第2位缬氨酸和第5位谷氨酰胺所对应碱基均替换成赖氨酸所对应的碱基，突变后重组质粒再经测序鉴定后，将二者转化至E．coli BL21（DE3）PlysS后经IPTG诱导表达，表达产物采用Western印迹进行鉴定后，用His—Tag亲和层析对融合蛋白进行纯化。结果两次测序结果显示人EBP25，和mEBP25重组序列和理论设计序列完全一致后，经IPTG诱导表达获得目的融合蛋白，通过SDS—PAGE电泳、Western印迹证实蛋白表达的特异性，并对蛋白进行纯化，获得EBP25和mEBP25融合蛋白。结论构建、表达纯化了EBP25和mEBP25融合蛋白，为进一步研究其中和内毒素／月旨多糖活性奠定了基础。

Objective To construct prokaryotic expression vectors expressing a new human endotoxin binding peptide consisting of 25 amino acid residues and its mutant and purify their fusion proteins. Methods The EBPEsgene was amplified by PCR and inserted into the prokaryotic expression vector pET-30. The recombinant plasmid pET-30-EBP25 was transformed into E. coll. DH5a and identified by DNA sequencing. Then the bases corresponding to the second valine and the fifth glutamine were replaced respectively with the bases of lysine by use of Quikchange site-directed mutation. The recombinant mutant plasmid pET-30-mEBP25 was again identified by DNA sequencing. Both EBP25 and mEBP25 were transfected into E. coll. BL21 （ DE3 ） PlysS and expressed under IPTG induction. Expressed EBP25 and mEBPE5 proteins were analyzed by SDS-PAGE and western blotting and purified with His-Tag affinity chromatography. Results Both EBP25 and mEBP25 recombinants were confirmed by DNA sequencing. Highly expressed and purified EBP25 and mEBP25 protein were obtained, and their specificities were verified by SDS-PAGE and western blot. Conclusion The preparation of EBP25 and mEBP25 fusion proteins may be useful in studying biological function of neutralizing endotoxin/lipopolysaccaride.