内毒素结合肽的改良、原核表达及纯化

目的：对人内毒素结合肽(endotoxin binding peptide,EBP)进行改良，获得突变体mEBP(mutate of endotoxin binding peptide,mEBP)基因并进行原核表达，纯化获得高纯度的mEBP。方法：应用PCR定点诱变方法获得mEBP基因，构建PinpointXa-3/mEBP融合表达载体,在BL21(DE3) pLysS宿主菌进行表达，溶菌酶裂解法提取包涵体，融合蛋白经PinpointTM Xa 纯化后，由factorXa酶切,获得目的肽，RP-HPLC纯化获得高纯度的mEBP。结果 应用PCR定点诱变技术完成了EBP第5.18位谷氨酰胺 赖氨酸的定点突变，且通过原核表达，色谱纯化获得了高纯度的mEBP。结论 成功获得高纯度的mEBP,为下一步的抗内毒素功能检测奠定基础。

Modification, Expression and Purification of Human Endotoxin Binding Peptide Gene

Objective To modify EBP(endotoxin binding peptide), clone and express the mutate of EBP gene and gain purified mEBP.Method mEBPgene was cloned by PCR site-directed mutagenesis. PinpointXa-3/mEBP expression vector was designed to express human mEBP as a fusion protein.in BL21 (DE3) pLysS, Digested engineering bacteria by lysozyme and collected inclusion bodies.Fusion protein was purified by pinpoint TM Xa purification system and cleaved by factorⅩa,mEBP was purified by RP-HPLC. Results. Mutations at residues 5 and 18(Gln Lys) was obtained by PCR site-directed mutagenesis,Expressed and purified mEBP successfully.Conclusions: Obtaining of purified mEBP lay a foundation for its biological activity research.