乳源抗菌-免疫调节融合蛋白原核表达和纯化  

Expression and purification of antibacterial-immunomodulating fusion protein

作　　者：江良梁[1] 秦宜德[1] 周艳[1] 王超[1] 刘滋康[1] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2008年第3期260-263,共4页Acta Universitatis Medicinalis Anhui

基　　金：安徽省教育厅自然科学基金项目(编号:2005JK257)

摘　　要：目的构建乳铁蛋白抗菌肽(LfcinB)与免疫调节肽(PGPIPN)融合肽表达质粒,并在大肠杆菌BL21中表达及纯化。方法采用重叠延伸PCR技术获得融合肽基因片段,并构建到原核表达载体pGEX-KG中,挑选阳性重组子,经限制性内切酶鉴定后转化到大肠杆菌BL21,然后用IPTG诱导表达,通过SDS-PAGE及Western-blot方法鉴定表达的融合蛋白。用Glutathione Sepharose4B亲和层析方法纯化融合蛋白。结果成功构建原核表达质粒pGEX-KG-FP,并在大肠杆菌BL21中诱导其高表达。SDS-PAGE及Western-blot分析显示,特异性的抗GST单克隆抗体所识别的融合蛋白分子量与理论值相近。经Glutathione Sepharose4B纯化后,得到较纯的GST-FP融合蛋白。结论构建了原核表达的融合蛋白质粒,并高效表达和纯化了该融合蛋白。Objective To construct the plasmids encoding Lactoferricin B and immuno-modulating peptide （PG- PIPN） fusion peptide （FP） in bacteria, to express and purify the fusion protein. Methods Fusion gene was obtained by overlap extension polymerase chain reaction （OE-PCR）. The expression plasmid was constructed by inserting fusion peptide cDNA into pGEX-KG. The positive recombinants were identified by restriction endonuclease digestion and expressed in E coli BL21 induced by isopropyl-beta D-thiogalactopyranoside （IPTG）. The desired fusion proteins were identified by SDS-PAGE and Western blot. The expressed products were purified by affinity chromatography using GST fusion protein purification beads. Results Antibacterial-immunomodulating fusion peptide cDNA was cloned into pGEX-KG vectors and expressed in E coli BL21 successfully. SDS-PAGE and Western blot showed the molecular weight of the fusion protein recognized by the specific monoclonal antibody GST was coincident with prediction. The fusion proteins were purificated prolifically. Conclusion The antibacterial-immunomodulating fusion protein is obtained by prokaryotic expression,which lays the foundation for study of its function.

关 键 词：大肠杆菌 肽类/遗传学 基因表达 重组融合蛋白 质类 

分 类 号：R341.6[医药卫生—基础医学] R378.2