鞭毛蛋白FliC突变体／HPV18L2N融合蛋白的表达与纯化

目的：人乳头瘤病毒（HPV）的持续性感染导致女性宫颈癌的发生。HPV的次要衣壳蛋白L2可以诱发交叉中和多种型别HPV的中和抗体，但是单独免疫L2诱发的抗体滴度较低。鼠伤寒沙门氏茵鞭毛蛋白FliC是一种有效的佐剂。删除FliC超变区域的突变体可与外源抗原融合表达并且显著增强外源抗原特异性抗体的产生。本研究旨在构建鞭毛蛋白FliC超变区删除突变体与HPV18L2N（aa．13—154）的融合基因，通过大肠杆菌原核表达系统表达F1ic突变体与HPV18L2N的融合蛋白并纯化，为研究鞭毛蛋白的佐剂活性及新型HPV18L2疫苗奠定基础。方法：以鼠伤寒沙门氏菌鞭毛蛋白编码基因fliC为模板，通过重叠PCR法构建删除fliCD3区域（fliCAD3）、D3＋CD2a区域（fliCAD3CD2a）、D3＋D2区域（fliCAD2D3）的突变体，同时将HPV18L2N基因插入置换突变体的超变区删除区域。含有重组基因的表达载体在大肠杆菌中诱导表达，经SDS—PAGE及Westernblot鉴定分析。表达的融合蛋白经Ni—Sepharose亲和层祈纯化及Q-Sepharose离子交换层析去除内毒素。纯化后的融合蛋白经Native—PAGE鉴定分析，通过鲎试剂凝胶法测量蛋白溶液中的内毒素含量。结果：构建了pET22b．fliCAD3／18L2N、pET22b—nic△D3cD2a／18L2N、pET22b—fliCAD2D3／18L2N重组载体。重组载体在大肠杆菌以包涵体形式高效表达，且主要以单体形式存在。结论：通过原核表达及层析法纯化，成功获得了无热源、高纯度的鞭毛蛋白FliC突变体与HPV18L2N的融合蛋白，为增强HPVL2免疫原性提供了一种新的途径，为进一步研制HPV18L2疫苗奠定了基础。

Expression and Purification of FliC Mutant/HPV 18 L2N Fusion Proteins

Objective： The persistent infection of human papillomavirus causes cervical cancer in women. The minor capsid protein L2 can induce neutralizing antibody that cross-neutralize different HPV genotypes, but L2 alone can only induce low-titer neutralizing antibody. The FliC of Salmonella typhimurium is a potent adjuvant. FliC mutant whose hypervariable region was deleted can express as fusion protein with foreign antigen and enhance production of foreign antigen-specific antibody. This study is to construct fliC hypervariable region deleted mutants and fliC mutant/18 L2N recombinant genes, express fusion proteins of fliC mutants with HPV 18 L2N in E.coil, further to purify them. It established a foundation for studying the adjuvant activity of FliC and a novel HPV 18 L2 vaccine. Methods： FliCAD3 （D3 region deleted）, fliCAD3CD2a （D3 and CD2a region deleted） and fliCAD2D3 （D2 and D3 region deleted） genes were constructed by overlap PCR based on Salmonella typhimurium fliC gene. HPV 18 L2N gene was fused to replace the internal deleted region of fliCAD3, fliCAD3CD2a, fliCAD2D3. The expression vectors containing recombinant genes were expressed in E.coil and analyzed by SDS-PAGE and Western blot. Fusion proteins were purified by Ni-Sepharose affinity chromatography and endotoxin was removed by Q-Sepharose ion-exchange chromatography. The purified proteins were analyzed by Native-PAGE and residual endotoxin was quantified by LAL test. Results： pET22b-fliC AD3/18 L2N, pET22b-fliC AD3CD2a/18 L2N, pET22b-fliC AD2D3/18 L2N recombinant genes were constructed and highly expressed in E.coil as inclusion body. Fusion proteins were validated to be monomeric by Native-PAGE. Conclusion： Through prokaryotic expression and purification by chromatography, we successfully obtained apyrogenic FIiC mutant/18 L2N monomeric fusion proteins with high purity. This implied a novel way to improve immunogenicity of L2 and laid a basis for its application in the research of a novel HPV 18 L2 vaccine.