| 包琦锋 刘洪洋 张婷 陈雪 许雪梅.鞭毛蛋白FliC 突变体/HPV 18 L2N融合蛋白的表达与纯化[J].,2014,14(1):7-12 |
| 鞭毛蛋白FliC 突变体/HPV 18 L2N融合蛋白的表达与纯化 |
| Expression and Purification of FliC Mutant/ HPV 18 L2N Fusion Proteins |
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| DOI： |
| 中文关键词: 鞭毛蛋白 人乳头瘤病毒18 L2 突变体 |
| 英文关键词: Flagellin Human papillomavirus 18 L2 Mutant |
| 基金项目:国家自然科学基金项目（2012151） |
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| 中文摘要: |
| 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。鼠伤寒沙门氏菌鞭毛蛋白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
HPV 18 L2N（aa.13-154）的融合基因, 通过大肠杆菌原核表达系统表达FliC突变体与HPV 18 L2N 的融合蛋白并纯化，为研究鞭
毛蛋白的佐剂活性及新型HPV 18L2 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fliC D3 区域（fliC△D3）、D3+CD2a 区域（fliC△D3CD2a）、D3+D2 区域（fliC△D2D3）的突变体，同时将HPV 18 L2N 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Western blot 鉴定分
析。表达的融合蛋白经Ni-Sepharose 亲和层析纯化及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-fliC△D3/18 L2N、pET22b-fliC△D3CD2a/18
L2N、pET22b-fliC△D2D3/18 L2N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HPV 18 L2N 的融合蛋白，为增强HPV L2 免疫原性
提供了一种新的途径，为进一步研制HPV 18 L2 疫苗奠定了基础。 |
| 英文摘要: |
| 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 typhimuriumis 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:FliC△D3 (D3 region deleted), fliC△D3CD2a (D3 and CD2a region deleted) and fliC△D2D3 (D2 and D3 region
deleted) genes were constructed by overlap PCR based on Salmonella typhimuriumfliC gene. HPV 18 L2N gene was fused to replace the
internal deleted region of fliC△D3, fliC△D3CD2a, fliC△D2D3. 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 △D3/18 L2N, pET22b-fliC△D3CD2a/18 L2N, pET22b-fliC
△D2D3/18 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 FliC 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. |
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