鼠伤寒沙门菌鞭毛蛋白FliC的原核表达及其对口蹄疫疫苗的佐剂作用  

Flagella from Salmonella typhimurium expressed in prokaryotic cells and its adjuvant effect on the vaccination against foot-and-mouth disease

作　　者：陈志华[1] 袁林[2] 翟丽娟[2] 李禹涛[2] 吴丽华[2] 王伟玉[1] 黄一帆[1] 胡松华[2] 

机构地区：[1]福建农林大学动物科学学院动物医学系,福建福州350002 [2]浙江大学动物科学学院动物医学系,浙江杭州310029

出　　处：《中国兽医学报》2011年第2期198-204,共7页Chinese Journal of Veterinary Science

摘　　要：用PCR扩增鼠伤寒沙门菌基因FliC,将PCR产物克隆至pMD18-T载体中,构建了克隆质粒pMD18-FliC。克隆质粒和pET-his载体用EcoR/Nhel双酶切,将得到的纯化基因FliC亚克隆至pET-his内,构建重组质粒pET-his-FliC。酶切、测序鉴定后,分别将重组质粒转染大肠杆菌BL21,经IPTG诱导表达,菌体蛋白超声处理,上清液用AKTA Explorer蛋白纯化系统纯化。用SDS-PAGE分析所得蛋白,发现于约53 000处出现了与目的蛋白一致的外源蛋白带。293-mTLR5细胞活性检测表明该融合蛋白能刺激TLR-5通路,引起NF-κB的活化。用小鼠模型检验FliC对O型口蹄疫抗原的免疫增强作用,证明其对口蹄疫疫苗具有佐剂作用。Gene encoding FliC was amplified from Salmonella typhimurium chromosomal DNA by PCR,and the PCR product was cloned into pMD18-T vector to construct plasmid pMD18-FliC.pMD18-FliC and pET-his were digested by EcoR and Nhel double enzymes,and the purified FliC gene was subcloned into vector pET-his.Thus,the prokaryotic expression vector pET-his-FliC was constructed.The reconstructed plasmid pET-his-FliC was transformed into E.coli BL21.Expression of FliC in E.coli BL21 was induced by IPTG,and the expressed FliC was extracted using Ultrusonic Cell Disrupter System.The FliC was purified by AKTA Explorer system,and analyzed by SDS-PAGE.Approximately 53 000 exogenous FliC protein was found.Bioassay suggested the FliC had strong biologic activity to induce TLR5-specific signaling.A mouse model study showed that FliC enhanced serum sepcific IgG and the IgG isotype response to vaccination against foot-and-mouse disease,indicating the vaccine adjuvant property of the FliC.

关 键 词：鞭毛蛋白 口蹄疫疫苗 佐剂 TLR5 

分 类 号：S852.612[农业科学—基础兽医学]