肠炎沙门氏菌SEF14菌毛体外表达条件研究  

Optimal condition for expressionion SEF14 fimbriae of S.enteritidis in vitro

作　　者：朱春红[1] 段晓丽[2] 厚华艳[2] 龚建森[1] 张江英[2] 姚丰华[2] 孟霞[2] 朱国强[2] 

机构地区：[1]江苏省家禽科学研究所,江苏扬州225125 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国预防兽医学报》2013年第12期976-979,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31101833;31101826;31270171);江苏省自然科学基金(BK2011430);国家科技支撑计划"动物源性食品产业链中重要化学危害因子控制技术及致病微生物溯源技术研究与集成示范"(2012BAK17B10);教育部创新团队(IRT0978);江苏高校优势学科建设工程资助项目;扬州市农业科技攻关计划项目(YZ2011066);扬州市社会发展项目(YZ2011091)

摘　　要：为筛选肠炎沙门氏菌(S.enteritidis)SEF14菌毛体外表达的最适培养条件,本研究对4种不同培养条件下S.enteritidis SEF14菌毛的体外表达情况进行了比较研究,并通过RT-PCR、透射电镜与免疫电镜观察和SDS-PAGE等方法进行验证。研究表明,其中以TSB肉汤25℃震荡培养24 h后转接20℃继续震荡培养24 h,最后转接粘附因子抗原培养基(CFA)(pH6.0)于37℃静置培养50 h^60 h的SEF14菌毛表达量最高。RT-PCR结果显示在该培养条件下SEF14菌毛主要亚单位蛋白编码基因sefA mRNA表达水平最高,在透射电镜和免疫电镜下均可观察到丰富的SEF14菌毛。SDS-PAGE结果显示,所抽提的菌毛蛋白大小为14.3 ku,与相关报道一致,而且等量菌泥的抽提量最大。Fimbriae are important determinant of immunogenicity of Salmonella. To optimized the condition for expressing SEF14 fimbriae of Salmonella serovars enteriditis in vitro, four different bacteria culture protocols were used and the expression of SEF14 fimbriae examined by RT-PCR, TEM assay and SDS-PAGE. The results showed that SEF14 fimbriae of S.enteritidis expressed more sufficiently by direct inoculation of TSB medium from stock cultures, followed by incubation at 25 ~C for 24 hours and transfer to fresh TSB medium with a proportion of 1：100 and cultured for a further 24 hours at 20 ~C, finally, the culture was grown in static colonization factor antigen broth （CFA, pH6.0） with a proportion of 1：50 at 37 ~C for 50 to 60 hours. RT-PCR assay showed that sefA mRNA expression level in this condition was higher than others, and the SEF14 fimbriae ware showed in the surface of S.enteritidis by TEM and immunoelectron microscopy assay. In addition, SEF14 fimbriae protein was about 14.3 ku detected by SDS-PAGE.

关 键 词：肠炎沙门氏菌 SEF14菌毛 表达条件 RT-PCR 电镜观察 匀浆抽提 

分 类 号：S852.61[农业科学—基础兽医学]