金黄色葡萄球菌GapC蛋白与鼠伤寒沙门菌鞭毛蛋白的融合表达及其活性  

Fusion expression of GapC protein of Staphylococcus aureus and flagellin of Salmonella typhimurium and activity of expressed product

作　　者：赵达[1] 王鹤[1] 梁宏儒[1] 胡旭[1] 姜东君[1] 高佳滨[1] 尹辉[1] 陈为宏[1] 乔波[1] 朱战波[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国生物制品学杂志》2014年第2期168-171,176,共5页Chinese Journal of Biologicals

基　　金：黑龙江省重大科技攻关项目(GA09B301-2)

摘　　要：目的在大肠埃希菌中融合表达金黄色葡萄球菌(Staphylococcus.aureus,S.aureus)GapC蛋白与鼠伤寒沙门菌鞭毛蛋白,并检测其甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)活性。方法采用PCR技术分别扩增鼠伤寒沙门菌鞭毛蛋白flic基因和S.Aureus GapC基因,通过overlap PCR将这两段基因拼接并克隆至载体pQE30a,构建重组表达质粒pQE30-flic-GapC,将重组表达质粒转化E.coli XL-blue,IPTG诱导表达,表达产物经镍柱亲和层析试剂盒纯化后,进行SDS-PAGE及Western blot分析,并检测其GAPDH活性。结果重组表达质粒pQE30-flic-GapC经双酶切及测序鉴定证明构建正确;表达产物相对分子质量约95 000,最佳诱导表达时间为5 h,主要以包涵体形式存在,占菌体总蛋白的31.4%;纯化产物可与小鼠抗S.aureus全菌体多抗血清和抗鼠伤寒沙门菌全菌体多抗血清发生特异性反应,GAPDH活性为(0.337±0.019)。结论成功表达并纯化了具有较高生物学活性的flic-GapC融合蛋白,为S.aureus亚单位疫苗的研究奠定了基础。Objective To express the fusion protein of GapC of Staphylococcus aureus and flagellin（flic） of Salmonella typhimurium in E. coli,and determine its glyceraldehydes-3-phosphate dehydrogenase（GADPH）activity. Methods The genes coding S. typhimurium flic and S. aureus GapC were amplified by PCR respectively,spliced by overlap PCR and inserted into vector pQE30a. The constructed recombinant plasmid pQE30-flic-GapC was transformed to E. coli XL-blue and induced with IPTG. The expressed product was nickel ion affinity chromatography,analyzed by SDS-PAGE and Western blot,and determined for GAPDH activity. Results Restriction analysis and sequencing proved that recombinant plasmid pQE30-flic-GapC was constructed correctly. The optimal time for induction of expression was 5 h. The expressed product,with a relative molecular mass of about 95 000,mainly existed in a form of inclusion body and contained 31. 4% of total somatic protein. The purified fusion protein was reacted with mouse polyclonal antisera against S. aureus and S. typhimurium,of which the GAPDH activity was 0. 337 ± 0. 019. Conclusion The fusion protein flic-GapC with high biological activity was expressed successfully,which laid a foundation of preparation of S. aureus subunit vaccine.

关 键 词：金黄色葡萄球菌 鼠伤寒沙门菌 GapC蛋白 鞭毛蛋白 融合蛋白 

分 类 号：R378.11[医药卫生—病原生物学] R378.23[医药卫生—基础医学]