重组幽门螺杆菌多表位疫苗工程菌株的构建及其微生物学特性研究  

The New Bacteria Expressing Recombinant Multi-epitope Vaccine against Helicobacter pylori and Its Microbiological Characteristics

作　　者：王保宁[1] 潘兴[1,2] 黄筱钧[1] 周永君 祝捷 高礼珍 牛晓娟 李婉宜[1] 李明远[1] 王红仁[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,成都610041 [2]四川万可泰生物技术有限责任公司,成都610041

出　　处：《四川大学学报（医学版）》2015年第3期354-358,共5页Journal of Sichuan University(Medical Sciences)

基　　金：四川省科技厅科技支撑计划项目(No.2014SZ0036)资助

摘　　要：目的构建含有幽门螺杆菌尿素酶(ureI、ureB)及霍乱毒素B亚单位(ctB)融合片段的多表位疫苗工程菌,并研究其微生物学特性。方法通过生物信息学方法从ureI和ureB基因中筛选出T细胞和B细胞优势表位,通过柔性Linker相连,并在其N端加入分子内佐剂序列ctB,按照大肠杆菌BL21(DE3)的密码子偏好性进行密码子优化,即为BIB序列。人工合成之后,插入原核表达质粒pET28a(+)中,构建pET28a(+)/ctB-ureI-ureB〔pET28a(+)/BIB〕重组质粒。经限制性内切酶酶切鉴定及DNA测序鉴定正确后,将质粒转化入大肠杆菌BL21(DE3)中。BIB工程菌经乳糖诱导表达后,SDS-PAGE检测重组蛋白BIB的表达情况,并对其N端氨基酸序列和相对分子质量进行测定,Western blot对其进行抗原性鉴定。结果原核表达质粒pET28a(+)/BIB经双酶切和测序鉴定构建正确,SDS-PAGE电泳显示在相对分子质量33×103处有一条明显条带,其N端氨基酸序列和相对分子质量与设计序列100%一致,Western blot结果显示BIB可以与抗幽门螺杆菌悉尼株(SS1株)小鼠血清及鼠抗CTB单抗产生特异性反应。结论成功构建了幽门螺杆菌多表位重组原核表达工程菌,该工程菌表达的BIB蛋白抗原性良好。Objective To construct the engineering bacteria with recombinant plasmid expressing the multiepitope vaccine which composed of Helicobacter pylori urea membrane channel protein（UreI）,Helicobacter pylori urease B subunit（UreB）and cholera toxin B subunit（CTB）,and then to study it＇s microbiological characteristics.Methods The sequence contains some dominant epitopes of Helicobacter pylori UreI and UreB was designed,and ctB was added at the N-terminal,all the sequence were linked by flexible linkers.Codon optimization was done according to Escherichia coli（E.coli）BL21（DE3）bias,the optimized sequence was designated BIB.BIB sequence was synthesized and cloned into plasmid pET28a（＋）.The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing.The recombinant protein BIB was expressed in E.coli BL21（DE3）and analyzed by Western blot.Results The plasmid of pET28a（＋）/BIB was constructed successfully,confirmed by restriction enzyme digestion and DNA sequencing.The recombinant protein BIB with relative molecular mass about33×103 could be produced by E.coli BL21（DE3）and was detected by Western blot.The relative molecular mass and N-terminal amino acid sequence of BIB were 100% identity with the design.Conclution The engineering bacteria with recombinant plasmid expressing the multi-epitope vaccine against Helicobacter pylori was constructed successfully.The recombinant protein BIB can be identified by anti-Sydney strain 1of Helicobacter pylori（H.pylori SS1）polyclonal antibody and anti-CTB monoclonal antibody,which demonstrated that BIB has the expected antigenicity.

关 键 词：幽门螺杆菌 多表位疫苗 尿素酶B亚单位 尿素酶I亚单位 霍乱毒素B亚单位 

分 类 号：R392[医药卫生—免疫学]