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

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.