幽门螺旋杆菌尿素酶多表位疫苗CTB-UE的生物信息学分析及其表达  

Bioinformatics and expression of Helicobacter pylori urease multi-epitope vaccine CTB-UE

作　　者：刘昆梅[1] 秦玉红[2] 奚涛[3] 郭乐[1,2] 

机构地区：[1]宁夏医科大学颅脑疾病重点实验室,宁夏银川750004 [2]宁夏医科大学临床医学院临床病原微生物重点实验室,宁夏银川750021 [3]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《中国生物制品学杂志》2015年第12期1238-1244,共7页Chinese Journal of Biologicals

基　　金：宁夏自然科学基金(NZ13274)

摘　　要：目的通过生物信息学软件评价幽门螺旋杆菌(Helicobacter pylori,Hp)尿素酶多表位疫苗CTB-UE的抗原结构,并在E.coli中表达、纯化融合蛋白CTB-UE。方法应用生物信息学软件DNAstar、Geno3D、MOE分析Hp尿素酶多表位疫苗CTB-UE的二级结构和三级结构;将人工合成的尿素酶多表位肽基因UE插入含CTB基因的重组质粒p ETC中,构建重组表达质粒p ETCUE,转化E.coli BL21(DE3),经IPTG诱导表达后,通过Ni-NTP镍离子亲和层析纯化重组蛋白CTB-UE,并进行Western blot分析。结果生物信息学软件分析显示,Hp尿素酶多表位疫苗CTB-UE具有合理的结构;重组表达质粒p ETCUE经双酶切和测序鉴定证实,融合基因CTB-UE与设计序列完全一致;表达的融合蛋白CTB-UE相对分子质量约为33 000,主要以包涵体形式表达,表达量占全菌蛋白的22.3%;纯化的融合蛋白CTB-UE纯度达95.6%,可与兔抗Hp多克隆抗体发生特异性结合。结论设计的Hp尿素酶多表位疫苗CTB-UE具有合理的结构,能在E.coli中高效表达,纯化后纯度较高,且具有较强的反应原性,为进一步研究针对尿素酶A、B双亚基的Hp多表位疫苗奠定了基础。Objective To evaluate the antigenic structure of urease multi-epitope vaccine CTB-UE against Helicobacter pylori（Hp） by bioinformatic software, express CTB-UE fusion protein in E. coli and purify the expressed product.Methods The secondary and tertiary structures of CTB-UE were analyzed by bioinformatic software DNAstar, Geno3 D and MOE. The synthetic urease multi-epitope gene UE was inserted into plasmid p ETC containing CTB gene, and the constructed recombinant plasmid p ETCUE was transformed to E. coli BL21（DE3）and induced with IPTG. The expressed recombinant fusion protein CTB-UE was purified by Ni-NTP nickel ion affinity chromatography, and analyzed by Western blot. Results Bioinformatic analysis showed a scientific and reasonable structure of multi-epitope vaccine CTB-UE.Restriction analysis and sequencing proved that recombinant plasmid p ETCUE was constructed correctly, in which the nucleotide sequence of CTB-UE gene was consistent with that designed. The expressed CTB-UE fusion protein, with a relative molecular mass of about 33 000, mainly existed in a form of inclusion body and contained 22. 3% of total somatic protein. The purified CTB-UE fusion protein reached a purity of 95. 6% and showed specific binding to rabbit polyclonal antibody against Hp. Conclusion The designed multi-epitope vaccine CTB-UE showed a scientific and reasonable structure, and was highly expressed in E. coli. The purified CTB-UE reached a high purity and showed strong reactogenicity, which laid a foundation of further study on multi-epitope vaccine targeting subunits A and B of Hp urease.

关 键 词：幽门螺旋杆菌 尿素酶 融合蛋白 多表位疫苗 生物信息学 表达 纯化 

分 类 号：R378.99[医药卫生—病原生物学] Q-332[医药卫生—基础医学]