rCtUBE的生物信息学分析及其表达优化(英文)  

Bioinformatics Analysis and Optimized Expression of the Epitope Vaccine rCtUBE

作　　者：郭乐[1,2] 刘昆梅[1,2] 秦玉红[1] 李小康[2] 段相国[1] 杨华[1] 徐广贤[1] 奚涛[2] 

机构地区：[1]宁夏医科大学检验学院,银川750021 [2]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国生物工程杂志》2013年第10期44-50,共7页China Biotechnology

基　　金：supported by a Grant from National Major Special Program of New Drug Research and Development(No.2012ZX09103-301-008)~~

摘　　要：目的:幽门螺旋杆菌(Hp)是慢性胃炎、消化性溃疡和胃癌的重要致病因子。研发基于Hp尿素酶的表位疫苗是一种很有前景的防治Hp感染的策略。方法:通过生物信息学软件对位于Hp尿素酶酶活性中心的表位肽F8和霍乱毒素B亚基(CTB)的连接顺序和间隔序列加以分析,设计出一种Hp表位疫苗rCtUBE。通过基因克隆技术构建含有融合基因rCtUBE的重组表达载体pETCUB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化融合蛋白rCtUBE,并进一步通过腹腔注射免疫BALB/c小鼠,鉴定Hp表位疫苗rCtUBE的免疫学活性。结果:经生物信息学软件分析,Hp表位疫苗rCtUBE具有科学合理的结构,并成功构建了重组表达载体pETCUB及其重组菌株BL21(DE3)/pETCUB,经蛋白表达优化及纯化,可获得高纯度(95.3%)的融合蛋白rCtUBE。融合蛋白rCtUBE以氢氧化铝为佐剂注射免疫BALB/c小鼠,经间接ELISA鉴定:小鼠能够产生针对CTB和表位肽F8的高滴度特异性抗体。结论:Hp表位疫苗rCtUBE具有科学合理的结构,能在大肠杆菌表达系统中获得较高水平的表达,且具有较高的免疫学特异性,因此为研发防治Hp感染的表位疫苗奠定了一定的实验基础。Helicobacter pylori （Hp） represents a major cause of gastroduodenal pathologies, such as chronic gastritis, peptic ulcer and gastric cancer. The aim is to design an epitope vaccine named rCtUBE with Cholera toxin B subunit （CTB） and an B cell epitope from Hp urease B subunit （UreB） by using the bioinformatics software and database, and get fusion protein rCtUBE with high purity after optimized expression and purification. Methods：An epitope vaccine rCtUBE composed of CTB and an epitope peptide named F8 involved in the active site of the urease was designed by analyzing the coupling sequence and linker between CTB and F8 through bioinformatics software. The recombinant expression vector pETCUB containing the fusion gene rCtUBE and the recombinant strain BL21（DE3）/pETCUB were constructed by gene cloning technology. After protein expression and optimization, the fusion protein rCtUBE was purified by Ni2＋-charged column chromatography and anion-exchange chromatography using DEAE Sepharose FF. Then, the activity of epitope vaccine rCtUBE was investigated by intraperitoneal immunization experiments in BALB/c mice. Results：The epitope vaccine rCtUBE had a scientific and reasonable structure through the analysis of bioinformatics software. The recombinant expression vector pETCUB and recombinant strain BL21（DE3）/pETCUB were constructed successfully. After protein expression optimization and purification, about 56 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of rCtUBE was 95.3%. Mice immunized with rCtUBE using aluminum hydroxide adjuvant could induce high level of antibodies specific for both CTB and F8 by ELISA. Conclusion：The epitope vaccine rCtUBE with a scientific and reasonable structure was expressed at a medium level in E. coli and had good immunological specificity. This will provide much experimental evidences for the development of epitope vaccines against Hp for human use.

关 键 词：幽门螺旋杆菌 尿素酶 表位疫苗 

分 类 号：Q819[生物学—生物工程]