幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究(英文)  

Purification and Immunologic Study of the Recombinant Urease B subunit from Helicobacter pylori

作　　者：秦玉红[1] 刘昆梅[2] 廖国玲[1] 杨华[1] 徐广贤[1] 李秀萍[1,3,4] 郭乐[1] 

机构地区：[1]宁夏医科大学检验学院宁夏临床病原微生物重点实验室,银川750021 [2]宁夏医科大学颅脑疾病重点实验室,银川750021 [3]宁夏医科大学 [4]宁夏医科大学总医院,银川750021

出　　处：《中国生物工程杂志》2014年第12期23-29,共7页China Biotechnology

基　　金：supported by a Grant from Ningnia High School Scientific Research Program(NGY2013065)~~

摘　　要：目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(Ure B),研发基于Ure B的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rU reB)基因的重组表达载体p ET-rU re B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重组尿素酶B亚基(rU re B),并进一步通过腹腔注射免疫BALB/c小鼠,研究rU re B的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体p ET-rU re B及其重组菌株BL21(DE3)/p ET-rU re B,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rU re B。重组蛋白rU re B辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和Ure B的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。Objective： Helicobacter pylori（ Hp） urease is an important colonization factor as well as a pathogenic factor,and the enzymatic activity sites of Hp urease locate in the Hp urease B subunit（ Ure B）.Research and development of Hp vaccine based on urease is a promising strategy for the prevention of Hp infection. The aims are to obtain recombinant Hp urease with high purity, and study its immunological properties. Methods： The Ure B gene was obtained from Helicobacter pylori standard strain SS1（ Hp SS1）,and the expression vector p ET-rU re B and the recombinant strain BL21（ DE3） / p ET-rU re B were also constructed by gene cloning technology. After protein expression and optimization,the recombinant protein rU re B was purified by Ni2 ＋-charged column chromatography and anion-exchange chromatography using DEAE Sepharose FF. Then,the immunological properties of rU re B protein was investigated by intraperitoneal immunization experiments in BALB /c mice with Freund＇s adjuvant. Results： Hp Ure B gene was obtained successfully from Hp SS1 through PCR,and the expression vector p ET-rU re B and recombinant strain BL21（ DE3） / p ET-rU re B were constructed successfully by gene cloning technology. After protein expression optimization and purification,about 69 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of rU re B was 96. 5%. Mice immunized with rU re B using Freund＇s adjuvant could induce high level of antibodies specific for both natural Hp urease and Ure B by ELISA, which can significantly inhibit the activity of Hp urease. Conclusion： The recombinant Ure B was expressed at a medium level in E. coli and had good immunological specificity. Besides,the antibodies induced by rU re B can effectively inhibit the activity of Hp urease. This will provide an experimental basis for the development of Hp vaccine based on urease.

关 键 词：幽门螺旋杆菌 尿素酶 尿素酶B亚基 

分 类 号：Q819[生物学—生物工程]