摘要
目的 对幽门螺杆菌（helicobacter pylori,Hp）的过氧化氢酶（catalase,KatA）进行重组表达和纯化,并进行免疫学特性研究.方法 采用PCR扩增幽门螺杆菌katA全长基因,构建重组表达载体pET-28a-katA,在大肠埃希菌BL21（DE3）中表达,通过包涵体洗涤和亲和层析纯化出重组蛋白,并采用Western blot和ELISA等方法对其进行免疫学特性研究.结果 克隆出幽门螺杆菌1 518 bp的全长katA基因,在大肠埃希菌中诱导表达出505个氨基酸组成、分子量约为58 000的重组KatA蛋白,其表达量约占细菌总蛋白的20%,且以包涵体形式表达.经亲和层析的重组蛋白纯度约为85%,并可与兔抗Hp抗血清发生特异性反应.通过重组蛋白的动物免疫可产生高效价抗血清,并特异性识别多种Hp菌株.结论 成功重组表达与纯化出幽门螺杆菌KatA蛋白,并具备良好的免疫学特性,为Hp致病机制、血清学诊断以及亚单位疫苗研究奠定重要的实验基础.
Objective To prepare recombinant catalase protein （rKatA） of Helicobacter pylori （H. pylori） expressed in E. coli,and investigate its immunological characteristics. Methods H. pylori catalase gene was amplified by PCR and constructed into pET-28a vector, rKatA was expressed in E coli BL21 （DE3） and purified by inclusion body washing and affinity chromatography （chelating sepharose fast flow）. The immunological characteristics of rKatA was investigate by Western blot and ELISA. Results The catalase gene of H. pylori cloned by PCR contained 1 518 base pairs molecule encoding 505 amino acids, rKatA was expressed as inclusion body in E. coli BL2I with about 20% expression rate and 58 000 molecular weight. The purity of rKatA was up to 85 % after affinity chromatography. Recombinant protein was specifically identified by rabbit antiserum against H. pylori, and high titer anti-rKatA antibody came from immunized rabbit with rKatA could recognized various H. pylori strains. Conclusion rKatA was successfully prepared and maintained good immunological characteristics,which would be useful for further research on pathogenic mechanism, serodiagnosis and subunit vaccine of H. pylori.
出处
《现代检验医学杂志》
CAS
2013年第1期7-11,共5页
Journal of Modern Laboratory Medicine
关键词
幽门螺杆菌
过氧化氢酶
重组表达
免疫学特性
helicobacter pylori catalase recombinant expression immunological characteristics
