炭疽芽胞杆菌BslA_((260-652))蛋白的表达纯化与黏附功能鉴定  

Recombinant expression,purification and adhesion function identify of Bacillus anthracis BslA_((260-652)) protein

作　　者：马坤[1,2] 王艳春[2] 陶好霞[2] 董杰[2] 曹诚[2] 张部昌[1] 刘纯杰[2] 

机构地区：[1]安徽大学生命科学学院,合肥230039 [2]军事医学科学院生物工程研究所,病原微生物与生物安全国家重点实验室,北京100071

出　　处：《微生物学报》2012年第3期360-366,共7页Acta Microbiologica Sinica

基　　金：国家科技支撑计划项目(2008BAI66B03);国家艾滋病和病毒性肝炎等重大传染病防治重大专项(2008ZX10004-015)~~

摘　　要：【目的】克隆表达炭疽芽胞杆菌BlsA的功能区片段并对其生物学功能进行鉴定。【方法】以炭疽芽胞杆菌A16R基因组DNA为模板PCR扩增bslA(260-652)基因片段,克隆至pET-28a(+)载体。将成功构建的重组质粒转化入大肠杆菌Rosetta(DE3)中,诱导表达后收集菌体经超声破碎后,对可溶表达部分用镍柱进行亲和层析纯化。以纯化后的蛋白为抗原,免疫BALB/c小鼠制备该蛋白的多抗,用ELISA和Western blot检测抗血清;使用间接免疫荧光实验和细菌黏附实验研究目标蛋白及其抗体的生物学功能。【结果】BslA(260-652)获得了可溶性表达,纯化后纯度约为87.4%。以纯化蛋白为抗原,免疫BALB/c小鼠制备的抗血清ELISA效价可达1∶20000。将BslA(260-652)蛋白与Hela细胞共孵育后,能够直接和Hela的细胞膜结合。细菌黏附实验表明BslA(260-652)蛋白及其相应的多抗血清都能够显著地抑制炭疽芽胞杆菌A16R对Hela细胞的黏附。【结论】大肠杆菌表达得到的炭疽芽胞杆菌BslA(260-652)蛋白具有与天然蛋白相似的生物活性,为深入研究BslA蛋白在炭疽芽胞杆菌致病过程中的作用奠定实验基础。[Objective]To obtain the recombinant BslA（260-652） protein of Bacillus anthracis and prepare its antibody for the adhesion activity studies.[Methods]The fragment coding BslA（260-652） was cloned into pET28a（＋） plasmid and induced to express recombinant protein in E.coli Rosetta（DE3） by Isopropyl β-D-1-thiogalactopyranoside（IPTG）.The expressed recombinant soluble protein was purified by a column packed with Ni Resin.Purified protein was used as the antigen to immunize BABL/c mice for three times to raise polyclonal antibody.The adhesion activity of BlsA（260-652） was detected by immunofluorescence experiments and bacterial adherence assays.[Results]The purity of the purified soluble BslA（260-652） was about 87.4%.ELISA assay titer of antiserum from vaccinated mice reached 1：20000.Western blot showed the antiserum could specifically recognize endogenous BslA protein.The purified BslA（260-652） displayed a typical adhesion-like function.Either the anti-BslA serum or the BslA（260-652） protein could inhibit A16R＇s Hela adherence.[Conclusion]The recombinant BslA（260-652） protein was successfully obtained,which would lay the foundation for further research of the anthrax vaccine and the role of this S-layer protein in the pathogenesis of anthrax.

关 键 词：炭疽芽胞杆菌 BslA蛋白 多克隆抗体 免疫荧光 黏附 

分 类 号：R378[医药卫生—病原生物学]