Bt Cry1类毒素共性结构域的分析、表达及鉴定

【目的】分析定位Bt Cry1类毒素Cry1Ab、Cry1Ac、Cry1B、Cry1C、Cry1F的共性结构域,克隆并表达共性结构域蛋白,为筛选Bt毒素广谱抗体及建立广谱检测方法打下基础。【方法】利用生物信息学和分子模拟技术,通过SWISS-MODEL同源建模分别对5种Cry1类毒素进行三维建模,并结合Ramachandran plot、ERRAT和Verify3D方法评价模型构象的合理性。通过分析比对5种Cry1类毒素的三维结构,确定DomainⅠ区域作为5种Cry1毒素的共性结构域。以含Cry1Ac基因的苏云金芽孢杆菌库斯塔克亚种为模板设计引物,PCR扩增获得共性结构域DomainⅠ基因,将其经NcoⅠ和NotⅠ双酶切连接至原核表达载体pET-26b(+),构建原核表达载体pET-26b-DomainⅠ。重组质粒经菌液PCR、双酶切以及测序鉴定验证正确后,转化至E.coli BL21(DE3),经终浓度为1 mmol·L~(-1)的IPTG在20℃下诱导表达16h后检测共性结构域蛋白的表达情况。离心收集诱导表达的大肠杆菌菌液,进行超声波破碎处理,收集上清及沉淀,采用SDS-PAGE分析融合蛋白的表达。利用His-Trap HP镍亲和柱纯化上清中的可溶性融合蛋白,经SDS-PAGE电泳、Western blot和ELISA试验验证纯化的共性结构域蛋白的生物活性。【结果】基于氨基酸序列及三维空间比对分析,发现5种Cry1类毒素的DomainⅠ的序列一致性最高,而且它们的DomainⅠ三维结构几乎完全重合,确定DomainⅠ区域作为5种Cry1毒素的共性结构域,通过PCR、双酶切及测序鉴定成功构建原核表达载体pET-26b-DomainⅠ,经IPTG诱导表达、His-Trap HP镍亲和柱纯化获得了可溶性的DomainⅠ共性结构域蛋白,SDS-PAGE和Western blot证实表达的共性结构域蛋白的分子量约为33.4 kD,且能与抗His标签鼠单克隆抗体发生特异性反应,ELISA试验证实共性结构域蛋白与5种Cry1类毒素特异性抗体均具有很强的结合能力,抗原表位分析结果显示共性结构域蛋白具有和完整的Cry蛋白存在多个潜在抗原表位位点的特征,抗原表位区域所占的比例分别为48.4%和63.6%,表明共性结构域蛋白具有良好的免疫原性和免疫反应性。【结论】基于分子模拟与分子克隆技术,成功定位及表达纯化获得共性结构域蛋白,为下一步利用共性结构域为靶标分子制备广谱特异性识别Cry1类毒素抗体打下基础。

Analysis,Expression and Identification of the Common Structural Domain of Bacillus thuringiensis (Bt) Cry1 Toxins

【Objective】The objective of this study is to express the optimal common structural domain through analyzing and locating the common structure of five Bacillus thuringiensis Cry1 toxins. This research will lay a foundation for producing the generic antibody and developing the detection method for Cry1 toxins. 【Method】Through bioinformatics, molecular simulation technique and homology modeling to build the three-dimensional structure models of five Cry1 toxins. The structures were evaluated using three programmes, Ramchandran plot, Verify3D and ERRAT. Domain I was identified as the common structure domain of five Cry1 toxins finally. In order to construct the pET-26b-Domain I vector, primers were designed according to the Cry1Ac gene of Bacillus thuringiensis ssp. kurstaki. As well as insert with Nco I and Not I digestion sites. When it was identified by PCR, restriction enzyme digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) which was induced with 1 mmol·L-1 IPTG, 20℃ for 16 h. The supernatant and precipitate were collected and verified by SDS-PAGE after E. coli BL21 (DE3) cells were collected and crushed by ultrasonic wave. The soluble fusion protein was purified by His-Trap HP nickel affinity column and verified by SDS-PAGE, Western blot and ELISA. 【Result】 According to the analysis of amino acid sequences and three- dimensional structures of the five Cry1 toxins, the sequences of Domain I were the highest identity part and its three-dimensional structure was very similar and then the Domain I was chosen as the common structure domain of the five Cry1 toxins. The expression vector pET-26b-Domain I was constructed successfully, and soluble Domain I protein was expressed and purified. The molecular weight of the fusion protein was confirmed to be 33.4 kD by SDS-PAGE and Western blot, which also showed specific activity to anti-6×His monoclonal antibody. The ELISA assay showed that the Domain I protein had a good sensitivity with the specific antibodies of the five Cry1 toxins, and the epitope prediction results showed that both the Domain I protein and the complete Cry protein existed multiple potential epitopes, and the percentage of their antigenic peptides were 48.4% and 63.6%, respectively. These results indicate that the Domain I protein has good immunogenicity and immune response. 【Conclusion】Based on molecular simulation and molecular cloning technology, the conserved structural domain protein was successfully expressed and purified. This study has established a foundation for the following studies and the common structural domain protein would as target molecule for the production of the generic antibody of Cry1 toxins in further research.