Bt Cry1类毒素共性结构域的分析、表达及鉴定

doi:10.3864/j.issn.0578-1752.2016.16.007

中国农业科学 ›› 2016, Vol. 49 ›› Issue (16): 3130-3139.doi: 10.3864/j.issn.0578-1752.2016.16.007

Bt Cry1类毒素共性结构域的分析、表达及鉴定

刘贝贝^1,2，张霄²，谢雅晶²，焦凌霞³，刘媛^1,2，张存政²，赵岩岩²，武爱华²，刘贤金^1,2

¹南京农业大学植物保护学院，南京 210095
²江苏省农业科学院食品质量安全与检测研究所/江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地/农业部农产品量安全控制技术与标准重点实验室，南京 210014
³河南科技学院食品学院，河南新乡 453003

收稿日期:2016-03-18 出版日期:2016-08-16 发布日期:2016-08-16
通讯作者: 刘贤金，E-mail：jaasliu@jaas.ac.cn
作者简介:刘贝贝，E-mail：2013102120@njau.edu.cn
基金资助:
国家自然科学基金（31371778、31301703）、国家公益性行业（农业）科研专项（201303088）、江苏省自主创新项目（CX（14）5068）刘贤金，E-mail：jaasliu@jaas.ac.cn

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

LIU Bei-bei^1,2, ZHANG Xiao², XIE Ya-jing², JIAO Ling-xia³, LIU Yuan^1,2, ZHANG Cun-zheng², ZHAO Yan-yan², WU Ai-hua², LIU Xian-jin^1,2

¹College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
²Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Science/Key Laboratory of Food Quality and Jiangsu Province-State Key Laboratory Breeding Base/Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Nanjing 210014
³School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, Henan

Received:2016-03-18 Online:2016-08-16 Published:2016-08-16

摘要/Abstract

摘要： 【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Domain I 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Domain I基因，将其经Nco I和Not I双酶切连接至原核表达载体pET-26b(+)，构建原核表达载体pET-26b-Domain I。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16 h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Domain I 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Domain I 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-Domain I，经IPTG诱导表达、His-Trap HP镍亲和柱纯化获得了可溶性的Domain I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。

关键词: Bt Cry1类毒素, 共性结构域, 原核表达, 纯化

Abstract: 【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.

Key words: Bacillus thuringiensis Cry1 toxins, common structural domain, prokaryotic expression, purification

刘贝贝，张霄，谢雅晶，焦凌霞，刘媛，张存政，赵岩岩，武爱华，刘贤金. Bt Cry1类毒素共性结构域的分析、表达及鉴定[J]. 中国农业科学, 2016, 49(16): 3130-3139.

LIU Bei-bei, ZHANG Xiao, XIE Ya-jing, JIAO Ling-xia, LIU Yuan, ZHANG Cun-zheng, ZHAO Yan-yan, WU Ai-hua, LIU Xian-jin. Analysis, Expression and Identification of the Common Structural Domain of Bacillus thuringiensis (Bt) Cry1 Toxins[J]. Scientia Agricultura Sinica, 2016, 49(16): 3130-3139.

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Bt Cry1类毒素共性结构域的分析、表达及鉴定

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

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