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

Abstract

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

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.

References

[1]    Hammond B G, Koch M S. A review of the food safety of Btcrops//Sansinenea E. Bacillus thuringiensis Biotechnology, Springer, 2012: 305-325.
[2]    Kaur S. Risk assessment of Bt transgenic crops//Sansinenea E. Bacillus thuringiensis Biotechnology, Springer, 2012: 41-85.

[3]    Yu H L, Li Y H, Wu K M. Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms. Journal of Integrative Plant Biology, 2011, 53(7): 520-538.

[4]    连丽君, 王雷, 张可炜. 转基因食品安全性的争论与事实. 食品与药品, 2006, 8(11): 12-15.

Lian L J, Wang l, Zhang K W. Debate and fact on safety of genetically modified foods. Food and Drug, 2006, 8(11): 12-15. (in Chinese)

[5]    Guertler P, Paul V, Albrecht C, Meyer H D. Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk. Analytical and Bioanalytical Chemistry, 2009, 393(6/7): 1629-1638.

[6]    Saxena D, Flores S, Stotzky G. Transgenic plants: Insecticidal toxin in root exudates from Bt corn. Nature, 1999, 402(6761): 480-481.

[7]    王广印, 范文秀, 陈碧华, 张建伟, 韩世栋. 转基因食品检测技术的应用与发展 I. 主要检测技术及其特点. 食品科学, 2008, 29(10): 698-705.

Wang G Y, Fan W X, Chen B H, Zhang J W, Han S D. Application and development of detection technology of genetically modified foods (GMFs)Ⅰ. main detection technologies of GMFs and their characteristics. Food Science, 2008, 29(10): 698-705. (in Chinese)

[8]    Spinks C A. Broad-specificity immunoassay of low molecular weight food contaminants: new paths to Utopia! Trends in Food Science & Technology, 2000, 11(6): 210-217.

[9]    刘媛, 余向阳, 梁颖, 祝金凤, 李顺玲, 张存政, 刘贤进. 农药广谱特异性抗体制备技术研究进展. 江苏农业学报, 2009, 25(2): 428-432.

Liu Y, Yu X Y, Liang Y, Zhu J F, Li S L, Zhang C Z, Liu X J. Recent advances in development of broad specificity antibodies for pesticides. Jiangsu Journal of Agricultural Sciences, 2009, 25(2): 428-432. (in Chinese)

[10]   Li Y L, Zhao F C, Zhao L Y, Yang Z Y. Development of a broad-specificity immunoassay for determination of organophosphorus pesticides using dual-generic hapten antigens. Food Analytical Methods, 2015, 8(2): 420-427.

[11]   Pardo-Lopez L, Soberon M, Bravo A. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiology Review, 2013, 37(1): 3-22.

[12]   赵新民, 夏立秋, 王发祥, 丁学知, 单世平, 张友明. 苏云金芽孢杆菌毒素Cry1Aa, Cry2Aa, Cry3Aa和Cry4Aa结构的计算机对比分析. 化学学报, 2008, 66(1): 108-111.

Zhao X M, Xia L Q, Wang F X, Ding X Z, Shan S P, Zhang Y M. Comparison and analysis of Cry1Aa, Cry2Aa, Cry3Aa and Cry4Aa of Bacillus thuringiensis toxins with computer. Acta Chimica Sinica, 2008, 66(1): 108-111. (in Chinese)

[13]   吴洪福, 郭淑元, 李海涛, 高继国. 苏云金芽孢杆菌杀虫晶体蛋白结构和功能研究进展. 东北农业大学学报, 2009, 40(2): 118-122.

Wu H F, Guo S Y, Li H T, Gao J G. Progress on structure and function of insecticidal crystal proteins from Bacillus thuringiensis. Journal of Northeast Agricultural University, 2009, 40(2): 118-122. (in Chinese)

[14]   谢小波, 舒庆尧. 用Envirologix Cry1Ab/Cry1Ac试剂盒快速测定转基因水稻Bt杀虫蛋白含量的研究. 中国农业科学, 2001, 34(5): 465-468.

Xie X B, Shu Q y. Studies on rapid quantitative analysis of Bt toxin by using envirologix kits in transgenic rice. Scientia Agricultura Sinica, 2001, 34(5): 465-468. (in Chinese)

[15]   Eteshola E. Isolation of scFv fragments specific for monokine induced by interferon-gamma (MIG) using phage display. Journal of Immunology Methods, 2010, 358(1/2): 104-110.

[16]   Roda A, Mirasoli M, Guardigli M, Michelini E, Simoni P, Magliulo M. Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize. Analytical and Bioanalytical Chemistry, 2006, 384(6): 1269-1275.

[17]   Zhang X, Liu Y, Zhang C Z, Wang Y, Xu C X, Liu X J. Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin. Ecotoxicology and Environmental Safety, 2012, 81: 84-90.

[18]   Grochulski P, Masson L, Borisova S, Pusztai-Carey M, Schwartz J L, Brousseau R, Cygler M. Bacillus thuringiensis CrylA (a) insecticidal toxin: crystal structure and channel formation. Journal of Molecular Biology, 1995, 254(3): 447-464.

[19]   刘卓明, 谢柳, 叶大维. 苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白及其分子设计. 基因组学与应用生物学, 2009, 28(2): 356-364.

Liu Z M, Xie L, Ye D W. Bt Cry1Ac insecticidal crystal protein family and its molecular design. Genomics and Applied Biology, 2009, 28(2): 356-364. (in Chinese)

[20]   Zhao X M, Zhou P D, Xia L Q. Homology modeling of mosquitocidal Cry30Ca2 of Bacillus thuringiensis and its molecular docking with N-acetylgalactosamine. Biomed Environmental Science, 2012, 25(5): 590-596.

[21]   Schwede T, Kopp P, Guex N, Peitsch M C. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Research, 2003, 31(13): 3381-3385.

[22]   Laskowski R A, MacArthur M W, Moss D S, ThorntonJ M. PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 1993, 26(2): 283-291.

[23]   Colovos C, Yeates T O. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Science, 1993, 2(9): 1511-1519.

[24]   Eisenberg D, Lothy R, Bowie J U. VERIFY3D: Assessment of protein models with three dimensional profiles. Methods in enzymology, 1997, 277: 396-404.

[25]   Guo S Y, Li J, Chen Z, He K L. Penetration of a single domain of Bacillus thuringiensis Cry1Ie-domain I to a lipid membrane in vitro. Journal of Integrative Agriculture, 2014, 13(5): 1043-1050.

[26]   George R A, Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Engineering Design and Selection, 2003, 15(11): 871-879.

[27]   闫璐颖, 陈建华, 张新国. 融合蛋白连接肽的研究进展. 生物技术, 2008, 18(3): 92-94.

Yan L Y, Chen J H, Zhang X G. Research progress in the linker of fusion protein. Biotechnology, 2008, 18(3): 92-94. (in Chinese)

[28]   Barlow D J, Edwards M S, Thornton J M. Continuous and discontinuous protein antigenic determinants. Nature, 1986, 322(6081): 747-748.

[29]   Jameson B A, Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Bioinformatics, 1988, 4(1): 181-186.

Related Articles 15

[1]	LIU ChuanXia, CHEN Xin, WANG Xiao, LI XueWen, LI TingTing, WENG ChangJiang, ZHENG Jun. Preparation and Application of Polyclonal Antibodies Against Pig CD1d Protein [J]. Scientia Agricultura Sinica, 2024, 57(8): 1620-1628.
[2]	WANG ZhiXiong, XU Dong, TIAN XiaoLi, WAN Peng, XIA Gen, SONG XuRong, WANG FuLian, GUI LianYou, ZHANG GuoHui. Cloning, Prokaryotic Expression and Ligand Binding Property of BminMinusOBP1 and BminPlusOBP1 from Bactrocera minax [J]. Scientia Agricultura Sinica, 2024, 57(24): 4894-4906.
[3]	QIAN YanHong, SONG Shuai, WEN XiaoHui, NIU RuiHui, YANG YanQiu, ZHENG BoBin, YUAN ZiGuo, LUO ShengJun. Establishment and Application of a Tube-Based Chemiluminescence Immunoassay Method for Detecting Antibodies Against Trichinella spiralis in Pigs [J]. Scientia Agricultura Sinica, 2024, 57(22): 4578-4588.
[4]	BIAN XianYu, LI SuFen, WANG JianXin, HAN Nan, LU HongTing, CHENG Xi, ZHOU JinZhu, TAO Ran, ZHU XueJiao, DONG HaiLong, ZHANG XueHan, LI Bin. Prokaryotic Expression, Antibody Preparation and Application of Major Non-Structural Proteins of Porcine Rotavirus [J]. Scientia Agricultura Sinica, 2024, 57(17): 3494-3506.
[5]	YANG Ling, TIAN XiaoLi, GUI LianYou, WANG FuLian, ZHANG GuoHui. Interaction Mechanisms Between Bactrocera minax Odorant-Binding Protein BminOBP6 and Its Ligands [J]. Scientia Agricultura Sinica, 2023, 56(7): 1311-1321.
[6]	YANG HuiZhen, YANG Huan, WU ZiXuan, FAN KuoHai, YIN Wei, SUN PanPan, ZHONG Jia, SUN Na, LI HongQuan. Prokaryotic Expression and Metal Binding Characterization of Metallothionein 1A and 2A of Sus scrofa [J]. Scientia Agricultura Sinica, 2023, 56(17): 3461-3478.
[7]	Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG. Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids [J]. Scientia Agricultura Sinica, 2021, 54(6): 1143-1153.
[8]	XiaoHe LIU,GuiSheng QIU,ZhaoGuo TONG,HuaiJiang ZHANG,WenTao YAN,Qiang YUE,LiNa SUN. Ligands Binding Characteristics of Chemosensory Protein CsasCSP16 of Carposina sasakii [J]. Scientia Agricultura Sinica, 2021, 54(5): 945-958.
[9]	QIN JianHui,LI JinQiao,ZHAO Xu,LI KeBin,CAO YaZhong,YIN Jiao. Expression, Purification and Functional Analysis of Odorant Binding Protein 11 (OBP11) in Anomala corpulenta [J]. Scientia Agricultura Sinica, 2021, 54(14): 3017-3028.
[10]	XIE KunLun,LIU LiMing,LIU Mei,PENG Bin,WU HuiJie,GU QinSheng. Prokaryotic Expression of dsRNA of Zucchini yellow mosaic virus and Its Control Efficacy on ZYMV [J]. Scientia Agricultura Sinica, 2020, 53(8): 1583-1593.
[11]	BI KeRan,LI Yin,HAN KaiKai,ZHAO DongMin,LIU QingTao,LIU YuZhuo,HUANG XinMei,YANG Jing. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein [J]. Scientia Agricultura Sinica, 2019, 52(23): 4429-4436.
[12]	Ling LI,Yao TAN,XiaoRong ZHOU,BaoPing PANG. Molecular Cloning, Prokaryotic Expression and Binding Characterization of Odorant Binding Protein GdauOBP20 in Galeruca daurica [J]. Scientia Agricultura Sinica, 2019, 52(20): 3705-3712.
[13]	LI Du, NIU ChangYing, LI FengQi, LUO Chen. Binding Characterization of Odorant Binding Protein OBP56h in Drosophila suzukii with Small Molecular Compounds [J]. Scientia Agricultura Sinica, 2019, 52(15): 2616-2623.
[14]	WANG Hui,CHAI ZhiXin,ZHU JiangJiang,ZHONG JinCheng,ZHANG ChengFu,Xin JinWei. Cloning and Identification of Long-Chain Non-Coding RNA Linc24063 and Its Correlation with the Expression Level of miRNAs in Yak [J]. Scientia Agricultura Sinica, 2019, 52(14): 2538-2547.
[15]	YI Min,LÜ Qing,LIU KeKe,WANG LiJun,WU YuJiao,ZHOU ZeYang,LONG MengXian. Expression, Purification and Localization Analysis of Polar Tube Protein 2 (NbPTP2) from Nosema bombycis [J]. Scientia Agricultura Sinica, 2019, 52(10): 1830-1838.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

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

RichHTML

PDF