Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (16): 3130-3139.doi: 10.3864/j.issn.0578-1752.2016.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

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

LIU Bei-bei1,2, ZHANG Xiao2, XIE Ya-jing2, JIAO Ling-xia3, LIU Yuan1,2, ZHANG Cun-zheng2, ZHAO Yan-yan2, WU Ai-hua2, LIU Xian-jin1,2   

  1. 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

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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

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