| Bacillus thuringiensis(Bt)act as one of the most widely used microbial insecticides,because of its environmentally friendly,harmless to human and livestock and wide insecticidal spectrum.Bt has attracted a lot of attention in the study of biological pesticides and genetically modified material.With Bt transgenic crops constantly appeared and has been wildly used,they bring very significant economic,social and ecological benefits to people.At the same time,the ecological security risks of the application and the safety problems to humans and other non-target organisms are also of concern;more and more countries has established the identification system for genetically modified products,in this way the security screening test for Cry toxin expression product is particularly urgent.At present,it is reported that the ELISA kit-single toxin antibody detection-was mainly used for the Bt genetically modified products detection methods and market application..But it is difficult to meet the needs of using one antibody to detect a variety of toxins in the practical application.Therefore,by virtue of the characteristics of Cry toxin three-dimensional structure with high similarity,it is a very valuable work to develop Cry toxin broad spectrum specificity detection technology based on common structure.This study is bsed on the three-dimensional structure of Bt Cry toxin is highly similar,five Cry1 toxins(Cry1Ab,Cry 1 Ac,Cry 1B,Cry 1C,Cry 1F)are used as the object,the common domain positions are analyzed,genetic engineering technology is used to achieve the common domain protein which prepared for the broad specificity antibody of Bt cryl toxins.Finally,it will apply to the detection of Bt toxin and lay the foundation for Bt toxin protein in genetically modified food(GMF)detection.1.The analysis and positioning of the common structural domain of five cryl toxinsThe bioinformatics software and network database resources were used.First of all,the amino acid sequence of five Cry1 toxins(Cry1Ab,Cry1Ac,Cry1B,Cry1C,Cry1F)were compared and analyzed,then homology modeling was taken to predict the five Cry1 toxins domain I three-dimensional structure model,and the related biological functions were analyzed,too.It was found that,in the amino acid sequence alignment of the three domains for the five Cry1 toxins the domain I peaked at 71%sequence identity,and in the three-dimensional structure the domain I also has a high similarity,which almost completely overlap.After the analysis of the model by Ramachandran plot,errat and Verify3D test with rationality of.A synthetical,Domain I acts as the common structural domain of five Cryl toxins.2.The cloning,prokaryotic expression and pulrification of common structural domain proteinBy using of genetic engineering technology,primers were designed to amplify domainI gene sequence.And construct the prokaryotic expression vector pET26b-Domain I by Ligation.The recombinant plasmid was identificated by restriction enzyme,sequence alignment,and transferred into Escherichia coli BL21(DE3)Compenent cells.The common domain protein expression was induced by IPTG,and the expression products were purified and identified.The antigenicity analysis combined with ELISA test proved that it has good immunogenicity and immune response.It will lay the foundation for the next step of the common domain to target molecules of broad specificity recognition of Cry1 toxins antibody.3.The screening,identification and activity analysis of broad-spectrum antibody of Bt Cryl toxinsThe phage display technology was used to produce the broad-spectrum antibody of Bt Cryl toxins using,the purification of common structural domain protein was selected as the antigen,according to subtractive panning from the humanized single domain phage antibody library(DAb).The results indicated that after four rounds of panning,nine positive clones were identified by monoclonal ELISA and PCR detection,and after the DNA sequencing and amino acid sequence alignment on the target band,five positive clones with different amino acid sequences were gained.Among them,E3 had a relatively high Broad specificity for five Cryl toxins,and established a new sandwich ELISA detection method based on toxin receptor BBMV and single domain antibody. |
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