Separation And Purification Of Cry1Ac,Cry2Ab And Cry51Aa1

Bacillus thuringiensis（Bt）produces Bt toxin in the process of spore germination.As a biological agent,Bt toxin is widely used to control pest.Among them,Cry1Ac and Cry2Ab are widely used,while Cry51Aa1 is only Bt parasporal crystal protein with hemipteran insecticidal activity.At present,Cry1Ac,Cry2Ab and Cry51Aa1 toxin are produced by using prokaryotic expression in the laboratory,but the production process is often accompanied by the appearance of hetero proteins.As a result,the insufficient purity affects the biological titer and restrict the study on the structure,function and mechanism of toxin proteins.In order to solve the problems of low purity of crude protein extract and unstable titer of bioassay,the purification methods of Cry1Ac,Cry2Ab and Cry51Aa1 toxins were established with gel filtration chromatography and anion exchange chromatography as the key technology in this paper.The application of this technology will improve the quality of Bt toxin products,and provide materials for subsequent studies on the structure,mechanism of Bt toxin proteins.1.Establishing the separation and purification system of Cry1Ac,Cry2Ab and Cry51Aa1In this study,the separation and purification system of Cry1Ac,Cry2Ab and Cry51Aa1 was established by screening chromatography technology and optimizing elution conditions.The separation and purification system of Cry1Ac was made up of ultrafiltration and gel filtration chromatography.After purification,the purity of Cry1Ac increased from 82.56%to 90.79%,and the recovery rate was 24.94%.The separation and purification technology of Cry2Ab was made up of ultrafiltration,gel filtration chromatography and anion exchange chromatography.The purity of Cry2Ab was increased from 40.85%to 83.95%after separation and purification and the recovery rate was 5.91%.The separation and purification system of Cry51Aa1 was composed of ultrafiltration,gel filtration chromatography and anion exchange chromatography.After purification,the purity of Cry51Aa1 increased from 81.34%to 96.25%,and the recovery rate was 7.47%.At the cost of recovery,gel filtration chromatography and anion exchange chromatography were the key steps to purify Cry1Ac,Cry2Ab and Cry51Aa1 toxins.2.The toxicity of purified Cry1Ac,Cry2Ab and Cry51Aa1 toxins to target pestsIn this study,the toxicity of Cry1Ac and Cry2Ab to Helicoverpa armigera and Cry51Aa1 to Apolygus lucorum were determined by diet overlay method and parafilm-capsule method,respectively.The LC₅₀ of Cry1Ac activated toxin to H.armigera was 0.042μg/cm²,that of purified Cry1Ac activated toxin was 0.037μg/cm².The LC₅₀ of Cry2Ab protoxin to H.armigera was 0.205μg/cm²,that of purified Cry2Ab protoxin was0.139μg/cm².The LC₅₀ of Cry51Aa1 protoxin to A.lucorum was 131.528μg/m L,that of purified Cry51Aa1 protoxin was 116.921μg/m L.The biological titer of the purified toxins against target pests were increased,and the quality of three product was improved.