Establishment Of A Preparation System For Cry1Ah And Cry1Ab/c Insecticidal Protein Reference Materials

The cry1Ah1 gene of Bacillus thuringiensis was cloned by our laboratory and obtained China's independent intellectual property rights.It has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer,and has obtained transgenic lines with good insect resistance traits.The cry1Ab/c gene is a hybrid Bt gene fused from cry1A(b)and cry1A(c).The hybrid gene has been successfully transferred into rice,and a new transgenic rice variety TT51-1 has been developed.With the rapid development of transgenic technology,the safety of genetically modified products has received extensive attention.Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products,but the development of genetically modified protein reference materials is relatively lagging,and it's urgent to establish a preparation system.In this study,the Bt expression system was used to prepare and purify Cry1 Ah and Cry1Ab/c proteins,and the reference materials preparation system was established.The main results are as follows:The preparation and purification system of Cry1 Ah and Cry1Ab/c proteins were optimized,and pure protein which can be used for preparation of reference materials was obtained.The activation conditions of Cry1 Ah and Cry1Ab/c proteins were determined as follows: protein: trypsin = 10:1(w/w),15% glycerol,water bath at 37°C for 2 h;protein: trypsin = 10:1(w/w),water bath at 37°C for 2 h.High-purity Cry1 Ah and Cry1Ab/c proteins(size exclusion chromatography purity: 99.66% and 99.68%,respectively)were obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification.Afterwards,the amino acid sequence of the activated Cry1 Ah and Cry1Ab/c proteins(50I-622 R and 24I-615 R,respectively)was determined using Edman degradation and mass spectrometry.The amino acid sequence of Cry1 Ah and Cry1Ab/c proteins after activation can be used to lay the foundation for the development of protein reference materials based on isotope dilution mass spectrometry.Cry1Ah and Cry1Ab/c proteins isotope dilution mass spectrometry analysis systems were established,and the candidate protein reference materials were tested for homogeneity,stability and valuation.The complete hydrolysis time of the two proteins was determined to be 24 h and 12 h,respectively,and a proteolytic-based Cry1 Ah and Cry1Ab/c proteins valuation traceability system was established.The results of analysis of variance showed that the homogeneity of the two protein reference materials was good;and the stability was good during storage at room temperature for 14 days,storage at 4°C for 28 days,storage at-20°C for 8 weeks,and storage at-70°C for 6 months.The concentrations of Cry1 Ah and Cry1Ab/c protein reference materials were(38.8 ± 1.4)?g/g and(99.9 ± 3.4)?g/g,respectively,after valuation and uncertainty evaluation.The reparation system of Cry1 Ah and Cry1Ab/c protein reference materials was established in this study.Two protein reference materials meeting the requirements of national secondary reference materials were prepared,which contributed to the development of the research and development of genetically modified protein reference materials,and provide reliable and effective guarantee for the detection of transgenic proteins in the future.