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目的探索一种能获得具豇豆胰蛋白酶抑制剂(CpTI)活性、免疫原性且氨基酸序列与天然蛋白一致的CpTI蛋白表达纯化方法,为评价转CpTI水稻的安全性提供重要材料。方法将CpTI基因克隆到PET41EK表达载体中,构建该基因的非融合表达载体PET41EK-CpTI,将表达载体转入大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,对诱导表达的时间、温度及IPTG浓度进行优化获得表达产物,对表达产物进行超滤和阴离子交换纯化从而获得纯化蛋白,对获得的纯化蛋白进行免疫原性、活性及N端测序鉴定等,并测定其浓度。结果在大肠杆菌Rosetta(DE3)、20～25℃、130r/min、0.5mmol/L IPTG、过夜诱导表达条件下,经过30kDa超滤和DAEE FF阴离子交换纯化,可以获得可溶表达量高、具有CpTI免疫原性和活性的纯化蛋白,其纯度达到95%。结论利用本研究建立的非融合表达方法可以得到在活性、免疫原性、分子量及氨基酸序列等方面与天然CpTI蛋白基本一致的CpTI蛋白,该法简单易行,能够满足大量生产表达的要求。
OBJECTIVE: To investigate a CpTI protein expression and purification method with the activity of cowpea trypsin inhibitor (CpTI) and its immunogenicity and the same amino acid sequence as the native protein, and to provide an important material for evaluating the safety of transgenic CpTI rice. Methods The CpTI gene was cloned into the PET41EK expression vector and the non-fusion expression vector PET41EK-CpTI was constructed. The expression vector was transfected into E. coli BL21 (DE3) and Rosetta (DE3) for expression. The expression of time, And IPTG concentration were optimized to obtain the expression product. The expression product was purified by ultrafiltration and anion exchange to obtain the purified protein. The purified protein was identified for its immunogenicity, activity and N-terminal sequencing, and the concentration was determined. Results After 30 kDa ultrafiltration and DAEE FF anion exchange purification in E. coli Rosetta (DE3) at 20-25 ℃, 130 r / min and 0.5 mmol / L IPTG for overnight expression, CpTI immunogenicity and activity of the purified protein, its purity of 95%. Conclusion The CpTI protein that is consistent with the native CpTI protein in terms of activity, immunogenicity, molecular weight and amino acid sequence can be obtained by the non-fusion expression method established in this study. This method is simple and can meet the requirements of mass production.