豇豆胰蛋白酶抑制剂的非融合表达纯化  

Non-fusion expression and purification of cowpea typsine inhibitor

作　　者：胡贻椿[1] 卓勤[1] 朴建华[1] 杨晓光[1] 

机构地区：[1]中国疾病预防控制中心营养与食品安全所卫生部微量元素营养重点实验室,北京100050

出　　处：《卫生研究》2012年第3期374-378,384,共6页Journal of Hygiene Research

基　　金：国家重点基础研究发展计划“973”项目(No.2007CB109207);国家转基因生物新品种培育科技重大专项(No.2008ZX08011-005,2011ZX08011-005)

摘　　要：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 establish a practicable method to express and purify Cowpea Typsine Inhibitor （CpTI）, which is comparable with native CpTI, for providing important materials to access the safety of genetically modified rice with CpTI. Methods The non-fusion expression vector PET41EK-CpTI was constructed by cloning the CpTI gene into expression vector PET41 EK. And the expression vector was then transformed into E. coli BL21 （ DE3 ） and E. coli Rosetta（ DE3 ）. Purified expression product was obtained through optimizing the induction temperature,induction time and IPTG concentration and then it was identified by western blotting,activity determination and N-terminal amino acid sequencing. Results The purified protein was obtained under the condition of E. coli Rosetta （ DE3 ） , 20 - 25℃, 130rpm, and 0.5mmol/L IPTG, overnight expression, 30kDa uhra-filtration and anion exchange purification. After identification, the CpTI protein with significant immunogenicity and CpTI activity was successfully obtained and its purity is up to 95%. Conclusion The CpTI protein obtained by the non-fusion method established inthis study is comparable with the native CpTI protein in terms of activity,immunogenicity, molecular weight and amino acids sequence. Moreover, the method is easy to operate and satisfied with the requirements of large-scale expression.

关 键 词：豇豆胰蛋白酶抑制剂 非融合表达 原核表达 

分 类 号：Q789[生物学—分子生物学] Q55