豇豆胰蛋白酶抑制剂和硫氧还蛋白的融合表达和活性测定  

Expression and Bioactivity Assay of Fusion Protein of Cowpea Trypsin Inhibitor(CpTI) and Thioredoxin

作　　者：畅文军[1] 王宇光[1] 雷禄旺[1] 

机构地区：[1]中国热带农业科学院热带生物技术研究所热带作物生物技术国家重点实验室,海口571101

出　　处：《农业生物技术学报》2006年第2期226-230,共5页Journal of Agricultural Biotechnology

基　　金：中国热带农业科学院产业化指令项目

摘　　要：采用融合蛋白表达技术,通过1个人工设计合成的柔性接头,将豇豆胰蛋白酶抑制剂(cowpeatrypsininhibitor,CpTI)基因与表达载体上的硫氧还蛋白(thioredoxin)基因连接,构建了融合表达载体。将表达载体转入大肠杆菌(Escherichiacoli)GI724,通过色氨酸诱导获得高效表达,产物以可溶状态存在。活性测定显示,融合蛋白具有抑制胰蛋白酶的生物学活性。以大豆胰蛋白酶抑制剂为参照,将融合蛋白热处理后进行活性测定,发现它具有较好的热稳定性。将肠激酶(enterokinase)与融合蛋白在37℃作用16h,可获得切掉thioredoxin的CpTI蛋白。活性测定显示,切除thioredoxin后CpTI的胰蛋白酶抑制活性比同样处理条件下的thioredoxin-CpTI明显下降,比活力仅为原来的80%,说明融合蛋白具有更高的稳定性。研究结果为thioredoxin-CpTI作为一种生物农药的应用奠定了基础。To construct fusion protein expression vector, cowpea trypsin inhibitor （CpTI） gene was linked with thioredoxin gene in the pTrxFus through a flexible linker designed with the software Chou-Fasman. The recombinant vector pTrxFus-CpTIwas identified and transformed into Escherichia coli GI724 to overexpress the fusion protein. The expression was induced with tryptophan. The fusion protein was confirmed to be soluble by SDS-PAGE analysis of the supernatant and precipitate of lysates. SDS-PAGE also showed that expressed protein accounted for about 20% of the total bacterium protein. With Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride （BApNA） as substrate, the bioactivity and heat resistance of fusion protein were assayed and the result showed, like soybean trypsin inhibitor, fusion protein performed relatively high thermal resistance. SDS-PAGE showed most fusion protein remained soluble after treated at 80 ℃, which greatly facilitates its purification and application. Thioredoxin-CpTI could be digested into thioredoxin and CpTI by incubating at 37 ℃ for 16 h with enterokinase. The CpTI inhibition activity was about 80% of thioredoxin-CpTI, indicating that the CpTI stability was increased via the fusion with thioredoxin. This work has laid a foundation for application ofthioredoxin-CpTI as a biological pesticide.

关 键 词：豇豆胰蛋白酶抑制剂 硫氧还蛋白 结构设计 融合蛋白表达 生物农药 

分 类 号：S188[农业科学—农业基础科学]