【作者】 杨毅；

【导师】 陈惠；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 胰蛋白酶抑制剂(TI)在荞麦中的含量非常丰富,属于丝氨酸蛋白酶抑制剂,可以抑制胰蛋白酶活性,拥有较高的热稳定性,并且在强酸性和强碱环境下都较为稳定；具备降血压、降血脂、降血糖、降低结肠癌及乳腺癌发生几率等功能；能够抗棉铃虫和抗真菌,具有抗衰老和抗艾滋病等作用。实验室已经在苦荞麦中分离得到一种胰蛋白酶抑制剂(FtTI), FtTI作为一种功能性蛋白,对其结构与功能的深入研究具有重要的理论意义及实际价值。通过将苦荞麦胰蛋白酶抑制剂(FtTI)65位精氨酸和67位天冬氨酸分别替换为亮氨酸和缬氨酸,研究胰蛋白酶抑制剂的活性位点,揭示FtTI结构与功能的关系。通过定点突变技术获得了三株突变菌株R65L, D67V, R65L/D67V。将突变前和突变后的胰蛋白酶抑制剂进行了分离纯化与抑制活性的研究。结果表明：(1) FtTI和突变体aFtTI-R65L, aFtTI-D67V和aFtTI-R65L/D67V经IPTG诱导培养5h,收集菌液经过超声波破碎得到粗产物,经过纯化后的胰蛋白酶抑制剂对胰蛋白酶的摩尔抑制比分别为1：1,1：1.15,1：1.3,1：1.2；抑制常数Ki分别为1.62nM,1.69 nM, 1.9 nM,1.8nM(BApNA作为底物)。突变后的胰蛋白酶抑制齐aFtTI-R65L, aFtTI-D67V(?)aFtTI-R65L/D67V的抑制活力与突变前相比分别降低了13.04%,23.85%和16.67%。(2) SDS-PAGE分析表明突变前和突变后表达产物胰蛋白酶抑制剂的大小一致,均为9.5kDa。(3)对突变体aFtTI-R65L, aFtTI-D67V和aFtTI-R65L/D67V抑制反应温度研究表明,其最适反应温度均为40℃。在10-80℃保温30min后,突变体对胰蛋白酶的抑制活性仍保留80%以上；在90℃保温30min,突变体的抑制活性开始显著下降,只保留其39%。突变体具有较高的耐热性。(4)将aFtTI在pH 3.0-10.0的不同缓冲溶液中放置30 min后,其抑制活性可保留90%左右。在pH 2.0条件下,aFtTI抑制活性丧失约31%；在pH 11.0条件下,aFtTI抑制活性丧失约43%。表明aFtTI与原始的胰蛋白酶抑制剂相一致,对强酸和强碱都具有相对稳定性。在pH 8.0左右,aFtTI抑制活性最大,与FtTI的表现一致,表明本实验对苦荞麦蛋白酶抑制剂FtTI的定点突变并不会改变它是一种偏碱性的胰蛋白酶抑制剂的性质,突变前后均保持了耐碱性的特点。(5)将突变前和突变后的胰蛋白酶抑制剂在气压为0.101 MPa、温度100℃条件下处理20 min,aFtTI迅速钝化；在气压为0.198 MPa、温度为120℃和气压为0.143 MPa、温度为110℃条件下处理20min, aFtTI失去抑制活性的96%和95%。aFtTI经超声波处理,其钝化效果与超声波功率和时间成正相关。以上研究为进一步揭示胰蛋白酶抑制剂结构与功能的关系、抗病虫害转基因植物的研究以及抗肿瘤药物的研制奠定基础。更多还原

【Abstract】 The buckwheat belongs to serine protease inhibitors, which contain rich trypsin inhibitor. Buckwheat protease inhibitors can inhibit trypsin activity, having a high thermal stability and a certain degree of pH stability. Also it can lower blood pressure, blood fat, lowering blood sugar, resistant to bollworm and anti-fungal, anti-aging and anti-AIDS action. One kind of FtTI was separated in early stage of laboratory studies. Studies have that it can inhibit pumpkin vine blight strongly, chayote leaf blight, tomato early blight and anthracnose and other pathogenic fungal hyphae pepper growth.The important theoretical significance and practical value that depth study of the structure and function has been confirmed, because of it’s a functional protein. We replaced the arginine (65 site) and aspartic acid (67 site) by Leu. and Val. The amino acid sequence and active center region of active groups are researched. Next, FtTI activity center was analyzed by bioinformatics.The FtTI gene from buckwheat was mutated by site-directed mutagenesis technology and final three mutant strains R65L, D67V, R65L/D67V were obtained. The expression products were isolated, purified and inhibition activity.(1) After induced culture by IPTG from mutant R65L, D67V, R65L/D67V, the moore rejection ratio to trypsin is 1:1,1:1.15,1:1.3 and 1:1.2. The inhibition constants (Ki) is 1.62nM,1.69 nM,1.9 nMand 1.8 nM (BApNA as substrate). Theinhibitors vitality of mutation aFtTI-R65L, aFtTI-D67V and aFtTI-R65L/D67V have decreased by 13.04%, 23.85% and 16.67%.(2) The SDS-PAGE analysis of expression products showed that mutation premutation and after of trypsin inhibitor have the same size both 9.5kDa.(3) The mutant aFtTI-R65L, aFtTI-D67V, aFtTI-R65L/D67V has more high heat resistance and the optimum temperature (40℃) does not change significantly. The thermal stability results showed that all three mutants have high heat resistance. After 10-80℃ for 30 min, aFtTI inhibitor activity to trypsin more than 80%. After 90℃ for 30 min, the aFtTI inhibitory activity begin to decreased significantly and only reserved it’s inhibitory activity about 39%. Thus, aFTtl has high heat resistance.(4) FtTI inhibitory activity could retain about 90%, after placed different buffer solutions (pH 3.0-10.0) for 30 min. Under pH 2.0 conditions, the FtTI inhibitory activity loss of about 31% and pH 11.0 conditions, the FtTI inhibitory activity loss of about 43%. It’s indicated that FtTI has a same feature with original trypsin inhibitor consistent, which are relatively stable under the strong acids and bases conditions. The FtTI has the maximum inhibitory activity in the pH 8.0. This study showed that FtTI mutagenesis of buckwheat inhibitors did not changed the alkaline nature and maintained the alkali resistance characteristics after mutations before and after.(5) Under high temperature and pressure (greater than 0.101MPaand 100℃) conditions, FtTI soon be passivated. FtTI lost its inhibitory activity of 96% and 95%, at 198MPa,120℃, at 0.143MPa,110℃ conditions for 20 min. However, ultrasonic inactivation FtTI shoed proportional to its amplitutude and duration action.In this study, lay the foundation for future study the relationship between structure and function of protease inhibitors, research of anti-pest plant gene transfer and development of anticancer drugs.更多还原