中文摘要：

荞麦胰蛋白酶抑制剂（BTI）属于丝氨酸蛋白酶抑制剂Potato I家族,典型构象中有1段暴露在分子外侧的结合区,该区内P1＇、P2、P6＇与P8＇位的氨基酸残基具有高度保守性.本文依据近期解析的rBTI晶体结构以及rBTI与胰蛋白酶复合物晶体结构信息,对rBTI中的P2和P8＇位氨基酸进行突变,构建了pExSecI-Bti-P44T和pExSecI-Bti-W53R重组质粒,转入大肠杆菌BL21（DE3）中进行表达,通过Resource Q阴离子交换层析和Superdex G 75 HR 10/300凝胶柱进行分离纯化后,测定了rBTI及其突变体对胰蛋白酶的抑制常数,以及它们对HepG2细胞内的蛋白酶体和细胞增殖的抑制作用.实验结果显示,rBTI的44和53位分别突变为Thr和Arg后,Ki分别为2.91×10-9mol/L和2.97×10-7mol/L,前者较rBTI（Ki=3.56×10-8 mol/L）降低1个数量级,而后者较rBTI升高1个数量级.功能分析显示,rBTI及2种突变体对HepG2细胞内的蛋白酶体基本没有抑制作用,但是它们都保留了对HepG2细胞增殖的抑制活性.这些结果揭示,作为一种特异的胰蛋白酶抑制剂,rBTI分子中的保守区域氨基酸残基虽然对胰蛋白酶的抑制作用有显著影响,但并不影响其抑制肿瘤细胞的增殖,仍能发挥其原有的生物学功能.

英文摘要：

Buckwheat trypsin inhibitor（BTI） is a serine protease inhibitor of the potato inhibitor I family.BTI contains a binding loop at the molecular surface,and the P1′,P2,P6′and P8′residues are highly conserved for the formation of internal hydrogen bonds.The pExSecI-Bti-P44T and pExSecI-Bti-W53R plasmids were constructed based on crystal data of recombinant buckwheat trypsin inhibitor（rBTI） and rBTI-trypsin complex.The proteins expressed by E.coli BL21（DE3） host cells were purified with Resource Q anion exchange chromatography and a Superdex G 75 HR 10/300 gel column.The proteasome inhibition of the purified rBTI and mutants were tested in HepG2 cells.The results showed that the Ki of rBTI-P44T was 2.91 × 10-9 mol /L,and that of the wide type is 3.56 × 10-8 mol / L.However,the Ki of rBTI-W53R was 2.97 × 10-7 mol / L,which was higher than that of rBTI.Our rBTI and the two mutants were all able to inhibit the proteasomes and suppressed HepG2 cell proliferation.These results suggested that the inhibitory effect of rBTI on tumor proliferation retained,even when its anti-trypsin activity was modified by change the conserved amino acid residues.