野生型和突变型荞麦蛋白酶抑制剂的活性比较及抗肿瘤功能分析

运用定点突变技术研究重组荞麦胰蛋白酶抑制剂(rBTI)的作用位点，先后构建了R45A-aBTI 和R45F-fBTI 两个突变体．抑制活性测定显示，aBTI 和fBTI 均丧失了胰蛋白酶抑制活性，却分别增加了对弹性蛋白酶和胰凝乳蛋白酶的抑制活性，确定Arg⁴⁵为野生型rBTI 的作用位点．稳定性分析表明，rBTI、aBTI 和fBTI 均具有很高的热稳定性及酸碱稳定性．采用MTT 比色法分别检测野生型和突变型抑制剂对肿瘤细胞生长的抑制作用．结果表明，突变前后的3种抑制剂对HL-60 和EC9706 细胞的生长均显示出很强的抑制作用，并且具有明显的浓度依赖性和时间依赖性．通过研究不仅确定了rBTI 的作用位点，而且获得了两种新型蛋白酶抑制剂，且作用位点的改变并不影响其对肿瘤细胞的生长抑制作用．为进一步研究胰蛋白酶抑制剂的结构与功能的关系以及抗肿瘤药物的研制提供了新的思路．

Analysis of Inhibitory Activity and Antineoplastic Effect of Wild Type rBTI and Its Mutants

To examine the active site of recombinant buckwheat trypsin inhibitor (rBTI), two mutants (R45A-aBTI, R45F-fBTI) were generated through site-directed mutagenesis. Activity analysis found that the aBTI and fBTI had lost trypsin inhibitory activity. However, aBTI and fBTI showed new inhibitory activities against elastase and chymotrypsin, respectively. This result suggested that Arg⁴⁵ is the active site of rBTI. These inhibitors showed remarkable stability to heat and pH. The possible effects of aBTI and fBTI on the proliferation of human HL-60 and EC9706 cell lines were investigated by MTT assays. It indicated that aBTI and fBTI could specifically inhibit the growth of HL-60 and EC9706 cells in a dose- and time-dependent manner. The active site of rBTI was determined and two new inhibitors were obtained in this research. Knowledge about the active site is useful for further clarifying the physiological mechanism of rBTI and other protease inhibitors. New inhibitory activities of aBTI and fBTI are potentially useful in the fields of health and medicine.