摘要
为了深入研究不同类型荞麦蛋白酶抑制剂在抑制肿瘤细胞生长及诱导其发生凋亡方面的作用,本实验在先前得到的重组荞麦胰蛋白酶抑制剂(rBTI)的基础上,运用定点突变技术将rBTI的活性位点进行替换,构建一种新型蛋白酶抑制剂aBTI。通过在大肠杆菌M15[pREP4]中表达,获得以可溶形式存在的aBTI目的蛋白。经Ni^(2+)-NTA亲和层析及superdex 75分子筛层析纯化,目的蛋白的纯度达到95%以上。抑制活性测定表明,aBTI具有专一性的弹性蛋白酶抑制活性,抑制常数K_i为3.34×10^(-7)mol/L。MTT比色法检测及细胞核形态学观察显示,aBTI在体外能够显著抑制HepG2肿瘤细胞的增殖(IC_(50):1.88μmol/L),并诱导其凋亡,具有较好的抗肿瘤效应。
Previous studies have shown that recombinant buckwheat trypsin inhibitor （rBTI） possesses potent antitumor activity in in vitro models. In this study, a mutant （R45A-aBTI） of rBTI was generated through site-directed mutagenesis, aBTI was expressed in a soluble state in E.coli. M15 [pREP4] and purified by affinity chromatography and gel filtration chromatography. The purity of the aBTI reached above 95%. Activity analysis indicated that aBTI had a specific inhibitory activity toward elastase, its inhibition constant （Ki） value was 3.34 × 10^-7 mol/L. MTT assay demonstrated that rBTI could inhibit the growth of HepG2 tumor cells in a dose-dependent manner （IC50：1.88 μmol/L）. Additionally, nuclear staining showed that aBTI could induce the apoptosis of HepG2 cells. Our results suggested that aBTI displayed more marked antitumor efficacy in in vitro and might be a novel candidate for cancer treatment.
出处
《中国细胞生物学学报》
CAS
CSCD
2010年第4期589-595,共7页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30671084
No.30870525
No.30970611)
山西省自然科学基金(No.200701 1077)资助项目~~
关键词
荞麦
蛋白酶抑制剂
定点突变
表达
抗肿瘤
buckwheat
protease inhibitor
site-directed mutagenesis
expression
antitumor
