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设计一种适合基因工程开发的无标签重组荞麦胰蛋白酶抑制剂rBTI-2,并研究其对肿瘤细胞的生长抑制作用。构建原核表达载体pExSecI-BTI-2,诱导表达获得可溶性目的蛋白,经Resource~(TM) Q纯化后作用于HL-7702、HepG2、EC9706和QBC-939细胞,MTT检测rBTI-2对其生长的影响,并与前期获得的几种融合蛋白酶抑制剂进行功能比对。结果表明:质粒pEXSecI-BTI-2构建成功,SDS-PAGE分析表明分子量约为7.8 kDa。MTT检测表明rBTI-2对几种肿瘤细胞的生长有明显的抑制作用,而对正常细胞HL-7702作用很小。几种蛋白酶抑制剂对肿瘤细胞的生长均有不同程度的影响,其中rBTI-2对肿瘤细胞的生长抑制作用要大于融合蛋白酶抑制剂rBTI,这为深入研究BTI诱导肿瘤细胞凋亡的分子机制及其应用开发提供了重要基础和研究依据。
Design a genetically engineered non-tag recombinant barley trypsin inhibitor rBTI-2, and study its growth inhibition of tumor cells. The prokaryotic expression vector pExSecI-BTI-2 was constructed and expressed in E.coli BL21 (DE3). The purified protein was purified and purified by Resource TM (TM) Q and applied to HL-7702, HepG2, EC9706 and QBC-939 cells. Effect, and functionally aligned with several fusion protease inhibitors previously obtained. The results showed that the plasmid pEXSecI-BTI-2 was successfully constructed and its molecular weight was about 7.8 kDa by SDS-PAGE analysis. MTT assay showed that rBTI-2 significantly inhibited the growth of several tumor cells, but had little effect on the normal cells HL-7702. Several protease inhibitors have different degrees of influence on the growth of tumor cells, of which rBTI-2 on tumor cell growth inhibitory effect than fusion protein inhibitor rBTI, which is to study the molecular mechanism of BTI-induced apoptosis in tumor cells and Its application and development provide an important foundation and research basis.