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目的原核表达、分离纯化毛首鞭形线虫(Trichuris trichiura)丝氨酸蛋白酶抑制剂1(TtSerpin1),并观察其对蛋白酶的抑制作用。方法从毛首鞭形线虫成虫cDNA中扩增TtSerpin1编码序列(Gen Bank登录号为MF401634),将其连接入原核表达载体,构建重组质粒pET32a-sumo/TtSerpin1。将重组质粒转化入大肠埃希菌(Escherichia coli)BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷诱导TtSerpin1融合蛋白表达。表达的包涵体蛋白经变性、复性、镍亲和层析纯化、SUMO蛋白酶酶切融合标签后获得rTtSerpin1。用发色底物法检测其对人组织蛋白酶G、中性粒细胞弹性蛋白酶、蛋白酶3、纤溶酶和胰蛋白酶,猪胰蛋白酶、胰弹性蛋白酶及牛α-胰糜蛋白酶的抑制作用。结果成功构建了重组质粒pET32a-sumo/TtSerpin1,并在E.coli中成功表达。表达产物主要为包涵体,复性、纯化后的rTtSerpin1具有较好的蛋白酶抑制活性。1 000 nmol/L的rTtSerpin1对人组织蛋白酶G(100 nmol/L)、中性粒细胞弹性蛋白酶(10 nmol/L)、蛋白酶3(200 nmol/L),猪胰弹性蛋白酶(10 nmol/L),牛α-胰糜蛋白酶(1 nmol/L)的蛋白酶活性抑制率分别为60.89%、82.84%、21.21%、58.32%、96.98%,但对人纤溶酶、胰蛋白酶及猪胰蛋白酶抑制活性较弱。rTtSerpin1对人组织蛋白酶G及中性粒细胞弹性蛋白酶的抑制常数(K_i)分别为(949.80±91.51)、(242.70±53.41)nmol/L。结论 rTtSerpin1对多种丝氨酸蛋白酶具有较强抑制作用。
Objective To isolate and purify Trichuris trichiura serine protease inhibitor 1 (TtSerpin1) and observe its inhibitory effect on prokaryotic expression. Methods The coding sequence of TtSerpin1 was amplified from cDNA of Acinetobacter plicata (GenBank accession number: MF401634) and ligated into prokaryotic expression vector to construct recombinant plasmid pET32a-sumo / TtSerpin1. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), and the expression of TtSerpin1 fusion protein was induced by isopropyl-β-D-thiogalactoside. The expressed inclusion body protein was denatured, refolded and purified by nickel affinity chromatography. The SUMO protease digested the fusion tag to obtain rTtSerpin1. Inhibition of human cathepsin G, neutrophil elastase, protease 3, plasmin and trypsin, porcine trypsin, pancreatic elastase and bovine α-chymotrypsin by chromogenic substrate method was tested. Results The recombinant plasmid pET32a-sumo / TtSerpin1 was successfully constructed and successfully expressed in E. coli. The expressed product was mainly inclusion body, refolding, purified rTtSerpin1 has better protease inhibitory activity. The effect of rTtSerpin 1 with 1000 nmol / L on the activity of human cathepsin G (100 nmol / L), neutrophil elastase (10 nmol / L), protease 3 (200 nmol / L), porcine pancreatic elastase ), And the inhibitory rates of protease activity of bovine α-chymotrypsin (1 nmol / L) were 60.89%, 82.84%, 21.21%, 58.32% and 96.98%, respectively, but the inhibition rates on human plasmin, trypsin and porcine trypsin Weak activity. Inhibitory constants (K_i) of rTtSerpin1 to human cathepsin G and neutrophil elastase were (949.80 ± 91.51) and (242.70 ± 53.41) nmol / L, respectively. Conclusion rTtSerpin1 has a strong inhibitory effect on various serine proteases.