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目的探究重组眼镜王蛇属胰蛋白酶和糜蛋白酶双重抑制剂(OH-TCI)在大肠杆菌中的表达与纯化及其对大鼠心肌缺血再灌注损伤(MIRI)的保护作用。方法将Pet32a-OH-TCI重组质粒转化到大肠杆菌中,通过异丙基硫代半乳糖苷(IPTG)诱导,经烟草蚀纹病毒(tobacco etch virus,TEV)酶切和镍亲和层析后获得具有天然活性的OH-TCI。健康SD大鼠,随机分为对照组和OH-TCI药物组。药物组于术前5 min腹腔注射OH-TCI,对照组给予等体积生理盐水处理。结扎大鼠的左冠状动脉前降支30 min后,复灌120 min构建大鼠MIRI模型。检测大鼠血清中超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、肌酸激酶(creatine kinase,CK)活性和丙二醛(MDA)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6的含量等指标,评估具有天然活性的重组蛋白酶抑制肽OH-TCI对大鼠MIRI的保护作用。结果重组蛋白酶抑制肽OH-TCI在pET32a/Rosetta-gami(DE3)表达系统中以融合蛋白形式得到可溶性表达,通过TEV酶切、两次亲和层析纯化后,得到具有天然活性的重组OH-TCI。与对照组相比,OH-TCI可使心肌MIRI大鼠血清中LDH活性、MDA含量和IL-6的释放量显著下降(P<0.01);炎性因子TNF-α释放量明显降低(P<0.05)。结论具有天然活性的重组蛋白酶抑制肽OH-TCI能够在大肠杆菌成功表达,并对心肌缺血再灌注样损伤具有一定的保护作用。
Objective To investigate the expression and purification of recombinant cobra cartanopsis tryptase and chymase double inhibitor (OH-TCI) in Escherichia coli and its protective effect on myocardial ischemia-reperfusion injury (MIRI) in rats. Methods The recombinant plasmid of Pet32a-OH-TCI was transformed into E.coli and induced by isopropylthiogalactoside (IPTG). After digested by tobacco etch virus (TEV) and nickel affinity chromatography OH-TCI with natural activity was obtained. Healthy SD rats were randomly divided into control group and OH-TCI group. The drug group was intraperitoneally injected with OH-TCI 5 min before the operation, and the control group was given equal volume of saline. The left anterior descending coronary artery of rats was ligated for 30 min, and the MIRI model was established after reperfusion for 120 min. Serum levels of superoxide dismutase (SOD), lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), tumor necrosis factor (TNF) (IL) -6 content and other indicators to assess the natural activity of the recombinant protease inhibitor OH-TCI rat MIRI protective effect. Results Recombinant protease inhibitor OH-TCI was expressed as a fusion protein in the expression system of pET32a / Rosetta-gami (DE3). After purified by two affinity chromatography with TEV, the natural OH- TCI. Compared with the control group, OH-TCI significantly decreased LDH activity, MDA content and IL-6 release in myocardium (P <0.01), and significantly decreased the release of TNF-α (P < 0.05). Conclusion The recombinant natural protease inhibitor OH-TCI can express successfully in Escherichia coli and has a protective effect on myocardial ischemia-reperfusion injury.