谷胱甘肽S-转移酶-肌钙蛋白I(28～110aa)表达纯化及稳定性的初步研究

Preliminary study on expressive purification and stability of glutathione S-transferase-troponin I(28-110 aa)

目的:评估重组融合蛋白谷胱甘肽S-转移酶(GST)-肌钙蛋白I(TnI)(28～110aa)的实用价值。方法:诱导表达菌株BL21-PGEX-4T-3/TnI(84～330bp)表达GST-TnI(28～110aa),谷胱甘肽Sepharose-4B亲合层析柱纯化融合蛋白,经自动免疫分析仪检测其免疫反应性。采用双抗体夹心ELISA法检测稳定性,并与天然提纯心肌肌钙蛋白I(cTnI)抗原进行比较。结果:GST-TnI(28～110aa)融合蛋白表达水平高,具较高免疫反应性。在37℃条件下,GST-TnI(28～110aa)融合蛋白第3天免疫反应性仍保留60%,而人心肌提纯的cTnI在第3天免疫反应性几近丧失。结论:GST-TnI(28～110aa)融合蛋白具较高的免疫反应性,与天然cTnI相比较,重组GST-TnI融合蛋白稳定性好,有望作为cTnI测定的候选参考标准品。

Objective To evaluate the practicability of glutathione S-transferase (GST)-troponin I (TnI) (28-110 aa). Methods The BL21-PGEX-4T-3/TnI (84-330 bp) was used to induce the GST-TnI (28-110 aa) expression. The fusion protein was purified by glutathione sepharose 4B affinity chromatography column, whose immunoreactivity was detected by commercial TnI autoimmunal analyzer. The stability of the fusion protein was studied by double antibody sandwitch ELISA in comparison with that of the native TnI. Results The GST-TnI (28-110 aa) had a high expression with good immunoreactivity. Stability studies showed that at 37℃, the fusion protein remained 60% activity after 3 days, while the nature cTnI lost almost all the activity. Conclusions In comparison with the native cTnI, the recombinant GST-TnI (28-110 aa) fusion protein show higher immunoreactivity and stability, which might be used as a standard candidate for cTnI immunoassay.