鲎C因子是一种对痕量内毒素高度敏感的丝氨酸蛋白酶原,在医药行业可替代鲎试剂用于内毒素检测。然而其重组表达困难,且对于环境中内毒素的控制要求极高。该文首先克隆了来源于东方鲎的全长C因子,并在N末端和C末端分别添加了Flag标签和6×His标签,同时用哺乳细胞表达载体中的分泌信号肽替换掉了原始C因子信号肽。构建好的重组质粒转染至人胚胎肾脏细胞Expi293F悬浮培养7 d后收获上清液,Western Blot检测蛋白与预测大小128 kDa一致,具有与内毒素结合以及结合后切割荧光底物的能力。该研究首次在人胚胎肾脏细胞Expi293F中成功表达出具有生物学活性的全长C因子,表达量达到19.18 mg/L,比DING在昆虫细胞SF9中的表达量提高了113%。该方法对于后续开发低成本内毒素检测试剂盒具有指导意义。
Tachypleus amebocyte lysate factor C, as a serine proteasogen, is highly sensitive to trace endotoxin. It can replace tachypleus amebocyte lysate reagent for endotoxin detection in the pharmaceutical industry. However, its recombinant expression is difficult, and the control of endotoxin in the environment is extremely demanding. Firstly, we cloned the full-length factor C from Tachypleus tridentatus and added the Flag tag and 6×His tag at the N-terminal and C-terminal. Secondly, we replaced the original factor C signal peptide with the secreted signal peptide in the mammalian cell expression vector. The recombinant plasmid was transfected into human embryonic kidney cell Expi293F for suspension culture, and the supernatant was harvested after 7 days of fermentation. The Western Blot detection protein was consistent with the predicted size of 128 kDa. It had the ability to bind with endotoxin and cut the fluorescent substrate after binding. In this study, we successfully expressed the full-length factor C with biological activity in human embryonic kidney cell Expi293F for the first time. This expression system produced 19.18 mg/L factor C, which was 113% higher than that of DING in insect cell SF9. This method has guiding significance for the subsequent development of low-cost endotoxin detection kits.
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