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中国精品科技期刊2020
刘鑫, 屈跃宽, 曹立民, 隋建新. 鲨鱼单域抗体-碱性磷酸酶融合蛋白的表达及热稳定性分析[J]. 食品工业科技, 2020, 41(21): 110-115. DOI: 10.13386/j.issn1002-0306.2020020051
引用本文: 刘鑫, 屈跃宽, 曹立民, 隋建新. 鲨鱼单域抗体-碱性磷酸酶融合蛋白的表达及热稳定性分析[J]. 食品工业科技, 2020, 41(21): 110-115. DOI: 10.13386/j.issn1002-0306.2020020051
LIU Xin, QU Yue-kuan, CAO Li-min, SUI Jian-xin. Expression and Thermal Stability Analysis of Shark Single Domain Antibody-Alkaline Phosphatase Fusion Protein[J]. Science and Technology of Food Industry, 2020, 41(21): 110-115. DOI: 10.13386/j.issn1002-0306.2020020051
Citation: LIU Xin, QU Yue-kuan, CAO Li-min, SUI Jian-xin. Expression and Thermal Stability Analysis of Shark Single Domain Antibody-Alkaline Phosphatase Fusion Protein[J]. Science and Technology of Food Industry, 2020, 41(21): 110-115. DOI: 10.13386/j.issn1002-0306.2020020051

鲨鱼单域抗体-碱性磷酸酶融合蛋白的表达及热稳定性分析

Expression and Thermal Stability Analysis of Shark Single Domain Antibody-Alkaline Phosphatase Fusion Protein

  • 摘要: 酶标抗体的稳定性对酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)至关重要,但常规化学偶联方法制备的酶标抗体具有热稳定性不足的缺点,这限制了酶标抗体在ELISA中的应用。本研究利用基因工程技术克隆表达出碱性磷酸酶与特异性鲨鱼单域抗体的融合蛋白,并对融合蛋白的亲和力和热稳定性进行了研究。结果表明:在16 ℃下,0.05 mmol/L的异丙基β-D-1-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导可实现融合蛋白的可溶性表达,表达出的融合蛋白分子量约为63 kDa,与目标抗原的亲和常数可达7.80×10-8 mol/L,具有良好的抗原结合能力;融合蛋白在58 ℃处理180 min后热稳定性良好,证明该融合蛋白可以作为ELISA检测中一种潜在的免疫诊断剂。

     

    Abstract: The stability of enzyme-labeled antibody plays an important role in enzyme-linked immunosorbent assay(ELISA),but the disadvantages in thermal stability of the conventional antibody-enzyme conjugation has restricted the application of enzyme-labeled antibodies in ELISA. In this study,the shark single domain antibody specific to myofibrillar-binding serine protease(MBSP)fused with alkaline phosphatase was expressed and its affinity and thermal stability were investigated. Results showed that when induced with Isopropyl-beta-D-thiogalactopyranoside(IPTG)at 16 ℃,the fusion protein which was about 63 kDa in molecular weight,which could be expressed in soluble with high affinity and thermal stability. The KD value of the fusion protein to MBSP could reach 7.80×10-8 mol/L and the binding activity of fusion protein could be kept well when treated at 58 ℃ even for 180 minutes. All the results implied that the fusion protein could be used as a potential immunodiagnostic agent in ELISA.

     

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