香鱼LECT2双抗体夹心ELISA法的建立及初步应用  

Establishment and Preliminary Application of Double Antibody Sandwich ELISA Method for LECT2 of Ayu(Plecoglossus altivelis)

作　　者：许立琼 史雨红 陈炯[1,2,3] XU Li-Qiong;SHI Yu-Hong;CHEN Jiong(State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products,Ningbo University,Ningbo 315211,China;Laboratory of Biochemistry and Molecular Biology,School of Marine Science,Ningbo University,Ningbo 315211,China;Key Laboratory of Aquacultral Biotechnology of Ministry of Education,School of Marine Science,Ningbo University,Ningbo 315832,China)

机构地区：[1]宁波大学农产品质量安全危害因子与风险防控国家重点实验室,宁波315211 [2]宁波大学海洋学院生物化学与分子生物学实验室,宁波315211 [3]宁波大学海洋学院水产生物技术教育部重点实验室,宁波315832

出　　处：《农业生物技术学报》2024年第7期1703-1714,共12页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(LY22C190003);宁波市自然科学基金(2021J115)。

摘　　要：白细胞衍生趋化因子(leucocyte cell-derived chemotaxin 2,LECT2)的表达量对鱼类健康状态具有指示作用。本研究利用毕赤酵母(Pichia pastoris)表达香鱼(Plecoglossus altivelis)重组PaLECT2蛋白(recombinant PaLECT2,rPaLECT2),分别免疫小鼠(Mus musculus)和新西兰白兔(Oryctolagus cuniculus)制备抗血清,再用Protein G亲和层析柱纯化获得抗体。以小鼠抗PaLECT2抗体作为捕获抗体以及辣根过氧化酶(horseradish peroxidase,HRP)标记的兔抗PaLECT2抗体作为检测抗体,通过条件优化建立双抗体夹心酶联免疫吸附法(double-antibody sandwich enzyme-linked immunosorbent assay,DAS-ELISA)。最后应用该方法检测嗜水气单胞菌(Aeromonas hydrophila)感染香鱼的模型和抗生素治疗的预后模型中血清PaLECT2蛋白含量。研究结果显示,小鼠和HRP-兔抗PaLECT2抗体效价分别为1∶12800和1∶25600。当捕获抗体浓度为8μg/mL而检测抗体稀释比例为1∶1600时,为最佳捕获抗体包被浓度和检测抗体工作浓度。所建DAS-ELISA法的最低检测限为11.22 ng/mL,具有良好的特异性和重复性。应用本方法测得感染组中PaLECT2的含量为(412±31.5)ng/mL,极显著高于健康组(54±7.2)ng/mL(P<0.001),而治疗组中PaLECT2的含量为(225±56.1)ng/mL,极显著低于感染组(P<0.01)。综上,本研究为PaLECT2蛋白表达水平的定量检测提供了一种更加快捷、标准化的方法,有望应用于香鱼健康状况诊断和监测。The expression level of leucocyte cell-derived chemotaxin 2(LECT2)reflects the health status of fish.In this study,the recombinant PaLECT2(rPaLECT2)protein,expressed by Pichia pastoris,was used to prepare anti-PaLECT2 antisera in mice(Mus musculus)and New Zealand white rabbits(Oryctolagus cuniculus).Then the antibodies were purified from antisera by Protein G affinity chromatography.The mouse anti-PaLECT2 antibody was used as the capture antibody and the rabbit anti-PaLECT2 antibody conjugated with horseradish peroxidase(HRP)as the detecting antibody.A double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)method based on PaLECT2 protein was established after optimization.Finally,it was applied to detect the PaLECT2 in serum the Aeromonas hydrophila-infected ayu model and the prognosis model of antibiotic treatment.The results showed that the titer of PaLECT2 antibody in mice were 1∶12800 and that of HRP-conjugated antibody in rabbits were 1∶25600,respectively.The DAS-ELISA method for PaLECT2 used the optimal coating concentration of 8μg/mL for the capture antibody and the best working dilution of 1∶1600 for the detection antibody.The minimum detection limit of the method was 11.22 ng/mL with good specificity and stability.The protein concentration of PaLECT2((412±31.5)ng/mL)in the infected group was extremely significantly higher than that in the healthy group(54±7.2)ng/mL(P<0.001).The protein concentration of PaLECT2 in the treated group(225±56.1)ng/mL was extremely significantly lower than that in the infected group(P<0.01).In conclusion,the establishment of this method provides a more efficient and standard method for the quantitative measurement of PaLECT2 protein levels,which will be applied to the diagnosis and monitoring of ayu health status.

关 键 词：白细胞衍生趋化因子(LECT2) 双抗体夹心ELISA法(DAS-ELISA) 香鱼 初步应用 

分 类 号：S917[农业科学—水产科学]