基于I-FABP的鲫肠道通透性检测方法的建立

肠道通透性与肠道稳态及个体健康状况直接相关，目前尚缺乏鱼类肠道通透性的定量评价方法及试剂。血清中肠脂肪酸结合蛋白（intestinal fatty acid binding protein，I-FABP）是肠道通透性的重要血清生物学指标，可用于肠道通透性的定量评价。为建立鲫（Carassius auratus）血清I-FABP的定量检测方法，克隆了鲫I-FABP基因，经原核表达和纯化，并利用重组蛋白分别免疫新西兰白兔（New Zealand white rabbit）及小鼠（Mus musculus），制备了兔源及鼠源多克隆抗体，其效价均为1∶80 000。以纯化的兔源多克隆抗体作为包被蛋白，以辣根过氧化物酶（horseradish peroxidase，HRP）标记的鼠源多克隆抗体作为酶标抗体，通过优化各反应条件建立了基于鲫I-FABP的双抗体夹心酶联免疫检测方法（double antibody sandwich ELISA，DAS-ELISA）。标准曲线的线性回归方程为y=0.003 5x+0.036 7，拟合度较高R²为 0.999 1，临界值为0.090 6。批内及批间变异系数分别为1.17%～5.50%及0.16%～4.78%，具有较好的重复性。以该方法对54份样品进行检测，与商业化试剂盒对比，符合率为100%。构建的DAS-ELISA检测方法可用于鲫肠道通透性的定量评估。鉴于鱼类I-FABP的保守性，该方法或可用于其他鱼类肠道通透性的定量评价。

DAS-ELISA method of detecting intestinal permeability based on I-FABP in crucian carp(Carassius auratus)

Intestinal permeability is directly related to intestinal homeostasis and individual health. At present, there is a lack of quantitative evaluation methods and reagents for intestinal permeability of fish. Serum intestinal fatty acid binding protein (I-FABP) is an important serum biological index for quantitative evaluation of intestinal permeability. In order to establish a quantitative detection method of serum I-FABP concentration in crucian carp (Carassius auratus), the I-FABP gene of crucian carp was cloned, expressed and purified in prokaryote. The rabbit and mouse polyclonal antibodies were prepared by immunizing New Zealand white rabbit and mouse with the recombinant protein, and their titers were 1:80 000. The rabbit polyclonal antibody was used as the coating protein, and the mouse polyclonal antibody labeled with HRP was used as the enzyme labeled antibody. By optimizing the reaction conditions, a double antibody sandwich ELISA (DAS-ELISA) method based on rI-FABP was established. The linear regression equation of the standard curve was y=0.003 5x+0.036 7, the fitting degree was high, R² was 0.999 1, and the cut-off value was 0.090 6. The intra- and inter-assay coefficient of variation was 1.17%-5.50% and 0.16%-4.78% indicating the good repeatability of established DAS-ELISA. Compared with a commercial kit, the coincidence rate among 54 samples was 100%. The DAS-ELISA detection method constructed in this study can be used for the quantitative evaluation of the intestinal permeability of crucian carp. In view of the conservatism of fish I-FABP, this method may be used for quantitative evaluation of intestinal permeability of other fish.