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冷小茜, 叶欢, 杜浩, 李创举, 危起伟. 中华鲟Vtg间接竞争ELISA检测方法的建立和应用[J]. 水生生物学报, 2018, 42(4): 779-785. DOI: 10.7541/2018.095
引用本文: 冷小茜, 叶欢, 杜浩, 李创举, 危起伟. 中华鲟Vtg间接竞争ELISA检测方法的建立和应用[J]. 水生生物学报, 2018, 42(4): 779-785. DOI: 10.7541/2018.095
LENG Xiao-Qian, YE Huan, DU Hao, LI Chuang-Ju, WEI Qi-Wei. ESTABLISHMENT AND APPLICATION OF AN ELISA TECHNIQUE FOR DETECTING CHINESE STURGEON VITELLOGENIN[J]. ACTA HYDROBIOLOGICA SINICA, 2018, 42(4): 779-785. DOI: 10.7541/2018.095
Citation: LENG Xiao-Qian, YE Huan, DU Hao, LI Chuang-Ju, WEI Qi-Wei. ESTABLISHMENT AND APPLICATION OF AN ELISA TECHNIQUE FOR DETECTING CHINESE STURGEON VITELLOGENIN[J]. ACTA HYDROBIOLOGICA SINICA, 2018, 42(4): 779-785. DOI: 10.7541/2018.095

中华鲟Vtg间接竞争ELISA检测方法的建立和应用

ESTABLISHMENT AND APPLICATION OF AN ELISA TECHNIQUE FOR DETECTING CHINESE STURGEON VITELLOGENIN

  • 摘要: 卵黄蛋白原(Vitellogenin, Vtg)被认为是一种理想的雌激素和类雌激素标志物, 通过建立一种中华鲟Acipenser sinensis血浆Vtg水平的检测方法, 进而开发一项中华鲟性腺成熟度的诊断技术。首先通过RACE-PCR方法扩增得到中华鲟vtg基因cDNA序列, 氨基酸序列分析预测其蛋白分子量大小为196 kD。构建Vtg功能区段融合原核表达载体pET32a(+)-vtg并表达纯化重组蛋白, 并以重组蛋白免疫兔子获得多克隆抗血清, Western blotting检测显示抗血清的特异性较好。以纯化的中华鲟重组Vtg蛋白为抗原, 中华鲟Vtg多克隆抗血清为抗体, 建立了中华鲟血浆Vtg的间接竞争酶联免疫检测方法(ELISA), 标准曲线线性回归方程为y= –0.2916x+0.6794, 相关系数R2为0.9976。该方法检测的灵敏度为4.12 μg/mL, 最低检测限为0.3 μg/mL, 批内和批间变异系数分别为2.52%和3.42%。通过对不同发育时期雌性中华鲟血样检测, 表明此ELISA方法可初步用于雌性中华鲟性腺发育时期监测。

     

    Abstract: In this study, the cDNA sequence of Vitellogenin (Vtg) gene in Chinese sturgeon Acipenser sinensis was amplified with the rapid amplification of cDNA end (RACE) method, and its theoretical molecular mass is 196 kD. The antigenic site of the encoded amino acid sequence was cloned, which was used to construct recombinant plasmid and express recombinant protein. The polyclonal antiserum was obtained by immunizing rabbits with purified recombinant protein, and its specificity to Vtg was examined by western blotting. A competitive Enzyme-linked immunosorbent assay (ELISA) was established to detect Vtg in the serum of A. sinensis using antiserum of Vtg as antibody and the purified recombinant protein as antigen. The linear regression of the standard curve was y=–0.2916x+0.6794 (R2=0.9976). The sensitivity of this method was 4.12 μg/mL, and the lowest threshold for detection was 0.3 μg/mL. The inter- and intra-assay coefficients of variation was 2.52% (n=3) and 3.42% (n=3), respectively. The test results of blood sample for the female A. sinensis at different developmental stages showed that the method could be used to monitor the gonadal development of female Chinese sturgeon.

     

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