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

作　　者：冷小茜 叶欢[1] 杜浩[1] 李创举[1] 危起伟[1] LENG Xiao-Qian;YE Huan;DU Hao;LI Chuang-Ju;WEI Qi-Wei(Key Laboratory of Freshwater Biodiversity Conservation,Ministry of Agriculture of China,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China)

机构地区：[1]中国水产科学研究院长江水产研究所,农业部淡水生物多样性保护重点实验室,武汉430223

出　　处：《水生生物学报》2018年第4期779-785,共7页Acta Hydrobiologica Sinica

基　　金：中国水产科学研究院基本科研业务费(2017JBF0102);国家重点基础研究发展计划(973计划,2015CB150702)资助。

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RACEPCR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 k D。构建Vtg功能区段融合原核表达载体p ET32a(+)-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R^2为0.9976。该方法检测的灵敏度为4.12μg/m L,最低检测限为0.3μg/m L,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。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 4.12μg/mL,and the lowest threshold for detection was 0.3μg/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.

关 键 词：中华鲟 卵黄蛋白原 ELISA 抗血清 

分 类 号：Q-31[生物学]