昆虫细胞表达兔出血症VP60蛋白间接ELISA方法建立及评价  

Establishment of indirect ELISA assay with recombinant VP60 protein expressed in insect cell for rabbit hemorrhagic disease and evaluation of its properties

作　　者：刘怀然[1,2] 曲连东[2] 刘家森[2] 单安山[1] 

机构地区：[1]东北农业大学动物科学技术学院,哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所、农业部实验动物质量监督检验测试中心,哈尔滨150001

出　　处：《东北农业大学学报》2010年第8期81-88,共8页Journal of Northeast Agricultural University

基　　金：黑龙江省博士后资金项目

摘　　要：采用Bac-to-Bac系统表达的兔出血症病毒（RHDV）结构蛋白VP60作为包被抗原,建立抗体检测间接ELISA方法,并对方法性能进行测定。试验以整合有VP60基因的重组杆状病毒接种Sf9昆虫细胞,感染细胞经裂解、离心初步纯化,作为包被抗原建立了检测RHD抗体的间接ELISA方法。经过对反应条件和试剂的优化选择,初步组装成间接ELISA试剂盒,并对其整体性能进行了评价。结果表明,含重组VP60蛋白的Sf9细胞裂解液最适包被稀释度为1：2000,换算为纯化VP60蛋白含量约为1.7μg·mL^-1;待检血清最适稀释度为1：100;羊抗兔IgGHRP标记抗体最适使用浓度为1：30000。初步组装的ELISA试剂盒检测RHD阴性血清无假阳性反应,与其他常见兔病阳性血清无交叉反应,特异性良好;敏感性高于RHDV全病毒间接ELISA和血凝抑制试验,低于进口间接ELISA试剂盒;试剂盒批内和批间变异率在10%以内。37℃加速破坏试验和4℃保存期试验结果表明试剂盒保存期可以稳定在6个月。该方法可应用于兔出血症抗体水平监测及实验兔等级检测中。To establish the indirect ELISA method for rabbit hemorrhagic disease virus （RHDV） antibody detection, the VP60 protein expressed in Bac-to-Bac system was used as coating antigen. In this test, Sf9 cells were inoculated with recombinant baculovirus, the cell lysate that containing the recombinant VP60 protein was used as the indirect ELISA coating antigen after initial purification by schizolysis and centrifugation. The indirect ELISA assay was established by reagents selection and reaction conditions opitimation. Tests were performed to evaluate the ELISA system after assembly into kits initially. The results showed that the opitimal dilution of Sf9 cells lysate was 1： 2 000, which equal to 1.7 μg·mL^-1 of purification recombinant VP60 protein, the opitimal dilution of the srea samples and goat anti-rabbit HRP labelled antibody was 1： 100 and 1： 30 000, respectively. The indirect ELISA kit has a proper specificity by no false positive reaction with RHD negative sera and no cross reaction with other rabbit disease sera. Itssensitivity was higer than RHDV coating indirect ELISA and Hemagglutinin Inhibition （HI） assay, and lower than the indirect ELISA kit fro m INGENASA company; the variability coefficient of intra-assay and inter-assay to the kits within 10%. 37 ℃ acceleration destroy and 4 ℃ conserve period assays showed that the stable conserve period of kits could last for 6 months. The indirect ELISA assay could be applied in antibody detection and laboratory rabbits quality monitoring to RHD.

关 键 词：兔出血症病毒 VP60基因 昆虫细胞 间接ELISA 

分 类 号：S852.61[农业科学—基础兽医学]