中蜂囊状幼虫病毒结构蛋白VP2杆状病毒表达与间接ELISA方法建立  

Development of an Indirect ELISA Based on the VP2 Protein of the Chinese Sacbrood Virus Using the Baculovirus Expression System

作　　者：李明[1,2] 张琳 马鸣潇 侯振中 LI Ming;ZHANG Lin;MA Mingxiao;HOU Zhenzhong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;College of Animal Husbandry and Veterinary,Jinzhou Medical University,Jinzhou 121001,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]锦州医科大学畜牧兽医学院,锦州121001

出　　处：《病毒学报》2019年第2期264-269,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31772760);题目:中华蜜蜂囊状幼虫病毒编码siRNA及其RNAi抑制因子在免疫保护中作用~~

摘　　要：为了检测中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)抗体水平,用Bac-to-Bac杆状病毒表达系统,构建含有CSBV VP2的重组杆状病毒rBac-VP2,并在昆虫细胞中获得表达。用经过纯化的重组VP2蛋白作为包被抗原,建立CSBV抗体的间接酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)检测方法。结果表明成功的表达了VP2蛋白。优化反应条件后,建立的ELISA检测方法仅与CSBV阳性IgY发生特异性反应,与其它蜜蜂病毒的阳性IgY不发生交叉反应,且检测阳性IgY的灵敏度达到了1∶6 400,证明其具有良好的特异性和敏感性。批内重复与批间重复变异系数均小于10%。与CSBV全病毒包被进行比较,检测结果差异不显著,说明该方法适用于抗CSBV抗体检测。To determine the level of antibody against the Chinese Sacbrood Virus(CSBV),the recombinant baculovirus rBac‐CSBV VP2 gene was constructed.The expressed VP2 protein in insect cells was obtained using the Bac‐to‐Bac- baculovirus expression system.An indirect enzyme‐linked immunosorbent assay(ELISA) was developed to detect anti‐ CSBV antibody using the purified VP2 protein as the coating antigen.The VP2 protein was expressed successfully.After optimization of reaction conditions,ELISA was highly specific to im ‐ munoglobulin(Ig)Y of the CSBV with no evidence of cross‐reactivity with the IgY of other bee viruses.The sensitivity of detection of IgY reached 1∶6,400 dilution,which showed that ELISA had good specificity and sensitivity.The coefficients of variation within and between batches were <10%.Compared with the virus coating of the whole CSBV,there was no significant difference in detection.The described method appears to be suitable for the detection of anti‐CSBV antibody.

关 键 词：中蜂囊状幼虫病毒(CSBV) 结构蛋白VP2 Bac‐to‐Bac杆状病毒表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学] S895[农业科学—兽医学]