鸭甲型肝炎病毒1型抗体CLEIA检测方法的建立  

Development of chemiluminescent enzyme immunoassay for detection of antibodies against duck hepatitis A virus type 1

作　　者：银凤桂 侯力丹[3] 魏艳秋[2] 杨宝芝[1] 李芸[2] 李晶[2] 张爽[2] 刘文军[2] 杨利敏[2] 

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]中国科学院微生物研究所病原微生物与免疫学重点实验室,北京100101 [3]中国兽医药品监察所,北京100081

出　　处：《中国预防兽医学报》2017年第6期451-455,460,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划"畜禽重大疫病防控与高效安全养殖综合技术研发"重点专项(2016YFD0500800);中国科学院重点部署项目(ZDRW-ZS-2016-4-4;KSZD-EW-Z-005-001)

摘　　要：为建立一种快速检测鸭甲肝病毒1型(DHAV-1)抗体的化学发光酶联免疫分析方法(CLEIA),本研究从DHAV-1中国流行株中扩增获得病毒结构蛋白VP1编码基因,构建重组原核表达质粒pET-32a-VP1,并进行重组蛋白的诱导表达。将纯化后的重组VP1蛋白作为检测抗原建立了CLEIA检测方法。结果显示,利用建立的CLEIA方法对AIV、AMPV、DPV、ARV、MPV、RAV等病毒血清进行检测,均无交叉反应,特异性强;灵敏度高于ELISA方法;重复性试验变异系数为2.18%~4.33%,具有较好的重复性;且与中和试验的阳性符合率和阴性符合率分别为100%和80%。本研究建立的CLEIA方法可以用于DHAV-1感染监测及疫苗接种后的免疫评价。A novel chemiluminescent enzyme immunoassay （CLEIA） was developed to detect antibodies against duck hepatitis A virus type 1（DHAV-1）. The VP1 gene fragment of DHAV-1 was amplified by RT-PCR using DHAV-1 RNA as the template. Recombinant expression plasmid pET-32a-VP1 was constructed, and the induced recombinant protein VP1 （rVP1） was purified using Nickel affinity chromatography. The CLEIA method was established using the purified rVP1 as testing antigen. Meanwhile, its specificity, sensitivity, repeatability and coincidence rate with the neutralization test were evaluated, the results showed that the rVP1 had no crossing reaction with the antibodies to AIV, AMPV, DPV, ARV, MPV, RAV, and the coefficient of variation of the repeatability experiment was 2.18% to 4.33%, indicating that the CLEIA method had high specificity and repeatability. Moreover, CLEIA was more sensitive than ELISA method. Compared with the serum neutralization test, the positive and negative coincidence rate was calculated as 100% and 80%, respectively. The CLEIA method could be used for infection monitoring and immune evaluation after vaccination with DHAV-1.

关 键 词：鸭病毒性肝炎 VP1 化学发光酶联免疫 

分 类 号：S852.65[农业科学—基础兽医学]