兔出血症病毒抗原捕获ELISA检测方法  

Development of an Antigen Capture ELISA for Detecting Rabbit Hemorrhagic Disease Virus

作　　者：秦海斌[1,2] 刘怀然[1] 刘家森[1] 姜骞[1] 韩凌霞[1] 司昌德[1] 曲连东[1] 

机构地区：[1]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 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《中国比较医学杂志》2007年第8期I0020-I0024,共5页Chinese Journal of Comparative Medicine

摘　　要：为了有效地预防和根除山羊痘，研制能够区分自然感染和疫苗免疫的标记疫苗。本研究在山羊痘病毒（goat pox virus，GPV）疫苗株AV41胸苷激酶（thymidine kinase，TK）基因克隆、鉴定的基础上，构建转移载体。将痘苗病毒（Vaccinia Virus，VV）晚期启动子P11和大肠杆菌β-半乳糖苷酶基因（LacZ）报告基因片段插入含有TK片段同源臂的载体pTK的KpnI酶切位点，经酶切、PCR鉴定出，命名为pTK-LacZ。用脂质体法转染感染了GPVAV41株的犊牛睾丸（bovine testes，BT）细胞，待出现80%以上的细胞病变时收获病毒，经过连续9代蓝色蚀斑筛选、纯化和PCR鉴定，获得纯化的表达LacZ基因、TK功能缺失的重组病毒，命名为rGPV-LacZ。生物学特性研究显示，LacZ基因的插入不影响重组病毒的增殖特性，其毒力、生长特性与亲本株相似，保持原有疫苗株的生长特性，而且在BT细胞和羔羊睾丸（lamb testes，LT）细胞连续传20代遗传性状很稳定。本研究筛选、纯化的重组病毒rGPV-LacZ可以区分自然感染和疫苗免疫动物，为进一步研制山羊痘病毒基因工程标记疫苗奠定了基础。An antigen-capture enzyme-linked immunosorbent assay （ELISA） for detecting rabbit hemorrhagic disease （RHD） virus was developed with a monoclonal antibodycapturing virus, while the rabbit anti-RHDV serum was used as the second antibody to identify virus. Working conditions of the Ag-ELISA were optimized and its capability was evaluated. In the present study, the optimum working concentration of McAb was 1μg/mL and that of rabbit anti-RHDV serum was 4 μg/mL. The Ag-ELISA displayed excellent specificity, sensitivity and repeatability. Using this test, 62.7% liver tissue samples from 67 probably infected rabbits had positive result, in contrast to 55.2% by HA test. Detection of five positive RHDV samples proved the highest dilution titer by ELISA was 3 - 13 times higher than that detected by HA test. The detection limit of this assay is 26 ng/mL to purified RHD virus. These results suggest that the Ag-capture ELISA is applicable to rapid diagnosis of RHD.

关 键 词：重组山羊痘病毒 标记疫苗 胸苷激酶基因(TK) β-半乳糖苷酶基因(LacZ) 

分 类 号：R-33[医药卫生]