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