鸡传染性法氏囊病毒南汇分离株cDNA探针的研制

采自上海郊县某发病鸡场的法氏囊病料 ,用单克隆抗体 (McAb )沉淀法从冻融的上清液提纯IBD病毒。用SDS 蛋白酶K法提纯核酸 ,得到高纯度的IBDVRNA后 ,再用特异性引物进行逆转录和PCR扩增 ,得到长度为 147bp的IBDVcDNA片段。将该片段回收、纯化和克隆 ,经测序鉴定 ,其结果与Genebank中同源率达 96 %。采用DIG非放射性标记系统 ,标记IBDVcDNA片段 ,得到足够量的探针 ,与不同毒株的IBDVRNA和送检病料RNA进行杂交 ,均有清晰斑点出现 ,其灵敏度为 0 1pg。

Preparation of DIG-labeled cDNA Probe for Infectious Bursal Disease Virus

The infectious bursal disease virus (IBDV) was purified with anti-IBDV monoclonal antibody,and the dsRNA of IBDV was extracted by SDS-protease K method. A pair of primers was designed to amplify a IBDV cDNA fragment of 147 bp by RT-PCR,and this cDNA fragment was inserted into the expression vector TG1 after purification. A clone was selected and identified by using of PCR technology. The result of cDNA sequencing showed that it was identified to that of Genbank. The fragment of IBDV cDNA was then labeled with non-radioactive DIG nucleic acid labeling and detection system. The labeled cDNA was used as a probe to perform hybridization with RNA from several IBDV isolates. 0.1 pg of IBDV RNA can be detected by this method.