马立克氏病毒meq基因缺失株SC9-1通过自然重组获得meq能力的分析

Capacity of the Meq-deleted Strain Marek's Virus SC9-1 to Acquire the Meq Gene by Natural Recombination

为了探究meq基因缺失的马立克氏病毒疫苗株SC9-1与超强毒株Md5是否能够通过自然重组获得meq基因的能力,将SC9-1疫苗毒和Md5超强毒共同感染鸡胚成纤维细胞(CEF),并在CEF上连续传三代,提取单个蚀斑的病毒DNA。同时将Md5超强毒接种免疫过SC9-1疫苗株的SPF鸡,在不同的时间点分离病毒,提取单个蚀斑的病毒DNA。将两种方式获得的病毒DNA进行PCR验证,并将香啤酒重组酶位点(FRT)残留序列克隆测序,比较其同源性。两种方式鉴定的病毒均为SC9-1或是Md5,没有检测到重组病毒,而且FRT残留序列同源性为100%。结果 SC9-1没有从野生毒株Md5获得缺失的meq基因,而且meq基因敲除区具有很好的遗传稳定性。

We wished to explore the ability of the meq-deleted Marek＇s disease virus （MDV） vaccine strain SC9-1 to acquire the rneq gene from the MDV wild strain Md5 by recombination. Chicken embryo fibroblast ceils （CEFs） were co-infected with the SC9-1 vaccine virus and Md5 virus, passaged to third generation, and viral DNA was extracted from a single plaque in the cell culture. Specific pathogen-free chickens pre-immunized with the SC9-1 vaccine virus were infected with the Md5 virus. Viruses were isolated from chickens at different time points. Then, viral DNA was extracted from a single plaque and amplification by polymerase chain reaction done to identify isolated viruses. The flip recombinase sites （FRT） residue region was cloned and sequenced. Results showed that the isolated viruses in cultured CEFs or in chickens were the SC9-1 or Md5 virus, and recombinant viruses were not detected. Sequence analyses revealed that the homology of the FRT residue sequence between the isolated virus and parent virus was 100~/00. Therefore, there is little chance that SC9-1 can acquire the meq gene from Md5 by natural recombination. Also, the meq-gene knockout region had good genetic stability during serial passages in vivo and in vitro.