摘要
利用体外定点突变技术,缺失vvIBDV HLJ-0504株VP5基因,通过多重PCR在基因组两端分别引入锤头状核酶序列(HamRz)和丁肝病毒核酶序列(HdvRz)。将带有核酶序列的IBDV基因组插入载体pCAGGs的β肌动蛋白启动子下游,构建了IBDV感染性克隆pCAGGHLJ0504A△ VP5HRT,将该感染性克隆与pCAGGHLJ0504BHRT共转染DF-I细胞。IFA,RT-PCR,电镜观察均显示获得重组病毒,将其命名为rHLJ0504HT△VP5。vvIBDV VP5基因缺失感染性克隆的成功构建为我们利用反向遗传操作技术快速致弱vvIBDV,构建IBD的候选疫苗株奠定了基础。
The recombinant clone of vvlBDV（ very virulent Infectious bursal disease virus） HLJ-0504 lacking of VP5 gene was constructed by silencing the start codon （ATG-ATC） with site-directed mutagenesis. The full length cDNA of segment A was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme, which was introduced into a eukaryotie expression vector PCAGGs under the strong chicken 13 actin promoter. The recmrlbinant plasmid, designated as pCAGGHLJ0504A A VP5HRT, was co-transfeeted into DF-I cells with pCAGGHLJ0504BHRT. The recombinant virus rHLJ0504 A VP5 was successfully rescued, which was verified by RT-PCR, indirect immnuofluorescence assay and electron microscope. The rescued virus could be a very helpful platform for further construction of candidate vaccine strain.
出处
《中国动物传染病学报》
CAS
2010年第2期1-7,共7页
Chinese Journal of Animal Infectious Diseases
基金
国家"973项目"(2005CB523202)
国家自然科学基金(30901083)
中国博士后科学基金(20080440921)
关键词
传染性法氏囊病病毒超强毒株
VP5
感染性克隆
重组病毒
Very virulent Infectious bursal disease virus （vvIBDV）
viral protein 5
infectious clone
recombinant virus
