摘要
为了建立重组鸭瘟病毒技术,构建了鸭瘟病毒转移质粒。在对鸭瘟强毒和弱毒株TK基因进行测序分析后,将鸭瘟病毒TK-UL24DNA片段克隆于pUC18载体中,构建了质粒pTK;将PCR扩增的GFP真核表达盒插入pTK质粒的TK基因内部,获得转移载体质粒pTK-GFP。鸭瘟病毒TK-UL24测序分析表明鸭瘟强、弱毒株TK基因序列完全相同;转移载体携带Pcmv-GFP-SV40pA表达盒,测序验证其序列与源序列一致。pTK-GFP在脂质体介导下,转染鸭胚成纤维细胞和鸭肾细胞,在荧光显微镜下观察绿色荧光蛋白表达情况。质粒转染细胞后,绿色荧光蛋白得到了有效的表达,为进一步开展重组鸭瘟病毒的研究和构建具有遗传标记的鸭瘟疫苗奠定了基础。
A transfer plasmid was constructed for the recombinant of duck enteritis virus（DEV）.Based on analysing of TK-UL24 DNA sequences of virulent and vaccine DEV strains,the TK-UL24 DNA segment from DEV was cloned into pUC18 to generate the pTK plasmid;An eukaryotic expression cassette containing the green fluorescene protein（GFP）gene was amplified by PCR and inserted into the TK gene in pTK plasmid to produce transfer vector pTK-GFP.The seqencing results showed that TK-UL24 sequence of the vaccine virus was identical to that of virulent strain.By restriction enzyme digestion and sequencing,it was proved that the transfer plasmid pTK-GFP included the DNA arms from DEV TK-UL24 segment and the expression cassette of Pcmv-GFP-SV40pA;The expression of GFP was observed with fluorescence microscope after the pTK-GFP was transfected into duck embryo fibroblasts（DEF）and duck kidney cells（DK）,indicating that the effective transfer plasmid was successfully constructed.The DEV transferring plasmid would be helpful for studying recombinant DEV and constructing new type of DEV vaccine virus with molecular marker.
出处
《动物医学进展》
CSCD
北大核心
2010年第3期25-29,共5页
Progress In Veterinary Medicine
基金
浙江省自然科学基金项目(Y306179)
关键词
鸭瘟病毒
TK基因
转移载体
Duck enteritis virus
TK gene
transfer plasmid
