摘要
为了建立新城疫疫苗株Mukteswar株的反向遗传操作系统,根据Mukteswar株的基因序列设计了9对引物,扩增获得基因组片段,通过Overlap PCR和In-Fusion无缝克隆的技术,将9个片段拼接和插入至pACNR-T7中,获得了Mukteswar株全长cDNA克隆质粒pAC-Mukteswar。将其和3个辅助质粒(pVAX-NP、pVAX-P和pcDNA-L)共转染至预先感染了痘病毒vTF7-3的BHK-21细胞,成功拯救出重组病毒rMukteswar。对rMukteswar的生长曲线、最小致死剂量的平均致死时间(MDT)等生物学特性进行测定,结果显示rMukteswar具有和野生株相似的增殖特性和毒力。成功建立了新城疫疫苗株Mukteswar的反向遗传操作系统,为研发高效率表达外源病原体蛋白的新城疫载体疫苗奠定了基础。
In order to establish the reverse genetic system of Newcastle disease vaccine strain Mukteswar,nine pairs of primers were designed according to the gene sequence of Mukteswar strain,and nine fragments were obtained by PCR amplification.The nine fragments were fused and inserted into vector pACNR-T7,and the full-length cDNA cloning plasmid pAC-Mukteswar of Mukteswar strain was obtained.pAC-Mukteswar was co-transfected with three supporting plasmids(pVAX-NP,pVAX-P and pcDNA-L)into BHK-21 cells pre-inoculated with vTF7-3,and the recombinant virus rMukteswar was successfully rescued from BHK-21 cells.The results of the biological characterization showed that rMukteswar had the same proliferation characteristics and virulence as its parental strain.In conclusion,the reverse genetic system of the vaccine strain Mukteswar was successfully established,which was valuable for future developing of Newcastle disease vector vaccines with with high efficiency expression of exogenous pathogen proteins.
作者
魏家阳
李丽
王国康
徐英英
曾哲
罗青平
邵华斌
温国元
商雨
潘兹书
WEI Jia-yang;LI Li;WANG Guo-kang;XU Ying-ying;ZENG Zhe;LUO Qing-ping;SHAO Hua-bin;WEN Guo-yuan;SHANG Yu;PAN Zi-shu(College of Life Science,Wuhan University,Wuhan,Hubei,430072,China;Key Laboratory of Prevention and Control Agents for Animal Bacteriosis,Ministry of Agriculture and Rural Affairs&Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology,Institute of Animal Husbandry and Veterinary,Hubei Academy of Agricultural Sciences,Wuhan,Hubei,430064)
出处
《动物医学进展》
北大核心
2023年第2期30-35,共6页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31873018)
国家现代农业产业技术体系建设专项资金(CARS-41-G13)
湖北省自然科学基金重点类项目(2021CFA019)
湖北省科技创新项目重大专项(2020ABA016)
湖北省农业科技创新中心重大科技研发(2020-620-000-002-01)。
