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兰州地区NDV的分离鉴定及M蛋白的原核表达
何智博,刘俊,胡永浩
0
(甘肃农业大学动物医学院,兰州 730070)
摘要:
为了分离出兰州地区鸡群新城疫病毒(NDV, Newcastle disease virus)并将M蛋白进行原核表达。通过鸡胚尿囊腔接种培养分离得到的兰州地区NDV,采用血凝及血凝抑制实验和Western bolt对病毒进行鉴定。利用分子克隆方法将NDV M蛋白进行原核表达,用Western blot技术分析其反应原性。根据NCBI发表的NDV M基因构建进化树。结果表明,成功分离的NDV悬液经RT-PCR技术扩增出M基因片段,重组菌pET-M经SDS-PAGE分析,在约42 kDa处有一明显的蛋白条带,Western blot技术证明其具有良好的反应原性。M基因进化树分析,分离毒株与La-Sota具有极高同源性。本研究成功分离出兰州地区NDV并将M蛋白进行原核表达,同时确定分离毒株与La-Sota毒株的同源性极高。为后续针对兰州地区新城疫流行病学调查和综合防控技术提供实验依据。
关键词:  NDV  血凝及血凝抑制  M蛋白  克隆  Western blot
DOI:10.13610/j.cnki.1672-352x.20160311.010
投稿时间:2015-08-05
基金项目:江苏省科技支撑计划—农业部分项目(BE2013315), 江苏省自然科学基金资助项目(BK2011184)和中央级公益性科研院所基本科研业务费专项(2015JBFM06)共同资助。
Identification of NDV in Lanzhou and prokaryotic expression of M protein
HE Zhibo,LIU Jun,HU Yonghao
(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070)
Abstract:
This study was to isolate Newcastle disease virus (NDV) of chickens in Lanzhou area and express M protein in prokaryotes. We isolated NDV through inoculated chick embryos and identified the virus using haemoglutination test, haemoglutination blocking test and Western bolt. We further used molecular cloning methods to express NDV M protein in prokaryotes and analyzed it’s reaction by Western blot. Phylogenetic tree was constructed based on NCBI published NDV M gene. The results showed that we successfully isolated the NDV and amplified the M gene by RT-PCR. The recombinant protein pET-M is about 42 kDa by SDS-PAGE analysis, and it has a great reactogenicity confirmed by Western blot. The isolated virus has a very high homology with La-Sota according to the phylogenetic analysis of the M gene. In conclusion, this study successfully isolated Lanzhou area NDV and achieved the prokaryotic expression of M protein and the isolated virus has a highly homology with La-Sota. It provides an experimental basis for the subsequent epidemiological investigation and comprehensive prevention control against NDV in Lanzhou area.
Key words:  NDV  hemagglutination and hemagglutination inhibition  M protein  cloning  Western blot

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