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ORFV-Yulin株的分离鉴定及其B2L基因在昆虫杆状病毒系统中的表达
冯 平,李云章,孙丰廷,等
作者单位
冯 平 内蒙古农业大学 兽医学院;榆林学院 生命科学学院 
李云章 内蒙古农业大学 兽医学院 
孙丰廷,等 武汉中拓康明生物科技有限公司 
摘要:
【目的】从榆林市具有典型羊口疮症状的陕北白绒山羊唇结痂病料中分离羊口疮病毒(Orf virus,ORFV),克隆分析其B2L基因序列,并通过昆虫杆状病毒表达B2L基因。【方法】利用牛肾传代细胞进行ORFV的分离培养,通过PCR的方法从分离到的病毒中扩增出B2L全长基因,测序后进行序列及遗传进化分析。利用扩增所得的B2L基因构建昆虫杆状病毒表达载体,包装出杆状病毒后侵染sf9细胞,并通过SDS-PAGE、Western-blot以及质谱鉴定等方法验证目的基因的表达情况。【结果】成功分离出1株羊口疮病毒,命名为ORFV-Yulin株;扩增的B2L全长基因长度为1 137 bp,与Hub13和GY-AHF10株亲缘关系最近,核苷酸序列和氨基酸序列的相似性分别为99%和99.2%。通过昆虫杆状病毒表达系统成功表达了B2L基因,获得了47 ku的重组蛋白。【结论】从榆林市陕北白绒山羊中获得了羊口疮病毒分离株(ORFV-Yulin);通过昆虫杆状病毒表达系统成功表达了ORFV-Yulin株的B2L基因。
关键词:  羊口疮病毒  B2L基因  昆虫杆状病毒表达系统
DOI:
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基金项目:陕西省科技厅农业科技创新与攻关项目(2016NY-120);陕西省科技厅重大统筹项目(2014KTDZ02-01);陕西省教育厅专项科研计划项目(14JK1860);榆林学院重点项目(14YK41)
Isolation and identification of ORFV-Yulin strain and expression of its B2L gene in baculovirus system
FENG Ping,LI Yunzhang,SUN Fengting,et al
Abstract:
【Objective】This paper isolated Orf virus (ORFV) from the lip scab of a lamb with typical orf symptoms in white cashmere goat from northern Shaanxi,analyzed the full length gene of ORFV B2L amplified,and expressed its full-length gene in baculovirus system.【Method】The ORFV strain was isolated with Madin Darby bovine kidney cell.At the same time,the full-length gene of ORFV B2L was amplified with specific primers,and its homology of amino acid and nucleotide was analyzed while the gene was analyzed by genetic evolution.The baculovirus expression vector was constructed using the amplified B2L gene to infect sf9 cells.The expressed product was identified by SDS-PAGE,mass spectrometry and Western blot.【Result】A ORFV strain,named as ORFV-Yulin,was successfully isolated.The length of B2L gene was 1 137 bp,and it was closely related to Hub13 and GY-AHF10 strains.The corresponding homologies of nucleotide sequence and amino acid sequence were 99% and 99.2%,respectively.The recombinant protein with 47 ku was obtained in baculovirus expression system. 【Conclusion】The B2L gene of ORFV-Yulin isolated from the northern Shaanxi white cashmere goat can be expressed through baculovirus expression system. 
Key words:  Orf virus (ORFV)  B2L gene  baculovirus expression system