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2007年7月至11月,中国开产前后的商品海兰褐蛋鸡群大面积暴发血管瘤,造成巨大经济损失。将病料接种DF1细胞,通过PCR和间接免疫荧光(IFA)确定此次暴发的血管瘤为J亚群白血病病毒(ALV-J)感染引起。从患病鸡群中分离到5株ALV-J(前4株已经报道),将第5株病毒命名为WS0705。为研究该毒株抗原性的特点,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示WS0705与英国ALV-J原型毒株HPRS-103同源性最高。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacH Tb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kD的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中得到良好的表达,并且其编码产物完全可以被外源性ALV-J的特异性单抗JE9识别,进一步证明本次暴发血管瘤的病原为ALV-J,并为进一步开发ALV-J相关诊断产品奠定了基础。
Abstract：During July to November in 2007,several outbreaks of Hemangiomas in Hy-line Brown laying hens were observed in China.The virus that infected these flocks was identified in cultured DF-1 cells by PCR and indirect fluorescent assay(IFA) with ALV-J specific monoclonal antibody JE-9.The gp85 gene of one strain named WS0705 of ALV-J was cloned and expressed.Phylogenetic analysis showed that gp85 amino sequences of WS0705 strain had the highest homology with that of the prototype HPRS-103.The gp85 gene from a constructed plasmid pMD18-T-WS0705gp85 was cloned into baculovirus transfer vector.rBac-WS0705gp85 was obtained by the Bac-to-Bac baculovirus expression system.The rBac-WS0705gp85 protein was analyzed by indirect immunofluor escence assay and Western blot.The results showed that positive green fluorescent was present in Sf9 cells infected with the recombinant virus and a 35 kD band was present in western blot.It is concluded that WS0705 gp85 gene was expressed in Sf9 cells infected with the recombinant virus and the SU protein of WS0705 can bind specifically to JE9 MAb of ALV-J.The expressed protein can be used to detect hemangiomas induced by ALV-J.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002157
中图分类号:S852.65
引用信息:
[1]邱波,刘功振,王晓伟,等.诱发血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达[J].病毒学报,2011,27(02):151-157.DOI:10.13242/j.cnki.bingduxuebao.002157.
基金信息:
国家自然科学基金(30871856); 教育部高等学校科技创新工程重点项目; 山东省自然科学基金(Y2007D46)
2011-03-15
2011-03-15