基于CMV启动子表达鹦鹉热衣原体PmpD-N蛋白的重组火鸡疱疹病毒的构建

摘要

为构建表达鹦鹉热衣原体多型外膜蛋白 D的 N端(PmpD-N)蛋白重组火鸡疱疹病毒(HVT),以鹦鹉热衣原体CB7 株DNA为模板,通过PCR扩增pmpD-N基因,构建带有巨细胞病毒(CMV)启动子的真核表达盒,采用细菌人工染色体技术构建 HVT重组子,转染细胞获得 HVT重组病毒,并在体外评价该重组病毒的相关特性.结果表明,转染细胞后成功获得 HVT重组病毒.通过 Western blot和 IFA分析表明,重组病毒表达了 PmpD-N 蛋白.Confocal 分析表明,PmpD-N 蛋白分布在细胞的胞核、胞浆和细胞表面. HVT重组病毒的复制特性和野生型 HVT一致. HVT重组病毒能够激活巨噬细胞促炎性细胞因子IL-6 和NO的产生.该试验成功构建了含有CMV启动子,能够表达PmpD-N蛋白的 HVT重组病毒,为下一步进行动物试验和分析该病毒的免疫效力奠定了基础.%In order to construct recombinant herpesvirus of turkeys (HVT)expressing the N-terminal frag-ment of PmpD (PmpD-N)of Chlamydia psittaci ,the pmpD-N gene was amplified from C.psittaci CB7 strain by PCR and the expressing cassette was constructed which containing the Cytomegalovirus (CMV) promoter.Recombinant HVT was generated by modifying the HVT genome within a bacterial artificial chromosome based on CMV promoter.Then,the characterization was evaluated in vitro .The recombinant virus with CMV promoter was recovered.The expression of PmpD-N of the recombinant virus was deter-mined by Western blot and IFA.Under immunofluorescence microscopy,PmpD-N protein was located in the nucleus,cytoplasm and on the cell surface.Growth kinetics of recombinant HVT was comparable to that of parental HVT.Macrophages could be activated to produce macrophage proinflammatory cytokine IL-6 and NO.In this study,a recombinant HVT expressing PmpD-N based on CMV promoter was con-structed successfully,and a further research was needed to analyze the vaccine efficacy.