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  

Construction of Recombinant Herpesvirus of Turkeys Expressing PmpD-N of Chlamydia psittaci Based on CMV Promoter

作　　者：刘杉杉 孙伟[1] 张稳 贾长青[1,2] 罗杰 沈易华 郁建生[1,2] 何诚 LIU Shan-shan;SUN Wei;ZHANG Wen;JIA Chang-qing;LUO Jie;SHEN Yi-hua;YU Jian-sheng;HE Cheng(Tongren Polytechnic College,Tongren,Guizhou,554300,China;National and Local Engineering Research Centre for Separation and Purification Ethnic Chinese Veterinary Herbs,Tongren,Guizhou,554300,China;Key Lab of Animal Epidemiology and Zoonosis of Ministry of Agriculture,College of Veterinary Medicine,China Agricultural University,Beijing,100193,China)

机构地区：[1]铜仁职业技术学院,贵州铜仁554300 [2]民族中兽药分离纯化技术国家地方联合工程研究中心,贵州铜仁554300 [3]中国农业大学动物医学院,北京100193

出　　处：《动物医学进展》2018年第7期13-18,共6页Progress In Veterinary Medicine

基　　金：铜仁市科技计划项目(铜市科研[2016]17号-5);国家自然科学基金项目(1172305)

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 fragment 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 determined 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 constructed successfully,and a further research was needed to analyze the vaccine efficacy.

关 键 词：鹦鹉热衣原体 多型外膜蛋白D的N端 火鸡疱疹病毒 细菌人工染色体技术 巨细胞病毒启动子 

分 类 号：S852.67[农业科学—基础兽医学] Q789[农业科学—兽医学]