小鹅瘟病毒VP3基因疫苗的构建及其诱导小鼠和鹅中和抗体的初报  

Construction of goose pavovirus VP3 gene vaccine and preliminary report on its elicitation of neutralizing antibodies in mice and geese

作　　者：韩新锋[1] 程安春[1,2] 汪铭书[1,2] 刘晓东[1] 卢菲[1] 黎敏[1] 车茜[1] 

机构地区：[1]四川农业大学动物科技学院禽病防治研究中心,雅安625014 [2]动物疫病与人类健康四川省重点实验室,雅安625014

出　　处：《高技术通讯》2008年第5期543-549,共7页Chinese High Technology Letters

基　　金：国家'十一五'科技支撑计划(2007Z06-017);教育部'新世纪优秀人才支持计划'(NCET-04-0906/NCET-04-0818);四川省重大基础研究(2006J13-049/2008JY0100);四川省重点建设学科(SZD0418)资助项目

摘　　要：PCR 扩增小鹅瘟病毒(GPV)CHv 株 VP3基因并克隆入 pMD 18-T-Simple 载体,亚克隆正向插入到真核表达质粒 pcDNA3.1(+)CMV 启动子下游多克隆位点,经 PCR 和HindⅢ与 BamH Ⅰ双酶切鉴定表明成功构建了 GPV VP3基因疫苗(pcDNA3.1-GPV-VP3)。该疫苗以200μg/只肌注免疫 Balb/c 小鼠和鹅,同时分别设肌注 PBS 和 pcDNA3.1(+)作为对照,于免疫后第30、45、60、75、90、105天应用鸭胚原代成纤维细胞(DEF)进行微量中和试验检测小鼠和鹅血清抗100 TCID50 GPV 的抗体效价。结果表明,pcDNA3.1-GPV-VP3免疫小鼠第30天可检测到抗 GPV 的中和抗体,抗体效价缓慢上升至90天达到高峰(平均1:135.8)后下降,105天仍高于75天;免疫鹅的中和抗体效价呈现与小鼠类似的规律,第90天达到高峰(平均1:98.5)。因此,pcDNA3.1-GPV-VP3具有良好的免疫原性,肌注免疫小鼠和鹅后能够诱发产生抗 GPV 的中和抗体,有进一步开展临床研究和应用的前景。VP3 gene of goose parvovirus （GPV） CHv strain was amplified by PCR and cloned into pMD18-T-Simple vector, then sub-cloned into the multiple cloning sites of the eukaryotic expression plasmid pcDNA3.1 （ ＋ ） under the control of CMV, and the identification of PCR and digestion with Hind In and BamH 1 proved the GPV VP3 gene vaccine （pc DNA3.1-GPV-VP3） was constructed successfully. Firstly the gene vaccines were inoculated intramuscularly into Balb/c mice and geese with the dose of 200μg each animal, then the neutralizing antibodies against 100TCIDso GPV were detected with duck embryo fibroblast （DEF） cells as indicators at 30, 45, 60, 75, 90, 105 days after immunization, and the mice and geese injected with PBS and pcDNA3.1 （ ＋ ） were set as controls. The neutralizing antibodies were detected 30 days after immunization, and virus neutralizing titers of mice averaged 1 ： 135.8 climbed the peak 90 days after immunization, then they went down; the virus neutralization titers at 105 days were higher than 75 days after immunization. The variation of geese neutralizing antibodies were similar as mice, the virus neutralization titers averaged 98.5 climbed the peak 90 days after immunization. The results showed that pcDNA3.1-GPV-VP3 possesses a good immunogenicity and could elicit anti-GPV neutralizing antibodies in mice and geese. Its further clinical applications should be developed in later studies.

关 键 词：小鹅瘟病毒 VP3基因 真核表达质粒 基因免疫 中和抗体 

分 类 号：S852.5[农业科学—基础兽医学]