南方医科大学学报2016，Vol.36Issue(4)：582-587,6.
狨猴血清中重组人5型腺病毒中和抗体滴度的测定
Detection of neutralizing antibody to human adenovirus type 5 in marmosets
摘要
Abstract
Objective To construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines. Methods Luciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets. Results The shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×101 .5 PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32;flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16. Conclusion Chemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.关键词
普通棉耳狨猴/腺病毒5型/荧光素酶/绿色荧光蛋白/中和试验Key words
Callithrix jacchus/human adenovirus 5/luciferase/green fluorescent protein/neutralization experiment引用本文复制引用
孙亚纯,李婷婷,王一琳,张玲,朱海,黎诚耀..狨猴血清中重组人5型腺病毒中和抗体滴度的测定[J].南方医科大学学报,2016,36(4):582-587,6.基金项目
国家自然科学基金青年科学基金（31500134）；广州市产学研协同创新重大专项（201508020061）；广州市珠江科技新星专项（201506010075）；广东省教育厅青年创新人才类项目（2014KQNCX048）；南方医科大学优秀青年教师培育计划；深圳市食品安全快速检测技术工程中心（GCZX20140509163151273） Supported by National Natural Science Foundation of China (31500134) （31500134）
