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

作　　者：孙亚纯 李婷婷[1] 王一琳[1] 张玲[1] 朱海 黎诚耀[1] 

机构地区：[1]南方医科大学输血医学系,广东广州510515 [2]深圳市易瑞生物技术有限公司,广东深圳518101

出　　处：《南方医科大学学报》2016年第4期582-587,共6页Journal of Southern Medical University

基　　金：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)~~

摘　　要：目的构建表达荧光素酶与绿色荧光蛋白报告基因的重组人5型腺病毒r Ad5/Luc/GFP,检测血清中和抗体最佳实验方法,分析小型灵长类动物普通棉耳狨猴体内腺病毒5型中和抗体水平,为评价腺病毒载体疫苗提供实验动物基础数据。方法利用ADMax包装系统,首先构建穿梭质粒p DC315-Luc-GFP,将所得的穿梭质粒和骨架质粒p BHGloxΔE1,3Cre共转染293A细胞,进行重组人5型腺病毒(r Ad5/Luc/GFP)的包装。通过3次空斑形成实验筛选出单克隆重组腺病毒,并在大量扩增单克隆腺病毒后用Cs Cl密度梯度离心法进行浓缩纯化。采用TCID50法测定纯化后病毒滴度,并挑选进行中和实验的最优病毒滴度。将合适病毒滴度的r Ad5/Luc/GFP与狨猴血清混合预孵育后感染293A细胞,分别采用化学发光法及流式细胞术,对14只普通棉耳狨猴血清中腺病毒5型中和抗体进行检测。结果 PCR、酶切和测序结果均表明穿梭质粒p DC315-Luc-GFP构建成功;GFP检测结果证明重组人5型腺病毒r Ad5/Luc/GFP包装扩增成功,病毒滴度可达6.9×1011.5PFU/m L。两种不同方法检测检测14只狨猴的中和抗体结果一致性较好(P<0.05,Kappa=0.811),化学发光法或流式细胞术检测阳性狨猴比率分别为28.6%(4/14)或21.4%(3/14),化学发光法检测得其中2只狨猴中和抗体滴度为1/16,另2只为1/32,而流式细胞术检测得滴度均为1/16。结论化学发光法是一种快速,准确,适用于多物种的方法,其灵敏度略高于以GFP为报告基因的流式细胞术(7.2%)。普通棉耳狨猴体内Ad5血清阳性率较低,有望应用基于此型或其他型别腺病毒作为载体进行基因治疗和基因疫苗研究。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 

分 类 号：R373[医药卫生—病原生物学]