摘要
目的制备人4型腺病毒（Ad4）绿色荧光蛋白（EGFP）报告载体。方法以我们前期制备的Ad4．GZ01株全基因组载体pBRAd4为基础，用常规分子克隆的方法将基因组E3区缺失并插人EGFP表达框。转染AD293细胞拯救得到重组病毒．测序、电泳和ELISA等方法鉴定，纯化病毒免疫小鼠，ELISA和细胞微量中和试验等方法检测其免疫原性。结果成功获得携带EGFP报告基因的重组人4型腺病毒rAd4EGFP，其可以被Ad4特异性单抗及抗血清所识别和中和，免疫小鼠可以诱导抗Ad4抗体及抗EGFP特异性抗体。结论成功制备人4型腺病毒绿色荧光蛋白报告载体，可用于疫苗研发、药物评价、转基因载体等研究。
Objective To prepare human adenovirus type 4 （Ad4） vector expressing enhanced green fluorescence protein （EGFP）. Methods This study used a previously prepared plasmid pBRAd4 containing the whole genome DNA of Ad4-GZ01 strain. The Ad4 genome E3 region of pBRAd4 was deleted and replaced with the EGFP expression frame by conventional molecular cloning method. Then the recombi- nant plasmid was transfeeted into AD293 cells to rescue recombinant virus which was identified by sequen- cing, SDS-PAGE and ELISA. The purified virions were injected to mice and the induced immune responses were detected by ELISA and microneutralization test. Results The recombinant Ad4 vector rAd4EGFP ex- pressing EGFP was obtained and could be recognized and neutralized by monoelonal antibody MN4b and an- tisera against Ad4. The Ad4-specifie and EGFP-speeific antibodies with high titers could be detected in mice immunized with rAd4EGFP. Conclusion Human Ad4 vector expressing EGFP was successfully obtained and could be used in research on vaccine development, drug evaluation and transgene vector.
作者
田新贵
陈咏
樊晔
周志超
陈诗颖
刘文宽
周荣
Tian Xingui;Chen Yong;Fan Ye;Zhou Zhichao;Chen Shiying;Liu Wenkuan;Zhou Rong(State Key Laboratory of Respiratory Disease, Guangzhou Institution of Respiratory Health, the First Affiliated Hospital of Guangzhoa Medical University, Guangzhou Medical University, Guangzhou 510182, China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2018年第4期268-273,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金面上项目（31570163）
关键词
人4型腺病毒
载体
加强型绿色荧光蛋白
中和作用
Human adenovirus type 4
Vector
Enhanced green fluorescence protein
Neutralization
