纤维顶球改造增强了人5型腺病毒载体对造血细胞的感染  

Fiber Knob ⁃ Modified Human Adenovirus Type 5 Vectors Enhance theTransduction of Hematopoietic Cells

作　　者：郭小娟[1] 梅玲玲 付鸿臣 邹小辉[1] 鲁茁壮[1] 洪涛[1] GUO Xiaojuan;MEI Lingling;FU Hongchen;ZOU Xiaohui;LU Zhuozhuang;HONG Tao(NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100052,China;School of Public Health and Management,Weifang Medical University,Weifang 261053,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会医学病毒和病毒病重点实验室,北京100052 [2]潍坊医学院公共卫生与管理学院,潍坊261053

出　　处：《病毒学报》2021年第5期1079-1088,共10页Chinese Journal of Virology

基　　金：国家科技重大专项(项目号:2018ZX10734404),题目:重要传染病病原标准化鉴定关键技术研究和参比库的建立。

摘　　要：基于人5型腺病毒(Human adenovirus type 5,HAdV⁃5)的腺病毒载体对造血细胞的基因转导效率低,将病毒fiber基因替换为HAdV⁃11p的同源基因F11p后,载体对造血细胞的感染效率增强。本研究拟在F11p纤维顶球(knob)结构域添加RGD4C多肽或HIV包膜糖蛋白(gp120)的V3结构域,观察重组HAdV⁃5对造血细胞感染效率的变化。在前期构建的pKAd5f11p153R⁃EPG腺病毒质粒基础上,结合限制性酶切和DNA组装技术,在F11p 153 aa后(knob AB loop,153位)、228位(FG loop)以及300位(IJ loop)插入RGD4C肽或者gp120的V3肽,构建了共6种重组腺病毒载体(F153RGD⁃EG、F228RGD⁃EG、F300RGD⁃EG、F153CV⁃EG、F228CV⁃EG和F300CV⁃EG),以fiber未改造的HAdV5⁃EG和改造为F11p的F11p⁃EG病毒作对照,观察了其对4种造血细胞系U937、K562、Jurkat和HL60以及人原代T细胞的感染效率。结果显示,对于U937细胞,当感染复数(MOI,vp/cell)为100时,HAdV5⁃EG感染效率最低,为2%;其次为F228CV⁃EG,感染率为45%;F300RGD⁃EG、F153CV⁃EG和F300CV⁃EG感染率为85%~90%;F153RGD⁃EG、F228RGD⁃EG高于阳性对照病毒F11p⁃EG,三者分别为99%、99%、95%。各病毒对于Jurkat细胞的感染率均较高,但HAdV5⁃EG明显低于F11p⁃EG、F153RGD⁃EG和F228RGD⁃EG,当MOI为100时分别为75%、93%、93%和96%。感染K562细胞的情况与U937细胞类似。各病毒对于HL60细胞感染效率最低,MOI为500时,F300RGD⁃EG和F300CV⁃EG的转导效率为28%和33%,是F11p⁃EG的10倍。对于人原代T细胞,F153RGD⁃EG和F228RGD⁃EG优于F11p⁃EG,当MOI为1000时,感染率分别为87%、90%和84%。研究结果表明,F11p knob插入RGD4C比单独F11p替换的HAdV⁃5对造血细胞的感染效率高,同时,本研究还发现HAdV⁃11p fiber knob的AB、FG或IJ loop可插入外源多肽,为腺病毒嗜向性改造增加了新靶点。Human adenovirus type 5(HAdV⁃5)⁃based vectors can barely transduce hematopoietic cells.The gene⁃transfer efficiency could be improved if a HAdV⁃11p fiber⁃pseudotyped HAdV⁃5 is used.We attempted to further increase the transduction of HAdV⁃5 to hematopoietic cells by inserting the RGD4C polypeptide or the V3 domain of glycoprotein(gp)120 of the human immunodeficiency virus(HIV)into the knob domain of pseudotyping fiber of HAdV⁃11p(F11p).The adenovirus plasmid pKAd5f11p153R⁃EPG was constructed previously,in which dual Cla I sites flanked the coding sequence of the fiber gene and an expression cassette of human EF1αpromoter controlling green fluorescent protein(EPG)was located in the deleted E1 region.Six recombinant viruses were generated by exchanging polymerase chain reaction(PCR)⁃amplified chimeric fiber genes with that inside pKAd5f11p153R⁃EPG using the restriction⁃assembly method.F153RGD⁃EG,F228RGD⁃EG and F300RGD⁃EG were the three viruses with RGD4C insertions behind the 153aa(AB loop),228aa(FG loop)and 300aa(IJ loop)of F11p protein;and F153CV⁃EG,F228CV⁃EG,and F300CV⁃EG were the rest three viruses with gp120 V3 insertions in the corresponding sites.HAdV5⁃EG was the HAdV⁃5 control virus carrying the original fiber,whereas F11p⁃EG was another HAdV⁃5 control virus carrying the pseudotyping fiber gene of HAdV⁃11p(F11p).Four hematopoietic cell lines(U937,K562,Jurkat and HL⁃60)and human primary T cells were employed to evaluate the gene⁃transfer efficiency.HAdV5⁃EG could barely infect U937 cells.Upon infection with F228CV⁃EG,F300RGD⁃EG,F153CV⁃EG,F300CV⁃EG,F11p⁃EG,F153RGD⁃EG and F228RGD⁃EG,at the multiplicity of infection(MOI;vp/cell)of 100,the percentage of green fluorescent protein⁃positive cells was 45%,85%,91%,91%,95%,99%and 99%,respectively.The results for K562 cells were similar to those for U937 cells.All vectors could transduce Jurkat cells effectively,but transduction of HL⁃60 cells was difficult.At an MOI of 500,the transduction efficien

关 键 词：人5型腺病毒(HAdV⁃5) 纤维顶球 造血细胞 

分 类 号：R373.9[医药卫生—病原生物学]