ITR缺陷对AAV病毒包装与感染力的影响  

Influence of Defect ITR on The Packaging and Infectivity of AAV

作　　者：曹佐武[1] 林羿[2] 程龙球[2] 邹飞雁[1] 

机构地区：[1]暨南大学生殖免疫研究所,广州510632 [2]暨南大学生命科学技术学院,广州510632

出　　处：《生物化学与生物物理进展》2008年第2期224-230,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金项目(30271370和30672231)~~

摘　　要：腺相关病毒(AAV)是一种复制缺陷性DNA病毒,其基因组两端的两个倒转末端重复序列(ITR)是rAAV包装复制所必需的自身结构.ITR容易缺失并影响病毒颗粒的包装和感染力.比较两个ITR完整的和一端ITR缺失的AAV病毒发现,一端ITR缺失的AAV载体质粒包装病毒的效率明显比ITR完整的质粒低.用这两种AAV病毒感染293细胞、HeLa细胞和小细胞肺癌细胞NCI H446细胞,显示两个ITR完整的AAV病毒感染细胞的能力明显比一个ITR缺失的病毒强.说明ITR缺失对AAV病毒的包装和感染力都有明显的影响.因此,筛选两个ITR完整的AAV载体质粒,并稳定其基因组的结构,对提高病毒生产的产量和增强病毒的感染力都有显著的价值.The inverted terminal repeat （ITR） is the only cis element of AAV genome essential for rAAV rescue, replication and packaging. It is prone to mutation or loss when it is latent in host cell or in plasmid. Plasmids with different ITR types were cloned to compare the influence of ITR types on the AAV packaging and infectivity. The vector plasmids were transformed the competent SURE cells to get different colonies. The ITR types of plasmids were screened by digestion with Sma I . AAV vector plasmid pScGFPud has two ITRs at both ends of AAV genome and plasmid pScGFPu has only one ITR at upstream end of AAV genome. When the two plasmids were co-transfected 293 cells to prepare rAAVs, 1.08×10m^3 viral particles （AAV1-GFPud） were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPud, 4.28 ×10m^12 viral particles （AAV1-GFPu） were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPu. Virus AAV1-GFPud infected 293, HeLa and NCI H446 cells more efficiently than did virus AAV1-GFPu. This suggests that defect ITRs in AAV genome is deleterious to AAV packaging and AAV infectivity and vector with complete ITRs is favorable to the yield and activity of rAAV.

关 键 词：腺相关病毒 倒转末端重复序列 病毒包装 

分 类 号：R394.8[医药卫生—医学遗传学] Q311.8[医药卫生—基础医学]