国产水痘减毒活疫苗株即刻早期基因ORF62的特征  

Characterization of immediate early gene ORF62 of live attenuated varicella vaccine Oka strains pro-duced in China

作　　者：吴宁俊[1] 廉成翔 黄灵[1] 卢珍玲[1] 王官清[2] 井上直樹 

机构地区：[1]厦门大学附属中山医院皮肤科,361004 [2]上海交通大学附属第一人民医院,201620 [3]日本岐阜药科大学微生物学和免疫学教研室

出　　处：《中华微生物学和免疫学杂志》2014年第6期417-422,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30671884,30840070）;福建省高校新世纪优秀人才支持计划资助项目（闽教科2007年20号）;厦门市科技计划资助项目（3502220134012）;厦门市医学科研基金资助项目（WSK0618）

摘　　要：目的：通过比较国产水痘减毒活疫苗株（ vOka ）与其父系株（ pOka ）即刻早期基因（ IE） ORF62启动子区序列及其启动活力、编码区序列及其反式激活力差别，探讨vOka株ORF62突变在其减毒机制中的可能作用。方法分别构建pOka株、长春百克生物科技股份公司vOka-BK株、上海生物制品研究所vOka-SH株和葛兰素史克公司vOka-GSK株（对照） ORF62启动子-报告子质粒和ORF62编码区表达质粒，通过测序分析ORF62启动子区和编码区序列差异，再应用基因转染瞬时表达技术分析3株vOka与pOka株ORF62启动子活力及ORF62编码IE62对即刻早期基因ORF4、早期基因ORF28和晚期基因ORF67启动子的反式激活力差异。结果与pOka株比较，3株vOka 一致存在ORF62启动子区110050位点T缺失突变，该突变未导致启动子区的转录因子结合基序改变，但3株vOka ORF62启动子的启动活力显著低于pOka株；3株vOka ORF62编码区存在一致的碱基置换突变并分别产生SmaⅠ、NaeⅠ和BssHⅡ新酶切位点；除vOka-SH株IE62在CV-1细胞中对ORF4启动子的反式激活力显著低于pOka株外，3株vOka IE62在CV-1和MeWo细胞中对ORF4、ORF28、ORF67启动子的反式激活力均显著高于pOka株。结论3株vOka ORF62启动子区110050位点T缺失突变可能是启动子活力降低和IE62反式激活力改变的原因之一，vOka与pOka株ORF62启动子活力和IE62反式激活力差异与转染细胞类型关系不大。Objective To investigate mutations in immediate early （ IE） gene ORF62 of three varicella vaccine Oka strains （ vOka ） including two strains produced in China and their parental Oka strain （pOka）, and then to further elucidate its possible roles in attenuation mechanism by comparing their ORF 62 promoter sequences and its activities , ORF62 coding regions and its transactivities .Methods ORF62 promoter-reporter plasmids and ORF62-expressing plasmids of pOka and three vOka strains （ vOka-BK from Changchun BCHT Biotechnology Co ., vOka-SH from Shanghai Institute of Biological Product Co .Ltd., and vOka-GSK from GlaxoSmithKline plc, as control） were constructed, respectively.ORF62 promoter regions and coding regions of the four strains were sequenced and then compared with each other .Differences of activities of the ORF62 promoter, and transactivities of the ORF62-encoded IE62 upon immediate early （ORF4）, early （ORF28） and late （ORF67） gene promoters between pOka and vOka strains were assayed with transient transfection technique .Results Compared with pOka strain , three vOka strains had a consistent T deletion mutation at site 110 050 in ORF62 promoters, which did not result in any change of tran-scription factor binding motif .However , activities of ORF62 promoters from three vOka strains were significantly lower than those of pOka strain .Three consistent substitution mutations were observed in ORF 62 coding regions of three vOka strains and three new enzyme restriction sites including SmaⅠ, NaeⅠand BssHⅡwere generated, respectively.Transactivities of IE62 from three vOka strains upon ORF4, ORF28 and ORF67 promoters were significantly higher than those of pOka both in CV-1 and MeWo cells , except that vOka-SH IE62 showed significantly lower transactivities upon ORF 4 promoter than those of pOka strain in CV-1 cells.Conclusion Consistent T deletion mutation at site 110 050 in ORF62 promoters of three vOka strains might be responsible for the reduced promoter activities and the

关 键 词：水痘-带状疱疹病毒 即刻早期基因62 启动活力 反式激活力 IMMEDIATE EARLY 62 

分 类 号：R392.1[医药卫生—免疫学]