摘要
目的分析水痘-带状疱疹病毒（VZV）疫苗株（vOka）ORF62碱基突变及ORF62编码即刻早期蛋白（IE62）对VZV基因启动子的反式激活力。方法分别以vOka及其亲本株（pOka）基因组DNA为模板，PCR扩增获得ORF62全序列，克隆到高效表达质粒pCAGGS中构建vOka和pOkaORF62表达质粒；采用双脱氧核苷酸链末端终止法对表达质粒中ORF62测序；应用基因转染瞬时表达技术比较vOka和pOkaIE62在MeWo和CV1细胞中对VzV基因启动子的反式激活力差别。结果成功构建vOka和pOkaORF62表达质粒pCAG-vOka62和pCAG-pOka62；测序结果发现，与pOka比较，vOkaORF62至少有9个碱基突变，其中106262、107136和107262位点碱基突变后分别产生SmaⅠ、BssHⅡ和NaeⅠ酶切位点。vOka IE62在MeWo细胞中对ORF4、ORF10和ORF61启动子的反式激活力低于pOka IE62，但在CV1细胞中对ORF9启动子的反式激活力高于pOka IE62。结论利用vOkaORF62碱基突变产生的酶切位点可鉴别vOka和pOka，vOka和pOkaIE62对VZV基因启动子的反式激活力差别可能与转染细胞类型有关。
Objective To analyze the point mutations in open reading frame 62（ORF62） of varicella-zoster virus（VZV） vaccine Oka（vOka） strain, and the transactivities of ORF62-encoding immediate early protein（IE62） on VZV promoters. Methods Complete ORF62 sequences of vOka and its parent Oka （pOka） strain were amplified by PCR, and then cloned into a highly efficient expressing plasmid pCAGGS, respectively, to construct vOka and pOka ORF62-expressing plasmids. The cloned ORF62 in the plasmids were then sequenced with a dideoxynucleotide chain termination method. Transactivities of vOka and pOka IE62 on VZV promoters were tested with transient transfection assays. Results vOka and pOka ORF62-expressing plasmids, pCAG-vOka62 and -pOka62, were successfully constructed. Compared with pOka, at least 9 point mutations were found in vOka ORF62, three of which resulted in new cutting sites of Sma Ⅰ , BssH Ⅱ , and Nae Ⅰ , respectively. Compared with pOka IE62, vOka IE62 had lower transactivities on ORF4, ORF10 and ORF61 promoters in MeWo cells, but higher transactivities on ORF9 promoter in CVI cells. Conclusion vOka ORF62 mutation-resulted enzyme cutting sites were useful to differentiate vOka from pOka. Differences between vOka and pOka IE62 in transactiviting VZV promoters might be cell-type dependent.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第10期953-957,共5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项目（闽教科2007年20号）
厦门市卫生局医学科研基金资助项目（WSK0618）
