应用长链RT-PCR法扩增我国登革2、4型病毒株全长cDNA  

Amplification of full-length cDNA of dengue virus type 2 and 4 by long RT-PCR

作　　者：范宝昌[1] 赵卫[1] 胡志君[1] 陈水平[1] 于曼[1] 秦鄂德[1] 杨佩英[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《中华微生物学和免疫学杂志》2002年第5期485-488,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 (3 0 0 0 0 14 4)

摘　　要：目的　采用长链RT PCR技术扩增登革 2型及 4型病毒基因组全长cDNA ,为构建登革病毒全长cDNA克隆、表达 ,深入阐明致病机理及探索新型疫苗奠定基础。方法　根据已测定的登革2、4型病毒全基因组序列 ,设计上下游引物。从感染登革病毒的乳鼠脑中提取病毒基因组RNA ,采用长链RT PCR技术进行扩增。为检验扩增产物的特异性 ,以PCR产物为模板扩增覆盖基因组的 10个片段。将含有复杂二级结构的 5′非编码区的扩增片段 ,在 377A型自动测序仪进行序列分析。结果　扩增出登革 2、4型病毒基因组全长近 11kbcDNA分子 ,非编码区测序结果表明扩增产物为登革 2、4型病毒所特有。结论　利用长链RT PCR首次成功扩增出登革病毒全长cDNA分子。Objective Construction of full length cDNA clone with long RT PCR for elucidating the pathogenesis of dengue virus and developing novel vaccine. Methods According to the published nucleotide sequences of D2 43 and D4 B5 strains, the primers were devised. After extraction of virus RNA from infected brain of new born mouse, the approximate 11kb full length cDNAs of dengue virus type 2 and 4 were amplified by long RT PCR. With PCR products as model, 10 fragments that cover the whole genome were amplified. The PCR fragments which contain the 5′ noncoding region were cloned into pGEM T vector, and the sequences of inserted fragments were determined by PRISM TM ABI 377 automated sequencer. Results The full length cDNAs of dengue virus type 2 and 4 were amplified by long RT PCR and their correctness were proved by partial nucleotide sequences analysis. Conclusion The genomic cDNAs of dengue virus were successfully amplified. [

关 键 词：登革病毒 全长CDNA 长链RT-PCR法 发病机制 登革热 基因克隆 基因表达 

分 类 号：R346[医药卫生—基础医学]