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目的 构建我国登革 2型病毒 43株基因组全长cDNA ,为进一步研究其体外RNA转录产物的感染性 ,阐明致病机理及探索新型疫苗奠定基础。方法 根据登革 2型病毒参考株NGC株的核苷酸序列 ,利用DNASTAR软件设计覆盖登革 2型病毒 43株基因组的 6对重叠引物。从感染登革 2型病毒 43株的乳鼠脑中提取病毒基因组RNA ,采用RT PCR分别扩增 6条基因片段 ,并将其分别与pGEM T载体进行连接。重组质粒用PCR进行快速鉴定 ,并在 377A型自动测序仪进行序列分析。然后利用单一酶切位点 ,分别自阳性重组子上切下各基因片段 ,在体外分别进行 5′半分子和 3′半分子的连接 ,最后将 5′和 3′半分子连接成基因组全长的cDNA。扩增各接头两侧长约 45 7～ 6 91bp的基因片段 ,连接至T载体后测序 ,从而对全长cDNA进行鉴定。结果 共扩增出 6条约 1 5～ 2 5kb的基因片段 ,并在体外进行连接 ,获得了全长cDNA。结论 通过测序证实成功地构建了我国登革 2型病毒 43株基因组全长cDNA分子。本研究结果将为阐明我国登革病毒株的毒力及致病机理奠定基础。
Objective To construct the full-length cDNA of dengue 2 virus in China, and lay the foundation for further study on the infectivity of RNA transcripts in vitro, elucidating the pathogenesis and exploring new vaccines. Methods According to the nucleotide sequence of dengue virus type 2 reference strain NGC, 6 pairs of overlapping primers covering 43 strains of dengue virus type 2 were designed by using DNASTAR software. The viral genomic RNA was extracted from the brains of 43 neonates infected with dengue virus type 2, and 6 gene fragments were amplified respectively by RT PCR and ligated with pGEM T vector respectively. The recombinant plasmids were rapidly identified by PCR and sequenced in a Model 377A automated sequencer. Then using a single restriction enzyme site, each gene fragment was excised from positive recombinants, 5 ’and 3’ half molecules were ligated in vitro, and finally the 5 ’and 3’ half molecules were ligated into the entire genome length CDNA. A total of 45 7 ~ 6 91bp DNA fragments were amplified on both sides of each linker, then ligated to T vector and sequenced to identify the full-length cDNA. Results A total of 6 gene fragments of ~ 15 ~ 25kb were amplified and ligated in vitro to obtain the full-length cDNA. Conclusion The full-length cDNA of 43 strains of dengue 2 virus was successfully constructed by sequencing. The results of this study will lay the foundation for elucidating the virulence and pathogenesis of Dengue virus strains in China.