我国登革2型病毒43株基因组全长cDNA的构建

Construction of the full-length cDNA of dengue type 2 virus isolated in China

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 Chinese strain 43 of dengue 2 virus, and thus to set up basis for investigating the infectivity of its in vitro RNA transcript, elucidating the mechanism of pathogenesis of dengue virus infection, and developing novel vaccine against dengue. Methods Using the software DNASTAR, we devised six pairs of over lapping primers which cover the whole genome of strain 43, according to the nucleotide sequence of international standard strain NGC. After extracting the RNA of virus from the infected brain tissue of the new born mice, we amplified six cDNA fragments of D2 43 strain by reverse transcription PCR. The cDNA fragments were cloned into vector pGEM T and then transformed into competent E.coli DH5α cells. Positive clones were screened by PCR and restriction enzyme digestion. The sequences of inserted fragments were determined by PRISM TM ABI 377 automated sequencer. Then the inserted cDNA fragments were cleaved down from the positive recombinants using unique endonuclease, ligated into the 5′ and 3′ halves of the genome cDNA in vitro , respectively, and then constructed a full length cDNA by ligation. The constructed full length cDNA was identified by PCR,which amplified the fragments spanning the ligation sites about 457 691bp, and the nucleotide sequence of the amplified fragments was determined by automated sequencer. Results Using RT PCR, six cDNA fragments, covering the whole genome of Chinese strain dengue virus 2, were amplified and ligated into the full length cDNA, which was proved by their nucleotide sequences. Conclusion The sequencing demonstrates that the full length of genome cDNA molecule of Chinese strain 43 of dengue 2 virus has been constructed successfully. The results obtained from this tesearch have set up the basis for illustrating the mechanisms of pathogenicity and virulence of Chinese strain 43 of dengue 2 virus.