1999年新分离的登革病毒福建株基因组非编码区的结构特征

Structure analysis of the sequences of the genome noncoding reg ions of dengue ty pe 2 virus Fujian strain isolated in 1999

目的 :测定和分析我国登革 2型病毒福建株 (FJ 11)基因组非编码区 (NCR)序列 ,为探讨其结构特征与功能的关系提供依据。方法 :从登革 2型病毒感染的C6/36细胞中提取总RNA ,采用RACE法扩增病毒基因组 5′和 3′端片段 ,分别将其克隆至pGEM T载体 ,挑取阳性克隆进行序列测定 ;利用RNAdraw软件对非编码区二级结构进行预测。结果与结论 :我国登革 2型病毒FJ 11株基因组 5′和 3′非编码区长度分别为 96nt和 4 5 4nt,具有黄病毒共有的保守序列和二级结构。与登革 2型病毒美洲基因型比较显示 ,5′NCR第 69位核苷酸的改变对其二级结构有影响 ;3′NCR端约 70nt能形成相同的保守结构 ,而前 380nt呈现多处核苷酸的差异而导致两者预测的二级结构差别较大 。

Objective: To determine and analyze the sequence s of the genome noncoding regions (NCR) of dengue type 2 virus Fujian strain (FJ 11) isolated in 1999 and to provide information about the relationship between viral NCRs and their functi o ns. Methods: With total RNA extracted from C6/36 cells infected by FJ11 virus s train, the specific fragments of viral 5~{!d~} and 3~{!d~} NCRs were amplified by RACE a nd cloned into pGEMT vectors, respectively.Positive clones were identified and t he inserted fragments were sequenced. The secondary structures of 5~{!d~} and 3~{!d~} NC R were predicted with RNAdraw software. Results and Conclusions: ~{!<~}WTBZ ~{!=~} The 5~{!d~} and 3~{!d~} N CRs of FJ11 strain are 96 and 454?nt in length,respectively and have the comm onl yconserved sequences and secondary structures of flaviviruses. Comparing with A merican genotype,the differences at nucleotide 69 of 5~{!d~} NCR had infl uence on the predicted secondary structure. A conserved region of 70?nt was fou n d at the 3~{!d~}terminus, which formed a characteristic stemloop structure. Howeve r, a striking size and sequence heterogenecity was observed in the 380?nt upstr eam re gion, which might be of great importance to the replication of dengue virus geno me RNA.