我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

Sequence Determination and Analysis of the 5′ and 3′ Terminal Regions of Two Dengue Type 2 Virus Strains Isolated in China

为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .

The 5′ and 3′ terminal sequences were determined in order to analyze the relationship between secondary structures and virulence of two dengue type 2 virus strains whose neurovirulence in suckling mice was different.Total RNA was isolated from C6/36 cells infected by D2\|04 and brains of suckling mice infected by D2\|44.With this RNA as template,the cDNAs of both 5′ and 3′ termini of D2\|04 and D2\|44 were amplified using RACE methods,respectively.The cDNAs were inserted into pGEM\|T vector and then the nucleotide sequences of the cDNA fragments inserted were determined.The secondary structures of 5′ and 3′ termini of D2\|04 were predicted with RNAdraw.The 5′ and 3′ untranslated regions of D2\|04 and D2 44 were 96 and 454 bases in length,respectively.A C to T change of 5′UTR that distinguished D2\|04 from D2\|44 at position 59 was predicted to change secondary structures.15 nucleotides of 3′UTR were different between D2\|04 and D2\|44.T to A(355) and T to G(326) that lay in CS1 and CS2A of 3′UTR were predicted to change secondary structures.The three sites may be relevant to virulence of dengue virus.