登革2型病毒广东流行株结构蛋白基因序列测定及分析

Sequencing and structural analysis of structural gene of three dengue virus type 2 isolated from Guangdong province

目的 对广东省 90年代以来流行的 3株登革 2型病毒 (DEN2 )的结构蛋白基因序列测定及分析 ,了解流行株之间的相互关系、变异及基因型。方法 应用RT PCR技术扩增广东省不同年份流行的 3株DEN2型病毒的结构蛋白基因 (C、PrM、E基因 )。分别克隆到pMD18T载体 ,转化JM10 9宿主菌 ,挑取阳性克隆进行鉴定及序列测定。结果 3株DEN2病毒结构蛋白基因序列长度均为2 32 5bp ,编码 775个氨基酸。三者核苷酸 (氨基酸 )的同源性分别是 :GD0 6 93与GD19 2 0 0 1为 96 %( 97%)、GD0 6 93与GD0 8 98为 94%( 97%)、GD0 8 98与GD19 2 0 0 1为 92 %( 94%)。其在相关毒力位点E383～ 385处均为GLU PRO GLY、E12 6处均为GLU。 3株DEN2与国际参考株比较表明 :GD0 6 93与GD19 2 0 0 1和澳大利亚TSV0 1株共享序列非常接近 ,核苷酸 (氨基酸 )的同源性为 98%( 98%) ;GD0 8 98与泰国株ThNH P2 8 93核苷酸 (氨基酸 )同源性为 98%( 98%)。此 3株DEN2二级结构与对乳鼠不致病的 0 4株比较 ,主要差别位于其 393～ 40 0氨基酸处 ,本室分离的 3株对乳鼠致病毒株为EEEEHHHH ,而 0 4株为EEEE ;337～ 343处本室分离的 3株对乳鼠致病毒株为HH HHHHH ,而 0 4株为 HHHHE ,恰好位于Dengue病毒E基因的Ⅲ区。结论 GD0 6 93?

Objective To study the interrelation, variation and origin of three strains of dengue virus prevailed in Guangdong province since 1990 by sequencing and structural analysis of their structural genes. Methods According to the genome of dengue virus type 2 NGC strain, two pairs of primers were designed. The structural protein genes (C, PrM, E) of three strains of dengue virus type 2 isolated from different years were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), then cloned into pMD18-T vector and sequenced, respectively. Results The structural gene of each of three strains of dengue virus was constituted by 2325 nucleotides encoding 775 amino acids. The nucleotide (amino acid) homologies were 96% (97%), 94% (97%) and 92% (94%) between GD06/93 and GD19/2001, GD06/93 and GD08/98, and GD08/98 and GD19/2001, respectively. In three strains of dengue virus, the amino acids at the virulence related site E383-385 were all GLU-PRO-GLY, and at E126 were all GLU. GD06/93 and GD19/2001 showed a high homology with international standard strain TSV01 (Australia), the nucleotide (amino acid) homology was 98% (98%). Meanwhile strain GD08/98 showed a highest homology with international standard strain ThNH-P28/93 (Thailand), the nucleotide (amino acid) homology was 98%(98%). Compared with the non-neurovirulence of strain 04 of dengue virus type 2, the variance of amino acid second structure mainly situated at 393-400 and 337-343 sites, 3 isolates in this laboratory were EEEEHHHH and HH-------HHHHH,while strain 04 was EEEE----and --------HHHHE. Conclusion GD06/93, GD19/2001 and TSV01 may belong to the same genotype; GD08/98 and ThNH-P28/93 may belong to another genotype. Compared with the strain 04 of dengue virus type 2, the second structures of 3 strains of dengue virus isolated from Guangdong province in the Ⅲ domain of E protein show a notable variance, these variances may be related with the virulence of different strains of dengue virus. [