登革2型病毒NGC株感染白纹伊蚊C6/36细胞中浓核病毒DNA的扩增和序列测定  

Amplification and Sequencing of Densonucleosis Virus DNA in C6/36 Cell Line of Aedes albopictus Infected by Strain NGC of Dengue Virus Type 2

作　　者：王娇[1,2] 左丽[1] 周永兵[1,3] 

机构地区：[1]贵阳医学院免疫学教研室,贵州贵阳550004 [2]贵州省中心血站 [3]江苏省镇江市第一人民医院

出　　处：《贵阳医学院学报》2008年第6期597-599,603,共4页Journal of Guiyang Medical College

基　　金：973计划前期研究专项项目(2008CB517408);国家自然科学基金资助项目(30360101)

摘　　要：目的:探索白纹伊蚊C6/36细胞对登革2型病毒(Dengue Type 2 virus,DEN2)不敏感的原因。方法:根据Genbank提供的DEN2新几内亚株(NGC株)NS1基因序列设计引物,设立病毒对照组、病毒DNA酶(DNase)处理组和细胞对照组,通过提取核酸、逆转录-聚合酶链反应(RT-PCR)、限制性内切酶(HindⅢ)酶切、1.5%琼脂糖凝胶电泳检测RT-PCR及其酶切产物,并对其扩增产物进行序列测定,用软件比对与白纹伊蚊C6/36细胞浓核病毒(Densonucleosis virus,DNV)基因的同源性。结果:经RT-PCR,细胞和病毒组均出现约1300bp的条带,不能被HindⅢ酶切,而病毒DNase处理组未能扩增出此条带;经GenBank比对,该片段序列与白纹伊蚊C6/36细胞DNV部分序列的同源性达95%以上。结论:C6/36细胞对DEN2不敏感的原因是由于伊蚊C6/36DNV的污染。Objective： To study the reason of insensitiveness of C6/36 cell line in Aedes albopictus to type 2 of dengue virus （DEN2）. Methods： The primers were designed according to the sequence of NS1 gene of dengue virus strain NGC. Three groups of C6/36 cell line were set up ： virus control group （ group A）, virus DNA enzyme （DNase） treated group （ group B）, and cell control group （ group C ）. Nucleic acid of C6/36 cell line was isolated, and RT-PCR, restriction enzyme（Hind Ⅲ）digestion, electrophoresis analysis with 0.8% agarosegels were carried out on it. The retrieval products were sequenced. The homology of the obtained nucleotide sequences with the Densonucleosis virus （DNV）in C6/36 cell line of Aedes albopictus was compared using the PHYLIP software. Results： A band of about 1 300 bp appeared in groups A and B, and it couldnt be cut by Hind Ⅲ. This band could not beamplified from group B. The comparison analysis result showed that the obtained nucleotide sequence had a homology of 95% with part of DNV sequence of C6/36 in Aedes albopictus. Conclusions： Aedes albopictus densonucleosis virus contamination might be the reason for the insensitiveness of C6/36 cell line to type 2 of Dengue virus.

关 键 词：登革热病毒 伊蚊属 白纹伊蚊C6/36细胞浓核病毒 逆转录聚合酶链反应 DNA序列 

分 类 号：R373.33[医药卫生—病原生物学]