贵州医科大学学报

2008, No.135(06) 597-599+603

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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

王娇;左丽;周永兵;
WANG Jiao,ZUO Li,ZHOU Yongbing(Department of immunology,Guiyang Medical College,Guiyang 550004,Guizhou,China)

摘要(Abstract):

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 couldn't 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(KeyWords): 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
dengue virus; Aedes; densonucleosis virus in C6/36 cell of Aedes albopictus; reverse transcriptase polymerase chain reaction; DNA order

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 王娇;左丽;周永兵;
WANG Jiao,ZUO Li,ZHOU Yongbing(Department of immunology,Guiyang Medical College,Guiyang 550004,Guizhou,China)

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DOI: 10.19367/j.cnki.1000-2707.2008.06.010

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