SSH技术筛选家蝇抗感染差异基因(英文)  

SSH screening on Musca domestica differential genes of defense against infection

作　　者：王艳[1] 金小宝[2] 朱家勇[2] 

机构地区：[1]广东药学院医药化工学院,中山528458 [2]广东药学院药物活性物质省级重点实验室,广州510006

出　　处：《中国人兽共患病学报》2014年第5期499-506,共8页Chinese Journal of Zoonoses

基　　金：Supported by grant from the National Natural Science Foundation of China(No.30671832);the Key Science and Technology Program of Guangzhou City(No.2005Z32E0211)~~

摘　　要：目的 为了解家蝇抗菌分子免疫机制及效应免疫分子的表达情况,运用ssh、real-time PCR,构建家蝇差异表达消减文库.方法 以金黄色葡萄球菌（S.aureus）、大肠杆菌（E.coli）和球孢白僵菌（Beauvoir bassinet bacteria）,感染家蝇幼虫构建金黄色葡萄球菌、大肠杆菌和球孢白僵菌感染的差减文库,成功构建了家蝇抗感染差异表达cDNA文库.结果 文库的阳性克隆进行PCR鉴定主要分布在250～750 bp之间,随机挑取252个白斑进行测序和同源性分析,应用反向Northern斑点杂交技术鉴定了35个基因,其中32个为真阳性,包括抗菌肽、酶、核糖体蛋白,以及一些功能不明的蛋白.结论 运用real-time PCR技术分析了三种蛋白基因的表达情况,结果显示诱导后不同时间点,不同发育阶段抗菌肽均普遍表达,但表达水平存在着明显的差异,诱导后表达明显升高.In order to identify immune-related genes in housefly （Musca domestica） larvae,suppression subtractive hybridization （SSH） was performed to generate subtracted cDNA libraries after bacteria-challenge by Staphylococcus aureus （S.aureus）,Escherichia coli （E.coli） and Beauvoir bassinet bacteria.Differentially screening was performed using the reverse northern dot blot method for further verification.Of 252 positive clones were obtained; identification of the inserted cD-NA fragments in subtractive library was done by using PCR.The results showed that there were inserted fragments of 250-750bp,which would provide useful baseline for the screening and cloning of specific anti-infection genes of immunity in Musca domestica.Quantitative real-time PCR was used to study the gene expression of AMPs as examples of immune-relevant mole-cules.It＇s suggested that the antibacterial peptide genes expression of Musca domestica is constitutive,and they are expressed at different development stages of Musca domestica,but the expressing levels are evidently different.

关 键 词：家蝇 抗感染免疫 抑制性消减杂交 实时定量荧光PCR 

分 类 号：R374[医药卫生—病原生物学]