检测家蝇抗菌肽基因diptericin时间表达模式的实时荧光定量方法的建立  

Establishing Real-time Quantitative PCR Assay for Detecting Expression Pattern of The Antimicrobial Peptide gene Diptericin from the Musca domestica

作　　者：杨小蓉[1] 金小宝[2] 朱家勇[2] 丁彩屏[1] 

机构地区：[1]广东药学院附属第一医院检验科,广东广州510080 [2]广东药学院病原生物研究所

出　　处：《现代预防医学》2012年第22期5893-5896,5900,共5页Modern Preventive Medicine

基　　金：国家自然科学基金(30672670)

摘　　要：目的建立荧光染料(SYBR Green I)实时荧光定量PCR(real-timePCR)方法检测的家蝇抗菌肽基因diptericin时间表达模式方法。方法基于已克隆出的diptericin基因,利用软件Prmier5.0设计上下游引物,对未诱导,诱导后2h、4h、8h、12h、24h、36h、48h及不同发育阶段的家蝇三龄幼虫进行real-timePCR设计引物,然后利用10倍系列稀释含有目的基因的质粒,进行实时荧光定量PCR反应,制作标准曲线。结果未刺激的三龄幼虫diptericin基因表达量非常低,与诱导后2h的相比,差1个数量级,诱导后2h开始缓慢上升,12h后达到最高峰,24h后缓慢下降,36h后回到诱后2h的水平,在家蝇三龄幼虫的卵、1龄及2龄期的表达量非常低,3龄期达到最高峰。结论实时荧光定量PCR检测家蝇抗菌肽基因diptericin的方法具有特异性好、灵敏度高,为进一步探讨家蝇抗菌肽生物活性,研究新的抗菌药物打下初步基础。OBJECTIVE To establish a fluorescent dye(SYBR Green I)real-time quantitative PCR(real-timePCR)to detect antimicrobial peptide gene diptericin expression patterns method from Musca domestica.METHODS Based on the cloned diptericin gene,software Prmier 5.0 was used for designing primers for non-induced,different developmental stages and different times(2h,4h,8h,12h,24h,36h and 48h)of Musca domestica were induced by real-time PCR.10 times of serial dilution of plasmid with target gene was used.Real-time PCR was used for standard curve.RESULTS The gene expression level that Uninduced third-instar was low compared with that induced.The greatest level of mRNA for all nine infection time was in 12 hours.After induced 2 hours,a significantly transcript level 107 was reflected.After 36 hours,mRNA levels were significantly descend.In different stages of development,the greatest level of mRNA for all six stages was in the 3rd-instars and adult.The lowest level of transcript detected was in the eggs and 1st-instars.A 102-fold increased in mRNA was revealed between the 3rd-and eggs,1st-,2nd-instar and pupae.Almost no change was found between the 3rd-instar and adult.CONCLUSION Real-time PCR detection method housefly gene diptericin specificity,high sensitivity,in order to further explore the biological activity of antimicrobial peptides housefly to the study of new antibiotics to lay the initial foundation.

关 键 词：家蝇 抗菌肽基因 diptericin 实时荧光PCR 表达模式 

分 类 号：R915[医药卫生—微生物与生化药学]