摘要
目的构建大劣按蚊抗约氏疟原虫感染相关cDNA文库,为进一步筛选、克隆按蚊免疫差异新基因奠定基础。方法以大劣按蚊-约氏疟原虫为实验模型,分别将饱吸约氏疟原虫感染鼠血和正常鼠血后24h的大劣按蚊作为T组和D组,采用抑制差减杂交方法和T/A克隆技术构建大劣按蚊抗约氏疟原虫感染相关cDNA文库。结果扩增差减cDNA文库获得2500余个白色阳性克隆,随机挑取白色克隆进行PCR扩增,92%的克隆中均有200~600bp的插入片段,测序显示,有与冈比亚按蚊未知功能基因同源的克隆。结论成功构建了大劣按蚊差异基因文库。
Objective To construct a differentially expressed cDNA library of Anopheles dirus, so as to lay solid foundation for screening and cloning new and specific innate immunity genes. Methods An. dirus infected with Plasmodium yoelii was used as Tester ( T ) group, while uninfected but normal blood fed as Driver ( D ) one. Engorged female mosquitos of two groups were collected separately at 24 hours after biting.The course of suppression subtractive hybridization (SSH) and T/A cloning techniques were used to construct a differentially expressed cDNA library of An. dirus. Results The amplified library contained more than 2 500 positive bacteria clones. Random analysis of 40 clones with colony PCR method showed that 92% clones contained 200-600 bp inserts, these inserts might be the cDNA fragments of differentially expressed genes of infected An. dirus. The BLAST search revealed that some genes were homologous to functionally unknown Anopheles gambiae EST. Conclusion A differentially expressed genes subtracted cDNA library of infected An. dirus is successfully constructed.
出处
《中国寄生虫病防治杂志》
CSCD
2005年第3期169-171,共3页
Chinese Journal of Parasitic Disease Control
基金
国家自然科学基金资助项目(No.30200237)。
关键词
大劣按蚊
疟原虫
约氏
抑制差减杂交
基因文库
Anopheles dirus
Plasmodium yoelii
suppression substractive hybridiztion
library