大劣按蚊抗约氏疟原虫感染相关cDNA文库的构建

CONSTRUCTION OF A DIFFERENTIALLY EXPRESSED cDNA LIBRARY OF ANOPHELES DIRUS AGAINST PLASMODIUM YOELII INFECTION

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.