棉铃虫中肠cDNA表达文库的免疫筛选及其克隆分析

Immunoscreening and Sequencely Organization of the Helicoverpa armigera cDNA Expression Library for cDNA Clones

昆虫围食膜是保护其中肠的一道物理屏障,破坏围食膜就可能使其中肠处于不正常的生理状态而最终导致死亡。本研究提取棉铃虫Helicoverpa armigera(Hbner)围食膜蛋白,成功制备棉铃虫围食膜蛋白多克隆抗体,并对棉铃虫中肠cDNA表达文库进行免疫筛选,共得到385个阳性克隆。对其中26个克隆进行鉴定并测序分析,结果表明19个克隆为肠粘蛋白。过碘酸-希夫试剂(PAS)显色法检测棉铃虫围食膜蛋白组成,发现围食膜蛋白大部分为糖蛋白。通过对棉铃虫中肠围食膜蛋白的分离鉴定,为进一步分析棉铃虫围食膜蛋白的生理功能,寻找生物防治新靶标,开发新型高效生物杀虫剂提供理论依据。

The peritrophic matrix （PM）is a physical barrier protecting the midgut. Destruction of peritrophic membrane can make the midgut in an abnormal physiological state, eventually leading to death. Helicoverpa armigera （Htibner）larval midgut eDNA expression library was immunoscreened with an anti-PM proteins polyclonal antiserum which has been successfully prepared by immuning a rabbit,385 positive clones were obtained from several screening. 26 purified eDNA clones of them were chosen for sequencing. The results showed that 19 cDNAs of the above 26 eD- NA clones encoded insect intestinal mucin （IIM）. The PM protein composition was then analyzed, and the result showed that there were a lot of glycopreteins by PAS staining analysis. By isolating and identifying the PM proteins of Helicoverpa armigera ,we could get a clear idea of the physiological function of PM protein. We could also hunt for no- vel target site in insect pest management and explore high efficient biological pesticide.