Gao Y, Hernandez V P, Fallon A M
Department of Entomology, University of Minnesota, 1980 Folwell Ave., St. Paul, MN 55108, USA.
Insect Mol Biol. 1999 Aug;8(3):311-8. doi: 10.1046/j.1365-2583.1999.83119.x.
An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7-10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12-36 h after induction.
一种埃及伊蚊蚊子细胞系Aag - 2，对免疫刺激的反应在性质上与来自相关蚊子白纹伊蚊的C7 - 10培养细胞相似。使用十二烷基硫酸钠聚丙烯酰胺凝胶，我们发现用热灭活的革兰氏阳性细菌处理生长中的细胞会优先诱导产生一种小肽。通过与其他研究类比，推测这种小肽是昆虫免疫肽防御素家族的一员。采用差异显示技术获得与诱导细胞中优先表达的mRNA相对应的部分聚合酶链反应产物。其中一个产物包含防御素基因的部分序列，用于筛选埃及伊蚊和白纹伊蚊细胞的cDNA文库。从埃及伊蚊细胞中，我们发现了两种先前描述的蚊子防御素A的同工型（A1和A4），以及一种新的同工型，我们将其定义为A5。从白纹伊蚊细胞中，我们发现了一种新的成熟蚊子防御素，命名为D，其最终氨基酸为脯氨酸和异亮氨酸。在埃及伊蚊和白纹伊蚊细胞系中，暴露于热灭活细菌后最早在3小时，Northern印迹上就可见防御素mRNA的表达，诱导后12 - 36小时防御素mRNA丰度最高。
