斯氏按蚊血淋巴前酚氧化酶差异表达与疟原虫卵囊黑化关系的研究

目的　探讨斯氏按蚊血淋巴前酚氧化酶与疟原虫卵囊黑化的相互关系。方法　斯氏按蚊分吸糖水组、吸正常小白鼠血组、吸约氏疟原虫感染血组和吸硝喹糖水组 ,挤压法收集雌斯氏按蚊血淋巴 ,解剖后取吸硝喹糖水蚊第 1、2和 3天斯氏按蚊中肠 ,超声提取中肠蛋白 ,Bradford法检测血淋巴总蛋白浓度 ,继而对血淋巴和中肠蛋白进行SDS -PAGE ,Western印迹电转移后检测前酚氧化酶。结果　四组血淋巴前酚氧化酶和用药后中肠前酚氧化酶均呈现一条阳性蛋白带 ,分子量分别均约 6 6kDa,与吸糖水组比较 ,吸正常小白鼠血组血淋巴前酚氧化酶表达增强 ,而感染后血淋巴前酚氧化酶表显著增强。硝喹诱导黑化后血淋巴前酚氧化酶表达水平呈逐日下调趋势 ,而硝喹诱导黑化过程中中肠前酚氧化酶量逐渐增加。结论　斯氏按蚊血淋巴前酚氧化酶参与疟原虫卵囊黑化反应。

Relationship between the differential expression of pro-phenoloxidase in the hemolymph of Anopheles stephensi and the melanization of Plasmodial oocysts

To explore the relationship between pro-phenoloxidase in the hemolymph of Anopheles stephensi and the melanization of Plasmodial oocysts,Anopheles stephensi mosquitoes were sorted out into sucrose solution-fed mosquitoes,normal-mouse-blood-fed mosquitoes,Plasmodial infected-blood-fed mosquitoes and nitroquine-adminstered mosquitoes,respectively. Hemolymph of adult female An.stephensi was collected by the expulsion method. Midguts were dissected out from Anopheles stephensi after the 1 st,2nd and 3rd day'administration. Protein from the midguts was abstracted by ultrosonic wave. The concentration of the total protein from the hemolymph and the midgut was examined with Bradford method and then was SDS-polyacrylamide gel electrophoresized. Different expressed pro-phenoloxidase was identified after electrotranferring by Western blotting.A single pro-phenoloxidase bind was shown in the hemolymph from the four groups of mosquitoes and in the midgut from the nitroquine-adminstered mosquitoes,in which molecular weight was all about 66kDa. But no significant difference was observed between the sucrose solution-fed mosquitoes and normal-mouse-blood-fed mosquitoes. Enhanced expression and marked increased expressionof pro-phenoloxidase in the hemolymph from the normal-mouse-blood-fed mosquitoes and infected-blood-fed mosquitoes were manifested respectively,compared with the succose solution-fed mosquitoes.Day-to-day down-regulated expression of pro-phenoloxidase in the hemolymph could be observed after melanization by the induction of nitroquine.But increased content of pro-phenoloxidase in the midgut was seen after the melanization by the induction of nitroquine.It concludes that pro-phenoloxidase in the hemolymph of An.stephensi is involved in the melanotic encapsulation of Plasmodial oocysts.