大劣按蚊差异表达蛋白与约氏疟原虫卵囊黑化关系的研究

Relationship of differentially expressed proteins in Anopheles dirus with Plasmodium yeolii oocyst melanization

目的 探讨大劣按蚊差异表达蛋白与约氏疟原虫卵囊黑化的相互关系。方法 将雌大劣按蚊成蚊分为吸糖水组、正常小白鼠血组和约氏疟原虫感染血组 ,以挤压法收集各组的血淋巴 ,解剖中肠并利用超声波提取其蛋白 ,继而对血淋巴和中肠蛋白进行SDS PAGE分离 ,Westernblot检测丝氨酸蛋白酶和前酚氧化酶的表达差异 ,并利用免疫组织化学进行血细胞丝氨酸蛋白酶与前酚氧化酶的定位检测。结果 SDS PAGE显示吸糖水大劣按蚊有 3 4条蛋白带 ,而吸正常小鼠血和吸感染血蚊血淋巴有 3 5条蛋白带 ,而且在分子量约 160× 10 3和 66× 10 3的蛋白差异带显著 ,吸正常血及感染血后表达增强。吸糖水和吸正常小白鼠血组血淋巴丝氨酸蛋白酶呈现 2条带 ,分子量分别约 160× 10 3和 15 0× 10 3,3组蚊血淋巴前酚氧化酶呈现 1条带 ,分子量约 66× 10 3,吸正常小鼠血和吸感染血组蚊 1d后血淋巴丝氨酸蛋白酶和前酚氧化酶表达均显著上调 ,尤其疟原虫感染后表达最强。感染 1d后中肠前酚氧化酶带型明显增宽。免疫酶化学显示 ,丝氨酸蛋白酶和前酚氧化酶在血细胞内均呈颗粒状分布 ,血细胞外血淋巴液内有少许散在颗粒分布。结论 约氏疟原虫卵囊黑化期间 ,大劣按蚊血淋巴丝氨酸蛋白酶和前酚氧化酶表达增强 ,提示二者可能参与了卵囊黑化包裹?

Objective To explore the relationship of the differentially expressed proteins in Anopheles dirus with the melanization of Plasmodium yoelii oocysts. Methods Anopheles dirus adult females were divided into sucrose solution feeding group (S), normal mouse blood feeding group (NB), and Plasmodium yoelii infected blood feeding group (IB), respectively. The hemolymph of Anopheles dirus in each group was collected by expulsion method. Tissues of the midguts of the Anopheles dirus from the three groups were collected and crushed by ultrasonic wave for protein extraction. The hemolymph and the midgut proteins were isolated by SDS polyacrylamide gel electrophoresis (SDA PAGE). The differentially expressed serine protease (SP) and the prophenoloxidase (PPO) were identified by Western blotting. Location of SP and PPO in the hemocytes was identified by immunohistochemical method. Results SDS PAGE revealed that there were 34 protein bands in mosquitoes in group S, 35 bands in NB and IB, respectively. The notably differential bands with molecular weight of about 160×10 3 and 66×10 3 were found. The two bands were up regulated after normal blood feeding and Plasmodium infection. There were 2 SP bands with molecular weight of 160×10 3 and 150×10 3 in S feeding and NB feeding mosquitoes. A single PPO band with molecular of about 66×10 3 was shown in the three groups. Enhanced expression levels of SP and PPO in the hemolymph of Anopheles dirus infected with Plasmodium yoelii were found at 1 d after feeding with normal mouse blood and Plasmodium infected blood. The strongest expression of the two enzymes was found in IB, but increased content of PPO in the midgut was found at 1 d after infection. Immunohistochemistry revealed that both SP and PPO were distributed as brown granules in the hemocytes, and a few particles were scattered in the hemolymph outside the hemocytes.Conclusion The enhanced expression levels of SP and PPO in the hemolymph of Anopheles dirus during the melanization of Plasmodium yoelii oocyst suggest that the two proteinases may be involved in the melanotic encapsulation of Plasmodium yoelii oocysts.