恶性疟原虫Pf332分子DBL、TM和WR功能区蛋白质的转运分析

Analysis of the role of the DBL,TM,and WR domains in trafficking of the Pf332 protein of Plasmodium falciparum

目的分析恶性疟原虫Pf332分子的DBL、TM和WR 3个功能区蛋白质在虫体内的表达和分布以及与肌动蛋白的结合情况,探讨其在感染红细胞内的转运过程及功能,为进一步研究疟原虫侵入机制及筛选红内期疫苗候选抗原提供理论依据。方法将构建融有GFP基因的恶性疟原虫Pf332分子的Pf332DBL-GFP、Pf332TM-GFP和Pf332WR-GFP重组质粒分别转染到3D7虫株恶性疟原虫,通过活体荧光实时观察DBL、TM和WR蛋白质表达及其在染虫红细胞内的分布,运用间接免疫荧光共定位方法观察蛋白质DBL、TM和WR与肌动蛋白的结合情况。结果转染试验表明,重组DBL-GFP、TM-GFP和WR-GFP基因能在虫体中正常表达蛋白质,该蛋白均分布在染虫红细胞的纳虫泡内,但未能转运到红细胞的胞浆内。间接免疫荧光共定位表明恶性疟原虫Pf332分子的DBL、TM和WR等3个功能区蛋白质均未与肌动蛋白(β-actin)结合。结论 Pf332分子的DBL、TM和WR等3个功能区蛋白在单独表达后自身均不能发生跨膜反应,即不能通过虫体的纳虫泡膜。由此推测,Pf332分子在虫体内合成后很可能通过多个功能基团协同作用转运到红细胞膜部位。

Objectives To analyze the role of the DBL, TM, and WR domains in molecular translocation of the Pf332 protein of Plasrnodium falciparum and to determine their interaction with actin in infected erythrocytes in order to pro- vide a theoretical basis for further study of the mechanism of invasion by P. falciparum and to screen potential antigens for use in P. falciparurn vaccines. Method Eukaryotic expression vectors were constructed to contain genes encoding the DBL, TM, and WR domains of Pf332 protein fused with green fluorescent protein （GFP）. These vectors were trans- fected into red blood cells infected with P. falciparum strain 3D7. Real-time fluorescence monitoring was used to deter mine the expression and distribution of the three functional domains of Pf332 protein within the red blood cells. Co-locali- zation of the expressed DBL, TM, and WR proteins with actin was studied using immunofluorescenee with specific anti- aetin antibodies. Results Transfection experiments revealed that recombinant plasmids encoding the DBL, TM, and WR domains were expressed in RBCs infected with P. Jhlciparum, but the expressed proteins were all trapped in the parasitophorous vacuole and failed to reach the cytoplasm of the infected RBCs. Results of indirect immunofluorescence localization revealed that the DBL, TM, and WR domains proteins did not interact with aetin. Conclusion The separately expressed domains （DBL, TM, and WR） of Pf332 protein were not transported across the membrane of the parasitopho- rous vacuole. No co-localization of the fusion proteins with aetin was observed. Thus, data have demonstrated for the first time that transportation and translocation of Pf332 protein inside infected RBCs are likely mediated by several do- mains of the molecule acting in concert.