Cloning and analysis of partial cDNA sequence of prophenoloxidase 4 gene of Anopheles dirus / 第三军医大学学报

Objective To clone the partial cDNA sequence of prophenoloxidase (PPO) gene of Anopheles dirus . Methods Degenerative primers were designed according to the conserved sequence blocks within the prophenoloxidase of insects. RNA sequence of the larva of Anopheles dirus was amplified by RT PCR to get the prophenoloxidase cDNA which was then cloned into T vector and sequenced. The partial cDNA sequence of prophenoloxidase gene was analysed and compared with other prophenoloxidase gene of insects. Results The partial cDNA sequence of AdPPO4 was 597 bp, and its deduced amino acid sequence was 199aa. The cDNA sequence homology and amino acid sequence homology was 84% and 90%, respectively, as compared with the PPO4 gene of Anopheles gambiae . Conclusion The AdPPO4, with high sequence homology with the PPO4 gene of Anopheles gambiae , is successfully cloned from the larva of Anopheles dirus .

Changes of prophenoloxidase in the midguts of Anopheles stephensi and Anopheles dirus before and after infection with Plasmodium yoelii / 第三军医大学学报

Objective To investigate the relationship between the immune defence reaction against Plasmodium infection and the prophenoloxidase (PPO) of the midgut by comparative analysis of the distributions and the changes of PPO in the midgut of Anopheles stephensi and Anopheles dirus before and after infection with Plasmodium yoelii . Methods Midguts were dissected out from both Anopheles stephensi and Anopheles dirus at 3, 5, 7, 11 and 15 d before and after infection with Plasmodium yoelii . Immunohistochemistry and Western blotting were performed respectively on the collected midguts using Manduca Sexta PPO IgG polyantibody. Results PPO in the midguts from both Anopheles stephensi and Anopheles dirus was mainly located in the circulation conduit of midgut before infection with Plasmodium yoelii , but aggregated and distributed at the interspace of midguts as pieced or/and stripped forms after infection. Furthermore, PPO in the midgut of Anopheles dirus was more concentrated than that of Anopheles stephensi . Western blotting revealed that the PPO band with about molecular weight of 67?10 3 was detected in the midguts of both Anopheles stephensi and Anopheles dirus before and after Plasmodium yoelii infection. There was significant difference before and after infection, and the PPO band was obviously enhanced after infection. PPO bands in the midgut of Anopheles dirus were more prominent than those of Anopheles stephensi . Conclusion PPO in the midgut of Anopheles mosquitoes may come from the hemolymph by the circulation conduit before Plasmodium yoelii infection. However, the different distributions and changes of PPO in the midguts resulted from the Anopheles mosquitoes infected with Plasmodia may be closely correlated with Plasmodia infection, which may be of important physiological significance and may be involved in the immune defensive reaction against Plasmodium .