PROTEIN EXPRESSION OF RAB5 AND RAB6 IN PROCAMBARUS CLARKII AND ANTIBODY PREPARATION ON PHAGOCYTIC ACTIVITY OF HAEMOCYTES

Rab5 and Rab6 are important regulatory factors in regulating trafficking organelles, especially in phagosome formation. In our previous study, the Rab5 and Rab6 gene were cloned from Procambarus clarkii (PcRab5 and PcRab6), and they were proved involving in the immune response to pathogenic infection. In this study, homologous recombination technology was applied to produce prokaryotic expression protein of PcRab5 and PcRab6, then the expressed protein was purified and used to immunize rabbits for the preparation of polyclonal antibodies. The expression proteins and polyclonal antibodies of PcRab5 and PcRab6 were injected into Procambarus clarkii to study the effects on phagocytosis activity of haemocytes. The results showed that the constructed prokaryotic expression vector pET-B2m-Rab5 and pET-B2m-TGF-Rab6 were successfully induced to expression, and the molecular weight of rPcRab5 and rPcRab6 were both 67 kDa. The anti-PcRab5 and anti-PcRab6 antiserum with potency of 1:2048K and 512K were obtained from the immunized rabbits, respectively. In addition, the antibodies could specifically recognize the prokaryotically expressed protein, respectively. After the PcRab5 and PcRab6 protein were injected into healthy Procambarus clarkii, the phagocytosis rate of haemocytes increased to 38% and 30%, respectively (P<0.01). On the other hand, both the proportion of phagocytes in haemocytes decreased (P<0.05) after the purified Rab5 and Rab6 polyclonal antibodies which were injected into Procambarus clarkii, respectively. The above results indicated that the PcRab5 and PcRab6 prokaryotic proteins and polyclonal antibodies were prepared in this study, and it was proved that Rab5 and Rab6 were involved in the phagocytosis of haemocytes in Procambarus clarkii. The results of this study could lay a foundation for the further study of the molecular functions of Rab5 and Rab6 in Procambarus clarkii, and also help to understand the role of Rab5 and Rab6 during the phagocytosis of haemocytes in crustaceans.