克氏原螯虾Rab5、Rab6蛋白及其抗体对血淋巴细胞吞噬活性的影响

为研究克氏原螯虾(Procambarus clarkii)Rab5(PcRab5)和Rab6(PcRab6)的生物学功能, 利用同源重组技术构建了克氏原螯虾pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体, 并进行了诱导表达和多克隆抗体的制备, 采用ELISA和Western Blot技术检测了抗体效价和特异性。将PcRab5和PcRab6的原核表达蛋白和多克隆抗体注射到健康克氏原螯虾体内, 研究了其对血淋巴细胞吞噬活性的影响。实验结果表明构建的pET-B2m-PcRab5和pET-B2m-PcRab6原核表达载体经诱导后可表达目的蛋白, 分子量均为67 kD, 纯化后蛋白条带单一, 纯度较高。重组表达纯化后的PcRab5和PcRab6蛋白免疫日本大耳兔分别获得效价为1:2048 K和1:512 K的兔抗血清, 制备的抗体可分别特异性识别PcRab5和PcRab6蛋白。将PcRab5和PcRab6蛋白注射健康克氏原螯虾后, 其血淋巴细胞中可吞噬荧光微球的细胞比例分别显著上升至38%和30%(P＜0.01)。而将纯化的PcRab5和PcRab6多克隆抗体分别注射至螯虾体内后, 其血淋巴细胞的吞噬细胞比例均出现显著下降(P＜0.05), PcRab5和PcRab6蛋白参与了血淋巴细胞吞噬功能。实验为进一步研究克氏原螯虾PcRab5和PcRab6分子功能奠定基础, 也可为理解甲壳动物Rab5和Rab6在血淋巴细胞吞噬功能中的作用提供帮助。

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.