Denis Maghil, Mullaivanam Ramasamy Sivakumar, Thayappan Karthigayani, Munusamy Arumugam
Laboratory of Pathobiology, Department of Zoology, University of Madras, Chennai, Tamil Nadu 600025, India.
Laboratory of Pathobiology, Department of Zoology, University of Madras, Chennai, Tamil Nadu 600025, India.
Int J Biol Macromol. 2017 Nov;104(Pt A):1212-1222. doi: 10.1016/j.ijbiomac.2017.07.034. Epub 2017 Jul 8.
Sialic acid specific lectin Pjlec isolated from serum of the freshwater crab Paratelphusa jacquemontii served as an antigen for the production of immunoglobulin (Ig) in Balb/c mice sera. Enzyme-linked immunosorbent assay (ELISA) of mice anti-sera with Pjlec lectin affirmed the induction and production of antibody. Anti-Pjlec antibody was isolated from the antisera of mice by Protein A Sepharose affinity chromatography and checked for purity by immunoblot with lectin. Mass spectrometry (MS/MS) of papain digethe peptide sequence of antigen binding fragment (Fab) and fragment crystallizable (Fc). Coatingsted anti-Pjlec revealed of anti-Pjlec to the target cell, rabbit erythrocyte failed to enhance in vitro phagocytosis in the crab. However, inoculation of anti-Pjlec in the hemolymph of the crab elicited in vitro phagocytosis. Proteins in hemocyte lysate supernatant (HLS) were separated by electrophoresis failed to immunoblot with Pjlec or anti-Pjlec. Peptide sequences of trypsin digested lectin protein appeared homologous to deuterostome chordate. The protostome crab that lack the ability to synthesize sialic acid however bind to sialic acid a deuterostome sugar to suggest the complexity in innate immune system of invertebrates. The application of lectin and its antibody require further study on application of pathological conditions associated with alterations in sialylated cell surface.
从淡水蟹杰氏仿相手蟹（Paratelphusa jacquemontii）血清中分离出的唾液酸特异性凝集素Pjlec，作为抗原用于在Balb/c小鼠血清中产生免疫球蛋白（Ig）。用Pjlec凝集素对小鼠抗血清进行酶联免疫吸附测定（ELISA），证实了抗体的诱导和产生。通过蛋白A琼脂糖亲和层析从小鼠抗血清中分离出抗Pjlec抗体，并用凝集素免疫印迹法检测其纯度。对抗原结合片段（Fab）和可结晶片段（Fc）的木瓜蛋白酶消化产物进行质谱分析（MS/MS）。包被抗Pjlec抗体后，该抗体对靶细胞兔红细胞的体外吞噬作用未能增强。然而，将抗Pjlec抗体接种到蟹的血淋巴中可引发体外吞噬作用。血细胞裂解物上清液（HLS）中的蛋白质经电泳分离后，未能与Pjlec或抗Pjlec进行免疫印迹。胰蛋白酶消化的凝集素蛋白的肽序列与后口动物脊索动物同源。原口动物蟹缺乏合成唾液酸的能力，但却能与后口动物的糖唾液酸结合，这表明无脊椎动物先天免疫系统的复杂性。凝集素及其抗体的应用需要进一步研究与唾液酸化细胞表面改变相关的病理状况的应用。
