Studies On Cloning And Expression Of The Duck Yolk Sac Membrane IgY Fc Receptor Gene

Ducks maternal antibodies can be vertically transmitted to the hatched ducklings through the yolk sac membrane and they would play an important role in immune response. Among all the egg yolk antibodies, IgY is the most affluent. It is the only maternal antibody immunoglobulin isotype which can be transferred to newly hatched ducklings. We speculate that the duck yolk sac membrane IgY receptor can transfer IgY. According to the previous researches, both receptors of mammalian FcRn and chicken FcRy were found to have the characteristic of pH-dependent which means the antibody would bind under acidic conditions and dissociate under alkaline conditions.The research contents are summarized as follows:1. Purification of FcRy from yolk sac membraneUsing the method of bitterness-ammonium sulfate and water soluble dilution, cruded extracts from duck egg yolk antibodies were acquired.The purified duck IgY heavy chain was67KDa and light chain was25KDa.Using duck IgY-affinity chromatography, duck FcRy was purified by adjusting pH values and the molecular weight was180KDa.2. Cloning and sequence analysis of the full-length FcRy geneTotal RNA was extracted from24-25days old Cherry Valley ducks yolk sac membrane. Five segments of the complete gene were amplified and sequenced. The complete sequence was6293bp and the nucleotide homology was99%when compared with Anasplatyrhynchos phospholipase A2receptor1.3. Subcloning, expression and purification of duck FcRy-CF geneFcRy-CF gene was amplified and cloned into the prokaryotic expression vectors pGEX-KG and pET-32a (+). The target proteins were successfully expressed. The GST-CF and His-CF fusion protein was39KDa and33KDa, separately. And the two proteins were purified from the single band.4. Preparation, purification and identification of rabbit anti-duck FcRy polyclonal antibodyPurified fusion protein His-CF was used to immune rabbit, rabbit anti-duck FcRy serum was successfully prepared. By the indirect ELISA assay, the titer of the antiserum against duck FcRy was as high as1:16000.Using antigen affinity chromatography hyperimmune serum was purified. The specific activity of the polyclonal antibodies were confirmed by Western blot.5.Expression of FcRy protein in duck organs and tissues was detected by Western blotWestern blot analysis for detecting FcRy on duck organs and tissues was established by using rabbit anti-duck FcRy-CF serum.The results indicated that FcRy was expressed in the tissues of duck ovarian, fallopian tube, trachea, lung, liver,duodenum, ileum and kidney.6. Immunohistochemistry was used to detect duck FcRy expression and distribution in vivoImmunohistochemistry method for FcRy detection in duck was established with the purified rabbit anti-duck FcRy polyclonal antibody. The results showed that FcRy was expressed in the tissues of ovary, oviduct, duodenum, lleum, liver and lung, and the main positive signals were observed in ovarian capsule (ovarian mesangial and superficial epithelium), tubal epithelium, duodenal mucosa, ileal mucosa epithelium goblet cells, hepatic central vein endothelial, alveolar tubes, bronchial epithelial and other parts.