The Expression And Distribution Of The Porcine FcRn In Mucous Epithelium

The mucosal surface of the body is an important gateway for many pathogenic bacteria invasion. The immunoglobulin IgG can be transported across their epithelial surfaces in several organs, in order to obtain passive immunity from the mother to the fetus or newborn. The neonatal Fc receptor (FcRn) plays an important role in this process. Researchers have shown that FcRn performs unique and important functions, which not only plays a critical role in IgG transcytosis across intestinal epithelium, mammary gland, placenta and yolk sac from mother to baby, mentain the homeostasis of serum IgG and albumin proteins as well, and it is also closely related with animal immune response. Research on FcRn can help to reveal IgG transfer mechanism and thus provides theoretical guidance for veterinary clinical application.The cytoplasma tail of porcine FcRn (FcRn-CT) was subcloned and expressed in E.coli by molecular biology, the specific rabbit anti-porcine FcRn polyclonal antibody was prepared, and the expression and localization of FcRn protein in porcine mucous epithelium was analysed by western blot and immunohistochemistry. These results will provide the foundation for further research on functions and applications of FcRn in mucous epithelium in the future. The research contents are summarized as follows:1. Cloning of procine FcRn geneAccording to the Sus scrofa FcRn mRNA sequence published in GenBank, FcRn gene was amplified from total RNA of porcine liver by RT-PCR and cloned into the pMD18-T. The recombinant plasmids were identified by PCR, restriction enzyme analysis and sequencing. The sequence analysis demonstrated that the FcRn gene had 99% homology with AY740682, and there is a deletion of 27 amino acids compared with AY740682.2. Subcloning and expression of procine FcRn-CT geneFcRn-CT gene were amplified from the recombinant plasmid pT-FcRn by PCR and cloned into the prokaryotic expression vector pGEX-KG and pET-32a(+).The recombinant plasmid was transformed into E. coli BL21 and successfully expressed. The expressed GST-CT and His-CT fusion protein in E. coli BL21 is about 29 KDa,23 KDa in the analysis of SDS-PAGE, respectively.3. Preparation and purification of rabbit anti-porcine FcRn polyclonal antibodyRabbits were immunized with purified GST-CT fusion protein which was emulsified with Frund's adjuvant, the titer of the antiserum against porcine FcRn was as high as 1:32000 detected by indirect ELISA assay using purified His-CT fusion protein.4. Expression of FcRn protein in porcine mucous epithelium was detected by Western blot Western blot analysis for detecting FcRn on porcine mucous epithelium was established by using rabbit anti-porcine FcRn-CT serum.The results indicated that FcRn was expressed in the tissues of trachea, lung, duodenum, uterus and vagina.5. Expression and distribution of FcRn protein in porcine mucous epithelium was detected by immunohistochemistryImmunohistochemistry analysis for detecting FcRn on porcine mucous epithelium was established by using purified rabbit anti-porcine FcRn-CT polyclonal antibody. The results showed that FcRn was expressed in the tissues of uterus, vagina, nasal cavity, trachea, lung, duodenum and jejunum, and the positive signals were in the uterine endometrial epithelium, vaginal epithelium, nasal vestibular mucosa epithelium and respiratory mucous epithelium, trachea mucosa and tracheal gland, duodenal mucosa, jejunum epithelium.