Intracellular Localization And Association Of Porcine Fc Receptor For IgG With Invariant Chain

Immune system is not yet perfect after the birth of mammals. It is very important that neonatal maternal obtain specific antibody from breast milk. Ig G is the most abundant immunoglobulins. The newborn Fc receptor(Fc Rn) was responsible remarkably for the transport of Ig G across fetal or neonatal epithelial barrier. The study found Ii molecules could play an additional role in directing Fc Rn trafficking within the endocytic compartments by physical association with Fc Rn. Whether can pig Ii associate with Fc Rn and what does its CLIP play? In order to solve these questions, this study was carried out to clone Ii gene and Fc Rn gene, and investigate cell localization and relationship of pig Ii chain and Fc Rn.Firstly, specific primers were designed based on the putative coding regions of the porcine Ii and Fc Rn registered in Gen Bank. Two gene fragments were amplified from pig spleen and duodenum by reverse transcription-polymerase chain reaction. And then the fragments were inserted into p MD18-T vector and the genes were sequenced and analyzed. The results indicated that the ORF of pig Ii contains 645 bp and encodes 215 amino acids and the ORF of pig Fc Rn contains1071 bp and encodes 357 amino acids. Comparing the cloned genes and Genbank genes, the similarities are approximately 99.8% in nucleotide sequence.Secondly, According to sequence of pig Ii gene and prokaryotic expressive vector p ET-32 a multi-clone sites. primer pairs were designed. The Ii was cloned into the p ET-32 a vector. After the identification of Eco R?and Sal?digestion, the recombinant plasmid p ET-32a-Ii was confirmed to construct. The p ET-32a-Ii was induced to express fusion protein in E.coli BL21 at 37? by IPTG. The 43 k D fusion proteins was analyzed by SDS-PAGE, and it exists in the form of inclusion bodies. simultaneously, the optimum induction time and IPTG concentration were optimized.Finally, we constructed the eukaryotic expression plasmids p EGFP-C1-Ii,p Mcherry-C1-Ii and p EGFP-N1-Fc Rn that fused greenor red fluorescent protein.Applying to transient transfection technology, These recombinant plasmids weretransfected into COS-7 cells. Fc Rn and Ii expression and cellular localizationwere visualized using the fluorescence photomicroscope. Immunoprecipitation was applied to analyze the association of Fc Rn and Ii or ?CLIP- Ii. The results showed that Ii or Fc Rn was successfully expressed and predominantly localized in cellular endomembrane system. Pig Ii with Fc Rn colocalized in cellular endomembrane system and CLIP region plays an important role in the course of the combination of Ii and Fc Rn.