Study On Expression Character Of Mutated Chicken Ii Gene And Fused Ii-NDV Gene

The invariant chain (Ii) is a non-polymorphic type ? transmembrane protein. A major function of Ii is to ensure the targeting of newly synthesized MHC ? to the endocytic pathway, the endosomal sorting signal for newly synthesizedαβIi complexes were identified as two independent motifs residues at positions Leu⁷/Ile⁸ and Met¹⁶ /Leu¹⁷ in the Ii cytoplasmic tail. Two similar signals, Leu⁸/Ile⁹ and Val¹⁷ /Leu¹⁸, were found in the cytoplasmic tail of chicken Ii. But the functional properties of the amino acids around the two motifs are not reported in chicken Ii so far. Two motifs and the amino acids around the motif residues were mutated to Ala by PCR-based megaprimer method for site-directed mutagenesis and ligated to the vector pEGFP-C1, the recombinant plasmid constructed respectively. These mutants were transiently transfected into the COS cells by Lipofectamine^TM2000, and then located fusion proteins were observed in the fluorescent microscope. Two Leu-based motifs both independently mediate efficient sorting to the endocytic pathway. When mutated Glu³, Glu⁴, Gln⁵, Iie⁹, Ser¹⁰, Ser¹⁵, Gly¹⁶ or Val¹⁷ to Ala, the GFP were detected on the plasma membrane, While Arg⁶, Asp⁷, Ser¹¹, Asp¹², Gly¹³ or Ser¹⁴ were mutated to Ala, GFP were located in the intracellular vesicles. Our results thus confirm that the cytosolic tail of chicken Ii comprises two independent endosomal sorting signals that function in internalization and a functional Leu-based sorting signal requires specific geometrically neighboring residues.The most conserved region of Ii is in the tranmembrane (TM) domain. It was reported that tranmembrane played an important role in Ii self-polymerizing to trimerization, even though absence cytoplasmic side of the membrane. It was confirmed that Gln⁴⁷, Thr⁴⁹ and Thr⁵⁰ in the tranmembrane were pre-requisite in the form of MHCII-Ii. Gln⁴⁷ and Thr⁵⁰ of chicken Ii were mutated to Ala by PCR-based megaprimer method for site-directed mutagenesis and ligated to the vector pEGFP-Cl, the recombinant plasmid C1-Q47A and C1-T50A constructed respectively. The recombinant vector N1-MHCII-αand N1-MHCII-βwere constructed by inserting PCR products into the plasmid pEGFP-Nl. C1-Q47A(C1-T50A) and N1-MHCII-α, N1-MHCII-βwere co-transfected into the COS-7 cell strain, using Western blot and Immunoprecipitation to examined the function of Ii tranmembrane.Ii was applied for constructing chimera gene containing antigen epitope. Ii as the basic framework would be used to substitute CLIP district for CD4+T cells epitope sequences, and the chimeric gene frangments were cloned into the eukaryotic expression vector. The construction of the chimera of epitope can be effectively presented to T cells. There is no research on the CLIP alternative DNA vaccine in chicken Ii, we used three overlapping PCR to remove the CLIP district of chicken Ii, at this time just formed a Hind? restriction site. And then by conventional PCR, NDV F343 epitope fragments were amplified with Hind? restriction sites, about 33 amino acids. F343 gene fragments were connected to the Ii by Hindlll and constructed the C1-Ii-F343 endogenous targeting epitopes chimeric gene vaccine. At the same time, construction of the C1-F343 gene vaccine is as for a non-targeting control. 30 6 to 8-week-old female BALB / C mice were divided into five groups targeted by endogenous gene vaccine (C1-Ii-F343) and non-targeting vaccine (C1-F343) in the quadriceps Immunization, saline, C1, and C1-â–³CLIP as a study control. After three times after immunization, blood from the humoral immunity in mice for testing, immunization results showed that only C1-Ii-F343 and C1-F343 immune mice can be detected for the F343-specific antigen antibody, antibody titers 6400 and 1600, respectively, within the target gene-derived vaccine was significantly higher than non-targeted vaccine group. Construction of epitope gene replacement chicken Ii CLIP fragment of the DNA vaccine was based on the two targeting experimental basis.