Immune Response Enhance Study Of Mouse Invariant Chain Short Peptide Ii-key

Major histocompatibility complex （MHC） is composed of a bunch of closely-chainedgenes groups, which locates in the specific sites in people and animal’s chromosomes.MHC is high polymorphism and distributed on the surface of different cells such as antigenpresentating cells. MHC molecules restrict mutual recognition among cells and guideimmune response. The MHC gene family is mainly divided into two subtribes: Class Igene and Class II gene. Aggregation between α, β chain of MHCII molecules and γ chainin endoplasmic reticulum forms nonamer. Contrast to the polymorphism of α and β chain, γchain is non-polymorphism, which is nominated as invariant chain （Ii）. Ii is very importantin MHCII molecules correct folding, combination, structure maintains, protein expressionand antigen presentation, it is also significant chaperone of MHC class II molecules. Insome study, the chimera which is reconstructed by the transmembrane segment of Ii chain,four N fringe-linked LRMK in CLIP segment and connecting epitope enhances theimmune responses. This paper mainly studied the functions of different segments of mouseIi chain in the immune response as carriers.First, a mouse Ii chain sequence in GenBank （login ID: NM₀01042605） wasachieved. Ii sequence could be divided into cytoplasm segment, transmembrane segmentand CLIP segments. Aseries of primers was designed according the three segments. Then apeptide containing three epitopes of Newcastle disease virus （NDV） fusion protein, namedF306kept in our lab, and the different Ii segments were linked, a series of chimeras wasconstructed. These chimeras were further inserted into pronucleus expressing carrierpGEX-4T-1and pET-32a. The results is identified by PCR and showed that all therecombined plasmids were successfully formed.Secondly, the ten fusion proteins were induced to express after optimizing theconditions. These expressed samples were repeatedly frozen and thawed and splitted withultrasonic them. Then the target proteins were extracted and purified with cutting the gelslices KCL solution. Electrophoresis detected by SDS-PAGE showed that ten recombinedplasmids expressed in E. coli. Molecular weights were29.8KD、36.6KD、37.0KD、37.2KD、44.8KD、45.0KD、57.5KD、23.8KD、24.8KD、23.0KD respectively, which wereconsistent with the theoretical prediction.Thirdly,6-8weeks old female Balb/c mice were immunized with the chimerascontaining different Ii segments and F306antigen peptide. After three times ofimmunization the antibody titers were detected in the mouse seren of six groups withindirect ELISA method optimizing the conditions. The results showed that the antibody titers induced by chimera containing Ii-key or Ii-key, DN and F306were2and4foldhigher than alone F306antigen peptide induced respectively. The chimera containingcytoplasm segment, transmembrane segment, Ii-key, DN and F306peptide was7timeshigher than F306, while the chimera im which CLIP was replaced with F306counld inducan antibody titter of1.5fold higher than F306. The F306contained three epitopes （F72、F161�'�F343） of NDV F protein. Further the antibody to each epitope was detected in the6groups. The results showed that antibody titter of F72is higher than that of F161andF343, which proved that the dominant antigenic epitope of NDV F protein was66-99（F72）amino acid.In conclusion, in this paper the different functional segments of mouse Ii weresuccessfully cloned and the chimera containing F306epitope was constructed. Thesechimeras were inserted into pronucleus plasmids pGEX-4T-1, pET-32a separately. Aftersuccessful expression, the targeting gene’s fusion proteins were achieved. And then theBalb/c mice were immunized with purified proteins and the antibody titers were detectedto analyze the role of mouse Ii chain’s various segments in the immune response as vectors.This could lay the foundation of clinic practice for the new-type invariant chain activefragment vector vaccine.