| The invariant chain (Ii) is a non-polymorphic type-II transmembrane glycoprotein, itconsists of three domains: cytoplasmic domain, transmembrane domain and lumenaldomain. Ii plays an important role as a chaperone for MHC II maturation and facilitatesantigen presentation in vertebrates. Ii is a single-copy gene but its cDNA and expressedprotein molecules have some isoforms. There are many functional fragments on Ii whichhave been known such as two Leu motifs in cytoplasmic domain which have the functionof endosome positioning, the sequence of transmembrane domain which has the functionof membrane anchoring and promote the formation of trimerization. CLIP in lumenaldomain can occupy the peptide-binding groove of MHC II molecule and prevent thecombination of endogenous antigen. Trimerization domain can form trimer and acts asscaffold for MHCII molecule assembly. A fragment, named as Ii-key lying just outside theN-terminals of CLIP, could promote antigen charge into the peptide-binding groove. Moreand more researches have proved that Ii is a versatile molecule, for example, theinteraction between Ii and MHC I, neonatal Fc γ receptor and angiotensin II receptor hasbeen discovered. Besides the immune regulation of Ii, more researchers also focus on itsenhancement function in immune response, for example, using Ii as an antigen carrierbased on the its endosome position or using dominant epitope to replace the CLIP of Ii orusing Ii inhibition and gene silencing technology. Currently, there are two mechanismshave been proposed to explain the function of Ii in enhancing the charging of antigenicpeptides to MHC class II molecules. First, Ii is digested remaining a fragment, CLIP, whichbinds in the antigenic peptide-binding groove, then DM is moved from the CLIP inexchange for an antigenic peptide. Secondly, Ii owns a sequence located in the upstream ofCLIP, which bind to an allosteric site, keeping the antigenic peptide-binding groove openuntil a new antigenic peptide is charged. This Ii segment has been named as the Ii-key,American antigen express inc. puts the Ii-key and antigenic peptide together by a linkerforming hybrid peptide vaccine which could induce more effective immune responses overthe native peptide in vaccination.Newcastle disease virus (NDV) can cause the poultry incidence. Once birds areinfected with NDV, it could give the poultry industry large losses. Fusion protein is oneimportant capsule protein of NDV and plays an important role in viral immune. Our Labmembers connect three potential epitopes derived from fusion protein of NDV virulentstrain F48E9and series into F306, which could enhance immunogenicity. But thewidespread use of peptide vaccine is limited by the MHC restriction, weak antigenicity,epitope replacement difficulty and short duration etc. So it is necessary to modify the F306.After the cloning of poultry Ii gene such as chicken, duck, goose, quail, we foundsome conservative sequences in Ii protein through sequence alignment, for example,similar sequence (LQRK) just like Ii-key(LRMK) sequence of human and mouse, whichwas called as poultry Ii-key. In order to research whether this Ii-key has the ability ofenhancing the immune function and the function of other fragments of chicken Ii, thepartial segments of chicken Ii were used to modify the string epitope (F306) derived fromfusion protein of NDV to compare the vaccine effectiveness between modified epitopes and unmodified epitopes. Briefly, the DNA sequence of tetrapeptide (LQRK) of Ii-keyand dipeptide (AP) from chicken invariant chain were designed on primers, usingpGEX-4T-1/F306as template, amplifying segments of Ii-key/F306, Ii-key/F306/APthrough PCR, then be inserted into plasmid pET-32a so recombinant plasmids pET-32a/Ii-key/F306and pET-32a/Ii-key/F306/AP were constructed. Connection between thesegments of former19amino acids of cytoplasmic domain and Ii-key/F306andIi-key/F306/AP were obtained by appending complementary endonuclease site of geneticengineering means. So plasmids pET-32a/cyt/Ii-key/F306, pET-32a/cyt/Ii-key/F306/APobtained, then all recombinant plasmids were transformed into host bacteria and inducedexpression, and all recombinant proteins were purified from the PAGE gel. Fiveprokaryotic expression plasmids were constructed and five proteins of F306, Ii-key/F306,Ii-key/F306/AP, CYT/Ii-key/F306and CYT/Ii-key/F306/AP, which connected by HIS-tagwere purified. Their purity is more than95%. All modified epitopes could be preparedthrough prokaryotic expression and have high purity which could be used for performingvaccination test on animals. |
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