| Major histocompatibility complex, which also named major histocompatibility complexed gene, is made up of a series of closely-chained gene groups. It is located in a specific sites in the chromosomes of people and animals’ DNA. MHC molecular has a high polymorphism. They can restrict mutual recognition between cells and induce the immune response. The MHC gene family is mainly consist of three subtribes: Class â… gene, Class â…¡ gene and Class â…¢ gene. MHC â…¡ gene is located in the surface of antigen presenting cells such as macrophages and so on. When the bacteria invader the organism, they can quickly present those exogenous antigen and recognize and phagocytose them. Then the bacterial debris would be presented to the CD4+ T cells with MHC molecular. As the mainly structure of MHC â…¡ molecules, y chain forms nonamer with a and β chain in endoplasmic reticulum. Different with a and β chain, y chain is non-polymorphism so that is nominated as invariant chain（Ii）. Ii has an effect on immune enhancement and antigen peptide directional transportation. Ii is also used in the treatment of tumor in clinic.Antigen epitope which also named antigenic determinant is a specific gene area of some antigens’existence. B lymphocyte antigen epitope could be recognized by antibody. T lymphocyte antigen epitope could be recognized by TCR/MHC complex. Epitope-based vaccine has an advantage that people don’t have to separate the infectious pathogens, but also could reduce the mutual interference between different antigen epitope.According to the antigen and antigen epitope prediction analysis of NDV HN and IBDV VP2 protein, we chose NDV HN（172-201AA） and IBDV VP2（197-209AA） as the target segments. Ii Cyt/TM is used to be the immune carrier to series with the target segments. Based on preparation above, we could study the immune enhancing effect of Ii functional fragments in different virus epitope series. We could also explore the influence of affect on immunogenicity when the different virus epitope’ sequence was exchanged.First of all, we acquired the sequence of mouse Ii （No.NM001042605）, IBDV-VP2（No.AY444873） and NDV-HN（No.AY510092） from GenBank. Then, we designed all the related primers with Primer Premier 5.0. These gene fragments, Ii segments and epitopes, NDV-HN （172-201） and IBDV-VP2 （197-209）, were cloned from the plasmids in Key Laboratory of Zoonoses of Anhui Province by PCR. Secondly the gene hybrids including NDV-HN（90bp）, Cyt/TM/HN（339bp）, HN/VP2（141bp）, Cyt/TM/HN/VP2（390bp）, IBDV-VP2（39bp）, Cyt/TM/VP2（288bp） and Cyt/TM/VP2/HN（390bp）, were constructed with a series of primers by overlaping extension PCR respectively. Finally all the constructed hybrids were inserted into prokaryotic expression vectors pET-32a for immunization antigen respectively. Additionally the both fragments, HN （172-201） and VP2 （197-209）, also were respectively inserted into pGEX-4T-1 for expression of the coating antigen used in the ELISA. All of the constructed hybrids were identified by sequencing.All the reconstructed plasmids were transfected into E.coli expression strain Rosetta, and then were induced by 1 mmol/L IPTG for expression respectively. The expressed recombinant proteins were purified with Native-PAGE and 0.25 mol/L KC1 gel cutting and identification by SDS-PAGE respectively. These proteins were examinated by western bloting to be improved the satisfied immunogenicity. The products were dissolved in solution buffer and stored at -70℃.Thirty two mice were divided into eight groups. Mice were anesthetized and injected intraperitoneally with 50 μg of each protein antigen respectively. One control group of mice was injected as above with normal saline water. The antisera were prepared after 3 times injecting from collecting mouse blood respectively, and were stored at -20℃ until used for estimation of the antibody titers. Detection of antibody with ELISA and put all the data in the SPSS software for statistical analysis. Result showed that the groups immunized by six hybrids containing Ii segments Cyt/TM had antibody titers 3.1 ～3.5-fold than the titers from the animals immunized by hybrids without Ii-segments （p<0.01）. It was interesting that no difference between the series multiepitopes and the single epitope, when they were linked with Ii-segment, were found in the production of specific antibodies （p>0.05）. This suggests that Ii segments （Cyt/TM） as a potential immune carrier could increase the immune response to single epitope as well as multiepitopes.In this work demonstrated that the multiepitopes, HN （172-201）/VP2 （197-209） and VP2 （197-209）/HN （172-201）, in various series in hybrids had the same immunogenicity, and Ii segments （Cyt/TM） could improve an immune response to single as well as multiepitopes. The Ii segments as an immune carrier potentiated an immune response to multiepitopes. |
