Comparison Of The Effect Between Two Segments In Invariant Chain CLIP On The Increasing Humoral Immune

Major histocompatibility complex (MHC) is composed of a genes group with same function, shows highly polymorphic and locates in a particular area of the chromosome. Its coding genes express on different cell surface in the process of immune response in antigen presented. The a and β chains of MHC class Ⅱ molecules with gamma chain polymerization form nine polymers (αPγ)3 in the endoplasmic reticulun,。 Jones et al. (1978) found that in the different strains of mice I-A and I-E molecules, y chain was different from the polymorphic a and β chans, it showed the nonpolymorphism, so it was called invariant chain (Ii). Ii is an important MHC Ⅱ chaperone protein molecule and named CD74 in leukocyte differentiation antigens. Ii plays an important role in MHC class Ⅱ molecule fold correctly, the assembly and its protein expression and antigen-presenting. Ii structure and the mechanism in antigen-presenting were more known. Ii has three domains, cytosolic domain, transmembrane domain and lumenal domain. Its main function is assistance of the MHC class Ⅱ molecules in exogenous antigen peptide presenting. The lumenal domain contains CLIP (class Ⅱ-associated invariant chain peptide,81-104 aa) and trimerization region (TRIM). Research shows that there is binding sites with MHCII (CBS, Class Ⅱ binding site) in the CLIP. The CBS is divided into two function segments, namely mixed with all polymorphism MHCII promiscuous binding site (PBS,81-87 aa) and groove binding sites (GBS,91-99 aa). When the CLIP binds to MHC class Ⅱ molecules, PBS locates outside of peptides combined groove in MHC class Ⅱ molecule related with the dissociation of Ii from MHC class Ⅱ molecules, while GBS locates in the peptide combined groove and interacts with MHC class Ⅱ molecules.First Ii structure was analyzed, which could be divided into cytosol domain, transmembrane domain(TM), CLIP and trimerization regions. CLIP could be divided into two segments of PBS and GBS subsection design primers. In previous work in our laboratory, it was found that the poison the advantage of NDV F protein antigen epitope F2. By Overlap PCR technology, the hybrids containing F2 antigen epitope and Ii different segments were constructed, then they were inserted into the pET-32a respectively, at the same time F2 antigen epitope was inserted into pGEX-4T-1. The results of sequencing with PCR showed that all the reconstructed hybrids and recombinant plasmids were expectant.Secondly, the condition optimization of induction temperature, IPTG concentration and inducing time in prokaryotic expression was carried out. Under the optimal conditions a large expression was performed. The results of identification in SDS-PAGE electrophoresis showed the eight recombinant plasmids could expressed in E. coli Rosetta (DE3) respectively, their molecular weights were 29.9 kDa (GST/F2),23.9 kDa (His/F2),32.8 kDa (His/Cyt/TM/F2),34.7 kDa (His/Cyt/ TM/F2/GBS),34.4 kDa (His/Cyt/TM/PBS/F2),45.1 kDa (His/Cyt/TM/F2/TRIM), 46.9 kDa (His/Cyt/TM/F2/GBS/TRIM) and 46.7 kDa (His/Cyt/TM/PBS/F2/TRIM) respectively. All were consistent with expected results. After that a large production of expression were treated with centrifuge, repeated freezing and thawing and ultrasonic crushing processing. The expression form was detected with SDS-PAGE electrophoresis, and then after the Native-PAGE (not degeneration reducing PAGE) and purification with cutting the gel slices 0.25 mol/L KCL solution rubber, the antigen proteins with the concentration and purity for experiment were received.Finally, the 32 mice, which were six weeks age and female of SPF KunMing, were divided into eight groups, in which one group was as control and seven were as experiment groups. They were immunized with fusion proteins that could satisfy the requirement for experiment. The second and third immunizations were carried out after ten and seven days respectively. After seven days of last immunization, the bloods were collected from eyeball of the mice, antiserums were then separated. Under the optimal experimental conditions, the indirect ELISA method was used for the test of antibody titers. The results showed that first the specific antibody titers of six groups, which were immunized with Ii segments/F2, were 1.5 to 4.9 times higher than the group with alone F2. group. Secondly, in the above six group, the titers of groups containing PBS or GBS of CLIP were 1.6-2.4 times higher than that of groups containing non both segments. Thirdly the titers of group containing PBS is 1.5 times higher than that of the group with GBS. In conclusion, Ii cytosol and transmembrane domains could a boosting immune for animals, and the role of PBS of CLIP in Ii carrier was better than that of the GBS.In summary, based on successful cloning mice Ii segments (Cyt/TM and two CLIP regions) and epitope F2 of NDV F protein respectively, they were inserted into the prokaryotic expression plasmid, pGEX-4T-1, pET-32a of the E. coli. After the the expression and purification of target proteins, SPF KunMing mice were immunezed, and their antibody titers were detected. Analysis of the titers of different experimental levels was carried out. The results indicated that Ii active segments (Cyt/TM and CLIP) could boost humoral immunity, in which the both regions of CLIP showed difference. All these provide a foundation for clinic practice of the new-type invariant chain active segment vector vaccine.