Polymorphism Of Murine H2-Eb And Specific Humoral Immune Response

Kunming (KM) mice are widely used in domestic immunological research and teaching. However, it is reasonable to assert the apparent individual variations in genetic background of KM mice, which are closed colony animals, have notable influence on the results of their immune response. To investigate the exact relationship between the two items, I detected the genetic polymorphism of 60 KM mice with the technique of Polymerase Chain Reaction- Sequence Specific Primer (PCR-SSP) and measured the production of Immunoglobulin G (IgG) against the protein antigen (ovalbumin) during secondary immune response by Enzyme-Linked Immunosorbent Assay (ELISA). It has been well established that Histocompatibility-2 complex (H-2 complex) located on Chromosome 17 of mice, i.e., Major Histocompatibility Complex (MHO , is the most complicated region concerning polymorphism. In addition, polymorphism is predominant in the E subregion, especially its second exon of beta locus (H2-Eb) , rather than the A subregion of I region in H-2 complex. Meanwhile, H-2 complex controls the specific humoral immune response as the class II gene-coding molecules present antigenic peptides to CD4+ helper T (Th) lymphocytes that are necessary for reaction against protein antigens. Therefore the second exon of H2-Eb was chosen for the detection of allelic polymorphism and the analysis of association between the polymorphism and anti-ovalbumin antibody production. Based on the nucleotide sequences of the second exon of Mus musculus domesticus (western European house mouse), six pairs of PCR primers (namely MudoEbl, MudoEb4, MudoEb5, MudoEb7, MudoEbl5 and MudoEbl7) were designed, each having one sequence-specific primer. The primer sequences of internal positive control were derived from murine tumor necrosis factor alpha (TNF alpha ). Therewere 78 positive cases of PCR-SSP in total (50 cases were from MudoEb5 and 28 from MudoEb7, while amplification with the other 4 primer pairs were all negative) as all the internal controls were successfully amplified, that is to say, 65% of alleles (78/120) were determined. Moreover, 26 cases were both positive for MudoEb5 and MudoEb7 detection, which means 43.3% of the genotypes of these mice are MudoEb5 /MudoEb7 on this locus. In brief, MudoEbS is predominant in the alleles of the second exon of H2-Eb and MudoEb5/MudoEb7 in genotypes of KM mice. Besides, the average anti-ovalbumin antibody level of KM mice with MudoEb7 is lower than that of those without MudoEb7 (p<0.05), which suggests the undefined alleles in KM mice contribute more to the production of anti-ovalbumin IgG antibody than MudoEb7 does. At the same time, there was no significant difference between the mice with MudoEb5 or not. In addition, no interactive effect was shown between MudoEb7 and MudoEb5 via two-way analysis of variance. The protein sequence variations in 3 1 domain of MHC class II molecules will result in structural changes of the peptide binding groove to present antigenic peptides to CD4+ T cells; therefore, the primary, secondary and the tertiary structures, which were predicted via homology modeling, of peptides coded by MudoEb5 or MudoEb7 were analyzed. With reference to the illuminated structure of MHC class II molecule (I-Ek), the shapes and electrostatic potentials of peptide binding pockets (P1, P4, P6, P7 and P9 pockets) were investigated, and the P1 pocket was inferred to determine the specificity difference in antigen presentation of these two peptides and correlate with different levels of humoral immune response. The possible formation of hydrogen bonds may be similar hi MudoEbS and MudoEb7 owing to the presence of conserved residuals.