Indentification Of Cynomolgus Macaque MHC-I B Gene Alleles And Analysis Of High Frequency Alleles

Objective: aims of this project are to obtain full-length cynomolgus monkey MHC-I B alleles, to determine whether there are high-frequency alleles exist in these new alleles exist, and to establish experimental basis for the following analysis of genes and traits association. Methods: designing cynomolgus monkey MHC-I B allele specific primers, amplified by PCR to obtain full-length alleles. The PCR products and plasmid vector are conjacted to form reconstructed plasmids, with the recombinant plasmid was transformed into DH5αcompetent cells, screened by blue and white positive clones, isolated and obtained full-length MHC alleles. Exons and introns in alleles will be analyzed through online software GenScan and Blast n, and translated into amino acid sequence. entropy value of amino acid sites by calculating, determine a conservative amino acid residues and variability, combined with alignment results of the existing fascicularis MHC-I B full cds in database, get highly variable sites in full-length alleles. Designing specific primers for a new allele of highly variable sites as the template, through PCR amplification, and calculation of the allele frequency distribution in large samples to determine whether the high-frequency allele of the gene. Results: The restriction enzyme digestion and sequencing results obtained fascicularis MHC-I B allele were 27, 20 of which are the same sequence with the database, seven new alleles, named by the International Non-human Primate MHC Committee (IPD) named: Mafa-B * 01702, Mafa-B * 12801, Mafa-B * 0380302, Mafa-B * 03809, Mafa-B * 12901, Mafa-B * 13001, Mafa-B * 13101; NCBI Database accession numbers were: GQ411543, GQ411545, GQ411546, GQ411547, GQ411550, GQ411551, GQ411552. The statistics in small samples of new high frequency allele Mafa-B*01702 in a large sample of the frequency of about 12.9% . Conclusion: gene-specific primers which are designed based on rhesus MHC genomic DNA can be amplified cynomolgus monkey MHC-I B full-length successfully, which indicates rhesus and cynomolgus monkeys have a similar MHC regulatory region. Obtained by comparing Mafa-B gene, found in theα1 andα2 more polymorphic loci, which is consistent with the MHC function. MHC-B alleles were amplified after cloning and sequencing of cynomolgus monkeys by each MHC-B allele type is about 8-10 species, the results of rhesus monkey MHC-B allele number of similar types, Macaca fascicularis MHC cDNA than the number of species may be a number of alleles genome silencing. Antigen binding site of the polymorphism of MHC-I molecules can make a large number of antigenic peptide recognition. The new allele frequencies were higher for the future connection of alleles and traits and the development of gene chips provide an experimental basis.