Indentification of Cynomolgus Macaque MHC-I B Gene Alleles and Analysis of High Frequency Alleles
|School||South China University of Technology|
|Course||Biochemistry and Molecular Biology|
|Keywords||Cynomolgus monkeys MHC-I B allele High frequency allele|
Objective: To obtain food cynomolgus monkey MHC-I full-length B alleles, the judge newly acquired alleles, whether there is a high frequency allele analysis to establish the experimental basis for subsequent genetic traits associated. Methods: Fresh the the cynomolgus monkeys MHC-I B allele specific primer, amplified by PCR, to obtain the full-length allele. Constituting the recombinant plasmid of the PCR product with a plasmid vector, transformed into DH5α competent cells with the recombinant plasmid, and positive clones were screened by blue and white sub-the isolated MHC full length allele. By GenScan software and Blast n ratio analysis allele in exon and intron, and the allele encoding the amino acid sequence translation. By calculating the entropy values ??of the amino acid sites, and to determine the amino acid residues conservative and variability, combined already in the database, the MHC-I B full length of the cynomolgus monkey CDS compared result, the full length alleles hypervariable loci . The hypervariable loci new allele specific primers were designed as a template was amplified by PCR, and the calculation of the allele frequency distribution in a large sample in order to determine whether this gene is the high frequency allele. Results: restriction enzyme digestion and sequencing results show 27 cynomolgus monkey MHC-I B allele, 20 of them the same as the sequence of database, seven new allele, named by 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 Indexed numbers were: GQ411543, GQ411545, GQ411546, GQ411547, GQ411550, GQ411551, GQ411552 statistics appear in a small sample of the high frequency of new alleles Mafa-B * 01702 large sample frequency of approximately 12.9% . Conclusion: rhesus monkey MHC genome as template the the cynomolgus monkeys MHC-I B full-length gene-specific primers could be amplified food proved rhesus monkey and cynomolgus monkey MHC regulatory region having similar successful design. The ratio of the obtained Mafa-B gene was found more polymorphic sites in the α1 and α2, which is consistent with MHC function. After MHC-B alleles amplified cloned and sequenced to give each cynomolgus monkey types of MHC-B alleles about 8-10 species, this result with rhesus monkey MHC-B allele is similar to the number of kinds, exceeds the number of food types of of cynomolgus monkeys MHC cDNA, may be the genome some alleles silencing. The antigen-binding site polymorphism of the MHC-I molecules to identify a large number of the antigen peptide. The new allele frequency is higher, provided the experimental basis for future alleles with traits linked gene chip development.