Polymorphism And Evolution Of Macaca MHCI Molecules Of Four Chinese Macaques

The major histocompatibility complex (MHC) is a set of cell surface molecules encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes, also called white blood cells (WBCs), which are immune cells, with other leukocytes or body cells. Tibetan macaques (M. thibetana), stump-tailed macaques (M. arctoides), Assamese macaques (M. assamensis), and northern pig-tailed macaques (M. leonina) are four major species of Macaca in China. In order to effectively use these species in biomedical research, thorough investigations of their MHC immunogenetics are required. In this study, we identified MHC class I sequences using cDNA cloning and sequencing on a cohort of6M. thibetana,3M. arctoides,3M. assamensis and3M. leonina derived from Sichuan and Yunnan of China. Mamu-B*007:03-svl is a splice variant of major histocompatibility complex class I in Chinese rhesus macaque devoid of α3domain, In this experiment the basic information about expression and cellular location of Mamu-B*007:03-svl was got and compared with the full-length Mamu-B*007:03genes. The main conclusion of this research as follows:(1)83new alleles were identified, including26MHC-A alleles,46MHC-B alleles and11MHC-I alleles. Among them, Math-Al*126:01, Math-Al*127:01, Math-B*190:01, Math-B*191:01, Math-B*192:01, Maar-Al*127:01and Maas-Al*128:01represent lineages that had not been reported earlier in Macaca. Phylogenetic analyses show that no obvious separation of lineages among these species of Macaca. This study provides important information about the MHC immunogenetics for the four major species of Chinese macaques, and adds value to these species as model organisms in biomedical research.(2) Eukaryotic expression vectors of Mamu-B*007:03-svl-myc-pEGFPN3and Mamu-B*007:03-myc-pEGFPN3were constructed and transfected to293T cells. The expressions of the two genes in cells were detected by Western blotting methods, and the cellular locations of the two genes were analyzed by immunofluorescence and laser confocal microscopy. Mamu-B*007:03-svl and Mamu-B*007:03were all expressed in the293T cell and were all glycosylation. While Mamu-B*007:03gene was expressed on the cell membrane, and Mamu-B*007:03-svl gene was expressed in both intra-cellular and cell membrane. The expression and cellular localization of Mamu-B*007:03-svl deviod of α3was significantly different from the full length Mamu-B*007:03gene. Further researches about functions of Mamu-B*007:03-svl need to be done.(3) In order to obtain baculovirus recombinant bacmids of synthetic gB and gC genes of macaque B virus. The gene fragments of synthetic gB and gC were cloned in the transposition plasmid pFastBac-HTA. The positive plasmids were transformed to the competent cells of DH1Obac.The recombinant bacmids were selected by PCR. The recombinant bacmids of gB and gC genes were constructed successfully. The experiment laid the foundation for the expression of gB and gC genes in the baculovirus expression system.