Characterization Of MHC Class Ⅰ Allele Polymorphism In Common Marmosets

Hepatitis C caused by the infection of hepatitis C virus （HCV） is a major liver disease that jeopardizes the world’s population. Approximately170million people are chronically infected with HCV worldwide. Although some great progresses were made in prevention and cure for chronic Hepatitis C infection, little is known about the molecular mechanism of HCV control. Due to the fact that HCV has a host range restricted to humans and chimpanzees and lacks suitable primate models, the development of vaccination and novel therapy for HCV has been hampered.GB virus-B （GBV-B） is a member of the Flaviviridae family of hepatitis viruses. GBV-B was first described over40years ago, following the inoculation of tamarins with sera from a patient suffering from acute hepatitis. Serial passage of sera from these animals into further tamarins resulted in hepatitis. This RNA virus is infectious to common marmosets （Callithrix jacchue jacchus）, a New World primates, which could be used as a surrogate model for hepatitic C virus （HCV） infection in human. In separate studies, chimeras of GBV-B carrying HCV5’-NCR or HVR1or p7even as the whole structural proteins were generated to infect marmosets for virological and immunological investigation.Common marmoset（Callithrix jacchue jacchus） is a small New World primate closely related to the tamarin which can infected by GBV-B and developed into classical viral hepatitis, so can be used as a surrogate model for hepatitic C virus （HCV） infection in human. This kind of primate presented smaller, less expensive, and more-readily available animal models than the chimpanzees. As an animal model, it is necessary to be known the information of histocompatibility complex（MHC）. However, little information is available on the major histocompatibility complex class I （MHC-I） that conditionned immune responses of cytotoxic T lymphocytes （CTL） of marmosets.Highly polymorphic major histocompatibility complex class I （MHC-I） molecules are involved in the cell-mediated immune responses to pathogens and foreign tissues. In addition, individual differences in CD8+T cell responses play a central role in viral clearance, which may be determined by their polymorphic nature of the MHC class I locus. Consequently the diversity of MHC-I is necessarily characterized. Marmosets are relatively inbred, which facilitates the study of MHC-restricted T cell immunity. Up to now, only few MHC-I sequences have been described from common marmosets and have not been comprehensively characterized. Here we have separated and analyzed the full-length MHC-I sequences from nine common marmosets, which are currently used in a separate study of chimeric HCV/GBV-B infected animal models. Data obtained from the study will immunogen etically enrich the background of the marmoset model.In this study, total cellular RNA was extracted from1-5*106lymphocytes using TRIzol （Life technologies, USA）, and eluted in50μl DEPC H2O and stored at-80℃. Full-length marmoset MHC （Caja） class I genes were amplified using two paired primers designed on rhesus macaque MHC class I sequences. Amplification of whole length MHC class I genes was performed with RT-PCR. Approximately1100bp amplicons were purified from1.0%agarose gel electrophoresis by using a gel extraction kit. Amplicons were ligated with pMD-20T vector and transformed into competent E. coli. All colones containing the predicative size inserts from each marmoset were selected for DNA sequencing by a commercial company. Identical sequence with contiguous forward and reverse sequences from two or more clones was considered a true MHC class I gene.Nucleotide and deduced amino acid （aa） sequences were analyzed by DNAMAN version5.2.2. Phylogenetic tree was constructed by MEGA version4.1using neighbor-joining method in alignment with reference sequences of primate MHC, which were retrieved from GenBank database with accession numbers as follows: common marmosets Caja-A （xr₀91099.1）, Caja-B （xm₀02764215.1）, Caja-C （xm₀02763560.1）, Caja-E （ef014284.1）, Caja-GOl （u59637.1）, Caja-G03（u59639.1）, Caja-G04（u59640.1）, Caja-G05（u59641.1）, HLA-A （nm₀02116.7）, HLA-B （nm₀05514.6）, HLA-C （nm₀02117.4）, HLA-E （nm₀05516.5）, HLA-F （nm₀18950.2）, HLA-G （nm₀02127.5）, rhesus macaque Mamu-A （gu592059.1）, Mamu-B （ef580173.1）, Mamu-E （nm₀01114966.1）, Mamu-F （nm₀01042770.2）, common chimpanzee Patr-A （dq539672.1）, Patr-B （dq539675.1）, Patr-C （afl65370.1）, Patr-E （nm₀01045498.1）, Patr-F （nm₀01129198.1）, Patr-G （nm₀01045512.1）. Novel sequences were submitted to GenBank and nomenclature were assigned by the Non-Human Primate of the Immuno Polymorphism DatabaseThree hundred and fifty five individual sequences were isolated from nine marmosets, in which15to92clones were obtained from amplicons with primers set for each animal. In order to eliminate the polymerase misincorporations or sequencing artifacts caused by sequence-based genotyping methods, a filtering step was adopted. Only identical sequence at least from two or more clones was considered as a putatively true gene. As a result,26novel sequences were initially identified from marmosets. To examine the phylogenetic relationship among MHC genes of marmosets, a phylogenetic tree was constructed by using the neighbor-joining method based on those26MHC sequences and24MHC references covering the whole gene region.Of26novel sequences,15clustered in Caja-G alleles were considered as true alleles because they were all full-length sequences encoding entire function proteins. Other11were class I pseudogenes, which did not express the functional protein with frameshift or deletion of nucleotide sequences in class I genes. Interestingly,6of those11pseudogenes lacked the part or whole a2domain. Fifteen novel Caja-G alleles with GenBank accession numbers and IPD nomenclatures were presented.Two alleles m01-caja-a16and ml3-caja-a09were found in three marmosets. Five alleles were identified in two animals. Other8novel class I sequences were individually detected from different animals. No alleles were likely present in most animals. The novel Caja-G alleles were over96%identities with the reference sequences. However, the Caja-G alleles should be further classified in accurate genotyping as only few MHC class I alleles from marmosets are available currently.Amino acid （aa） sequences were deduced from15novel Caja-G alleles, which presented two different sizes of molecules with359or362amino acids, respectively. Similarities of nucleotide and amino acid sequences among15Caja-G alleles range from89.54%to99.91%or83.15%to100%respectively, Sequences m13-caja-a09and m15-caja-a06are differed in only one base nucleotide, but the deduced amino acid sequences are same.MHC class I heavy chain is functionally divided into five distinct regions:three extracellular domains （α1, a2and α3domain）, a transmembrane domain and a cytoplasmic domain. Within α1and a2domains, polymorphisms are mainly located in three regions:aa position62to83,92-121and152-179,which are hypervariable regions （HVR） compared to aa position62to83,92-121and135-157in human. Diversity in al and a2domains is very important in these two domains specifically form the peptide binding region （PBR） of MHC-I molecule. The a3domain contains a conserved seven amino acid loop which serves as a binding site for CD8cells.In summary, we identified15novel class I G （Caja-G） alleles encoding whole function molecules,6class I C （Cqja-C）-like pseudogenes and5class I G （Caja-G）-like pseudogenes from nine common marmosets. A high degree of Caja-G polymorphism in this species of common marmosets was revealed. The data will provide useful genetic materials and helpful information to explore the CD8+T cell responses in HCV/GBV-B chimera infected marmoset models.