Charaterization Of MHC-I Restriction Allelic Antigen Of Comm Marmosets

Common marmoset （Callithrixjacchus） is a small New World primate closely related to the tamarin, whose natural habitat is northern Central America.Currently, owing to the small size, easy handling, high fecundity, low cost compared with chimpanzees and rhesus macaques, and more easily cage-feeding in research, common marmoset is widely use in brain development, teratology, lipid metabolism, behavioral science,endocrinology, immunology, virology, reproductive biology and so on. Previous studies have shown that common marmoset can be infected by GBV-B and develop into classical viral hepatitis. And GBV-B is close to HCV, both of them are hepatotropic flaviviridae. So our lab had a hypothesis:chimeric HCV/GBV-B viruses will mimic HCV infection in small primates? In recent years, our results showed that marmosets can be infected with two chimeras of GBV-B containing HCV structural proteins coding for core and envelope protein genes （coreElE2p7）, full envelope protein genes （ElE2p7） substituted for the counterpart elements of GBV-B,respectively. Substantial levels of viremia were found in two HCV chimera-infected marmosets and chimeric RNA were detected in the liver tissue. Infected marmosets model might become a major tool to study the functions of HCV proteins in vivo and provide a suitable primate model replaced the chimpanzee to evaluate vaccine and antiviral drugs for HCV. But the results showed that the viral load, duration, degree of liver injury were different between the individuals although every of them had a peisistent infection. The ELISOPT tested for the specific T-cell response to HCV epitopes results also showed that the immune responses to a same epitodes was dfferent in chimera-infected marmosets. Synthesis analysis the phenomenon we found it must related to the immune responses in individual. The polymorphism of the major histocompatibility complex（MHC） detemine the cellular immune response that against the antigen peptide. Marmosets with unknown genetic background may cause different immune response when infected with HCV/GBV-B chimeric viruses. If they have the same MHC-I alleles, maybe it will present the same antigen peptide, so they will have same T-cell response to HCV. We can choose the marmosets which have the same MHC-I alleles to ensure the stability and repeatability of the experiment. But until now only few MHC-I sequences have been described from common marmosets while the study of MHC-I of human,mouse and rhesus macacus were deeply. So it is important to reveal the characteristics of the allele and the antigen molecules of marmoset MHC class I to improve the stability and repeatability of the experiment, enriched the background information of MHC-I in marmoset, know more about the molecular mechanism on HCV specific cellular immune protective reaction, and then provide important information for vaccine research.Major histocompatibility complex（MHC） is the most complex gene with the highest density. They showed abundant polymorphism, and all of the genes are codominant expression. MHC plays a role in immune response and immune regulation, mainly participate in processing, handling and presenting the antigen. In T cell-mediated immune response cause by the virus infection,cancer antigen and other endogenous antigen, MHC class I combines with the antigen peptide, and transfer it to the cell surface with the help of golgi apparatus, and then present to the CD8+T cells. Because of the different molecular structure of the MHC allele product, each individual may have different response to the antigen. So the MHC class I is important in virus infection and tumor disease. As a animal model, clear MHC background data will facilitate the use of common marmoset in medicine research. However, only few MHC-I sequences have been described from common marmosets and have not been comprehensively characterized.Here we have separated the full-length MHC-I sequences andβ2m from three common marmosets, which are currently used in a separate study of chimeric HCV/GBV-B infected animal models, and analyzed the sequences detailedly. Also we illustrated the protein structures of MHC-I of marmosets, tried to prepare the MHC-I/peptide soluble monomer. They were expressed in E.coli, purified, and their structures were mensurated with the protein analysis software and circular dichroism spectrum. Our research enriched the background information of MHC-I of marmoset and lay the foundation of the application of marmosets. We hope to increase the samples so we can find the MHC-I alleles that exist in most of marmoset, so we can construct the MHC-I/peptide tatramers to study the specific T-cell for HCV.Objective:Our research revealed the characteristics of the marmoset MHC-I alleles and β2m gene, and illustrated the protein structures of them, which enriched the background information of MHC-I of marmoset. As an important imformation it willimprove the stability and repeatability of the experiment, know more about the molecular mechanism on HCV specific cellular immune protective reaction, and then provide important information for vaccine research, also they will lay the foundation for the construction of MHC tetramer.Method:1:Experimental subjectThree commonmarmosets （Callithrix jacchue jacchus） used in thisstudy were juvenile males with an average weight of 350 g.2:Experimental method（1） preparation of marmoset MHC class I heavy chain:Full-length marmoset MHC （Caja） class I genes were amplified from total RNA in PBMC by RT-PCR. Identical sequence with contiguous forward and reverse sequences from two or more clones was considered a true MHC class I allele. Nucleotide and deduced amino acid （aa） sequences were analyzed by DNAMAN version 5.2.2. Phylogenetic tree was constructed by MEGA version 4.1 using neighbor-joining method. Chose the most frequent genes in these marmosets as the gene to be expressed. Subcloning the extracellular domains of MHC class I gene, and BSP sequence was linked to the C-terminus of the extracellular domains of MHC class I genes by PCR. The fragment was expressed using prokaryotic expression system pET-28a（+） E.coli. The expression of the fusion protein was confirmed by SDS-PAGE and Western-Blot. Proteins was purified by the affinity chromatography of Ni-NTA.（2） preparation of marmoset MHC class I β2-microglobulin:β2m mRNA gene was amplified from total RNA in PBMC by RT-PCR. Sequences was analyzed by DNAMAN and DNAstar, and the amino acid sequence was compared with the P2m in other primates and different species to make homology comparisons and evolution analysis. Subcloning the mature peptide of β2m. The fragment was expressed using prokaryotic expression system pET-28a（+） E.coli. The expression of the fusion protein was confirmed by SDS-PAGE and Western-Blot. Proteins was purified by the affinity chromatography of Ni-NTA.（3） Structure analysis of fusion proteins and Soluble monomer preparation and identification:The primary structure of fusion proteins were analyzed by the BioEdit and DNAstar. Hydrophobicity analysis was performed using the Protscale. The protein physical and chemical properties analysis were performed using the ANTHEPROT. Phosphorylation site analysis was using NetPHos2.0 Server. The secondary structure contents of fusion proteins were estimated by circular dichroism（CD） spectrum, SOPMA and PredictProtein. The three-dimentional（3D） structures of proteins were estimated by homology modeling. Soluble MHC-I/peptide monomers were constructed by the method of Altman.Results:（1） preparation of marmoset MHC class I heavy chainFull-length marmoset MHC （Caja） class I genes were amplified from total RNA from the PBMC in three common marmoset. Amplicons of MHC class I were about 1100bp, similar to the result in the previous study. Identical sequence with contiguous forward and reverse sequences from two or more clones was considered a true MHC class I allele. As a result,7 novel sequences that had all full-length sequences encoding full length putative protein and 1 aberrant sequences were initially identified from this three marmosets. All the alleles were divided into Caja-G After the translation, Caja-Gshowed the highly polymorphic when compared with the human HLA-G sequences. MHC class I of common marmoseta contained three extracellular domains （al-a3）, a transmembrane domain, and a cytophlasmic domain. Diversity in α1 and α2 was very important in these two domains which specifically form the binding pocket of the peptide-binding region of MHC-I molecule. Peptide-binding region was the recognition site of TCR. The a3 domain contained a conserved aa loop which serveed as binding site for CD8 cells. We chose Caja-G*09:03 as the gene to be expressed, which was the most frequent in these twelve marmosets. Subcloned the extracellular domains of Caja-G*09:03 gene, and BSP sequence was linked to the C-terminus of the extracellular domains of Caja-G*09:03 gene by PCR. The fragment of Caja-G*09:03-BSP was expressed in pET-28a（+） E.coli. The molecular weight of Caja-G*09:03-BSP（MHC-I-BSP） fusion protein was about 34kDa. After washing and dissolving of the inclusion body, SDS-PAGE results showed that the inclusion body was pure. Due to lack of the antibody specific for marmoset MHC class I gene, we used the antibody specific for the His tag at the end of the fusion protein to detect the protein. Western Blot results showed that the molecular weight was same as the SDS-PAGE.Protein was purified by the affinity chromatography of Ni-NTA.（2） preparation of marmoset MHC class Ⅰ β32-microglobululinAmplicons of β32m was about 1100bp, the first 20 aa in β2m gene were the signal peptide, and the next 99 aa were mature peptide. By analyzing the sequence we found that the sites of 25 and 80 aa for the mature peptide were cysteine, and the aa sequences nearby of 80 were " YACRVSH", which were correspondence to the motif sequence "YxCxVxH" in immunoglobulin superfamily. Compared with the β2m mRNA sequence（XM₀02753411.2） of common marmoset in GenBank, the homology between them was 100%. Subcloned the mature peptide ofp2m, The fragment of mature peptide of β2m was expressed in pET-28a（+） E.coli.The molecular weight of the mature peptide of β2m fusion protein was about 12kDa, which was identical to the theoretical size that prognosticated. We use two antibody specific for the His tag and the marmoset β2m protein to verify the β2m fusion protein. Results showed no band before the induction while we can see the specific band after the induction. The fusion protein was purified by the affinity chromatography of Ni-NTA.（3） Structure analysis of fusion proteins and Soluble monomer preparation and identificationThe analysis results showed that the theoretical value of the molecular weight of the MHC-I-BSP and β2m were 33.63KDa and 11.60KDa, identical to the product expressed. The theoretical assumption pI was 5.085 and 7.355 respectively. The hydrophobicity of the MHC-I-BSP was 1.389 while β2m was 1.122, weaker than the MHC-I-BSP. MHC-I-BSP had 21 phosphorylation site while β2m had 8. SOPMA results showed that MHC-I-BSP protein was based on a-helix, P-sheet and random coli while PredictProtein indicated that it contain 22.3% of the a-helix and 32.6% of theβ-sheet. SOPMA and PredictProtein analysis of β2m indicated that it based on β-sheet, but no a-helix. Analysis of the purified fusion proteins by CD spectrum was different from SOPMA and PredictProtein. The three-dimentional（3D） structures of MHC-I-BSP and β2m proteins were estimated by homology modeling. The homology modeling of 3D structure of MHC-I-BSP and β2m were similar as human’s. MHC-I-BSP was composed of two a-helix and eight P-sheet, and had a peptide-binding groove, a3 domain was consist of seven β-sheet. β2m only had seven β-sheet, no a-helix, which is the same as the human P2m. Soluble MHC-I/peptide monomers were constructed by the method of Altman. The monomer was refolded in the presence of (32m and HCV peptide El-282, CV9 to form a soluble Caja-G*09:03 monomers. But after refolding, no monomers form according to the result of HPLC and ELISA. The reason why no monomers form was analyzed.Conlusions:（1） The full-length MHC-I sequences and P2m were amplified from three common marmosets by RT-PCR. The sequences of those were analyzed completely, which enriched the common marmoset MHC background data.（2） Subcloned the extracellular domains of Caja-G*09:03 gene and the mature peptide of β2m, and BSP sequence was linked to the C-terminus of Caja-G*09:03, so it can bind the biotin. Both of them lays the foundation of the construction of MHC-I/peptide tetramer.（3） The extracellular domains of MHC class I genes and the mature peptide of β2m were expressed in prokaryotic expression system and purified. The proteins might be a powerful tool to furtherly study the tetrameric technology to identify T-cell epitopes.（4） Their primary structure and secondarystructure of MHC-I-BSP and β32m proteins were analyzed by bioinformatics software and circular dichroism（CD） spectrum. The three-dimentional（3D） structures of MHC-I-BSP and β2m proteins were estimated by homology modeling. The analysis of proteins will provide useful genetic materials and helpful information to explore the CD8+ T cell responses in HCV/GBV-B chimera infected marmoset models.（5） MHC-I/peptide monomers were constructed by the method of Altman. But after refolding, no monomers form according to the result of HPLC and ELISA. The reason why no monomers form was analyzed and provides a clear direction for subsequent experiments.