Isolation, Identification And Expression Analysis Of CD25 Gene In Tupaia Belangeir

Hepatitis B infection has been a global health problem. It was estimated that more than 500,000 people die annually due to HBV associated liver diseases such as cirrhosis and hepatocellular carcinoma. Research on chronic viral hepatitis B infection has been faced with difficulties that deficiency of suitable animal model hinder the proceeding on the approach of HBV pathogenesis as well as antiviral drug screening and vaccine research. Chimpanzees can be efficiently infected with HBV, however, chimpanzees are restricted in their use as experimental animals in hepatitis research due to low numbers and ethical concerns. Tree shrew( Tupaia belangeri), a squirrel-like animal close to primates in evolution, which was found to be susceptible to a variety of human viruses, including herpes simplex, rotavirus, HCV and HBV. It represents an excellent animal model for studying human infectious diseases such as HBV infection.How effectively clear HBV and cure chronic viral hepatitis B infection is still a major issues in the global medical community. The key is to break the body's immune tolerance to HBV and arouse virus-specific immune response. CD4+CD25+ regulatory T cells play a important role in suppressing virus-specific CD8+ T cell immune responses during chronic HBV infections. CD25(interleukin-2 receptor alpha chain) is an important cell surface marker for CD4+CD25+ Tregs. The use of regulatory T cells as target and block surface proteins of Treg cell such as CD25 with anti-CD25 monoclonal antibody could inhibit activation and function of Treg, stimulating the effective immune response could provided a research platform for HBV pathogenesis, treatment and prevention in chronic HBV infected tree shrew animal model.To fully utilize tree shrew as chronic HBV infection animal model. A full-length cDNA of tree shrew interleukin-2 receptor alpha chain(tsCD25) gene was obtained from tree shrew spleen by RT-PCR. The tsCD25 cDNA contains an ORF of 816 bp, encoding a 30.9 kD of peptide with a predicted 271 amino acids. The mature tsCD25 peptide contains two Sushi domain, two N-linked and multiple O-linked glycosylation sites.We compared the phylogenetic relationship between tsCD25 and other species CD25. The tsCD25 has high similarity to those of primates at the amino acid level, ranging from 65.3% to 66.4%. However, tsCD25 protein shares a lower identity with that of pig (59.5%), horse (56.9%) and cow (52.4%). Among known mammalian sequences, the rodent CD25 proteins are the most distant from tree shrew with the amino acid identity of mouse at 57.4% and rat at 46.4%.We compared tsCD25 amino acid sequence with hman CD25. The mapped domains within tsCD25 is similar to those present in human CD25. All of them have signal peptide with 21 amino acids at the N-terminus of proteins. The two molecules have common characteristic in their extracellular regeion forming the Sushi domain. In addition to 19 amino acids hydrophobic transmembrance domain, at the C-terminal of proteins have a only 13 amino acids intracellular domain.Tissue transcription analysis indicates that tsCD25 mRNA is expressed in peripheral blood, spleen and lung. We compared the effects of different mitogens to tree shrew lymphocytes proliferation. PMA and ionomycin induced significant lymphocytes proliferation compared to ConA and PHA. Lymphocytes expression analysis demonstrates that tsCD25 mRNA is up-regulated by PMA and ionomycin stimulation. Furthermore, we also detected tsCD122 and tsCD132 mRNA in various tissues and activating lymphocytes. These two subunits of the IL-2R is also detectable in lung, spleen and peripheral blood. In addition to these tissues, tsCD132 gene is also expressed in liver, kidney and bone marrow. The expression of tsCD122 and tsCD132 mRNA could be augmented by activating lymphocytes with PMA and ionomycin.We constructed recombinant expression vector by inserting coding sequence of tsCD25 gene into eukaryotic expression vector pcDNA3.1, then transfected BALA/3T3 cell to express desired genes. By G418 screening and limiting dilution to pick out monoclonal cell, we established stably express tsCD25 protein cell lines. Our results has provided a foundation for generating monoclonal antibodies to this cell surface protein, which will in turn facilitate functional studies on CD4+CD25+ Treg cells when using tree shrew as animal models during chronic infection.