Characterization of bioactive recombinant woodchuck interleukin-2 amplified by RLM-RACE and produced in eukaryotic expression system.
Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable laboratory model of hepatitis B virus (HBV) infection, in which molecular, immunological and pathological events occurring in infected humans are adequately reflected. To advance studies on T cell immune responses and propagation of hepadnavirus in T lymphocytes in this animal model, we determined the complete sequence of woodchuck interleukin-2 (wIL-2) cDNA by utilizing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) reaction. The wIL-2 sequence revealed a single open reading frame encoding for the predicted precursor protein comprised of a signal peptide and a 134 amino acid-long mature protein. The mature wIL-2 protein produced in the Escherichia coli expression system, designated as ec-rwIL-2, was found to be immunogenic but not biologically active. In contrast, precursor wIL-2 protein cloned into baculovirus transfer vector and expressed in Sf9 cells, designated as bac-rwIL-2, demonstrated functional competence. Further, bac-rwIL-2 was able to stimulate proliferation and to induce multiple daughter cell generations in woodchuck T cells, as well as facilitated the survival of standard IL-2-dependent mouse CTLL-2 cells in culture. Western blot analysis of bac-rwIL-2 using antibodies generated against ec-rwIL-2 revealed a single protein band of 15.5kDa. The availability of biologically active recombinant wIL-2 should facilitate ex vivo studies on functional competence of woodchuck T lymphocytes derived from different stages of hepadnaviral hepatitis and assist in recognizing their contribution to the pathogenesis of liver injury in the woodchuck model of hepatitis B.
Gujar
Shashi A
SA
Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, Nfld A1B 3V6, Canada.
Michalak
Tomasz I
TI
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D008213:Lymphocyte Activation; D008392:Marmota / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000235:genetics / Q000821:virology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
D018076:DNA, Complementary; D007376:Interleukin-2; D012333:RNA, Messenger; D011994:Recombinant Proteins