The gene-encoding mature porcine interleukin-18 (pIL-18) was amplified by PCR and cloned into a prokaryotic expression vector pET-30a(+). The resulting recombinant plasmid pET-30a-pIL-18 was transformed into Escherichia coli. Expression of recombinant pIL-18 protein was induced by 1 mM isopropyl β-D-thiogalactoside at 37°C. The purified recombinant protein was used to generate a hyperimmune antiserum in a rabbit. The anti-pIL-18 serum was evaluated for its specificity and titer through indirect enzyme-linked immunosorbent assay. The specific reactivity of the anti-pIL-18 antibody was further confirmed by Western blot and immunofluorescence assays. Moreover, the pIL-18 was able to stimulate the proliferation of pig peripheral blood mononuclear cells in vitro, indicating it may have certain biological activity.
Get full access to this article
View all access options for this article.
References
1.
OkamuraH, TsutsiH, KomatsuT, YutsudoM, HakuraA, TanimotoT, TorigoeK, OkuraT, NukadaY, HattoriK, AkitaK, NambaM, TanabeF, KonishiK, FukudaS, KurimotoM. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature, 1995; 378:88–91.
2.
MunetaY, MoriY, ShimojiY, YokomizoY. Porcine interleukin 18: cloning, characterization of the cDNA and expression with the baculovirus system. Cytokine, 2002; 12:566–572.
3.
MicallefMJ, OhtsukiT, KohnoK, TanabeF, UshioS, NambaM, TanimotoT, TorigoeK, FujiiM, IkedaM, FukudaS, KurimotoM. Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production. Eur J Immunol, 1996; 7:1647–1651.
4.
NakanishiK, YoshimotoT, TsutsuiH, OkamuraH. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol, 2001; 19:423–474.
5.
WeiXQ, NiedbalaW, XuD, LuoZX, PollockKG, BrewerJM. Host genetic background determines whether IL-18 deficiency results in increased susceptibility or resistance to murine Leishmania major infection. Immunol Lett, 2004; 94:35–37.
6.
AkhianiAA, SchonK, LyckeN. Vaccine-induced immunity against Helicobacter pylori infection is impaired in IL-18-deficient mice. J Immunol, 2004; 173:3348–3356.
7.
KinoshitaK, YamagataT, NozakiY, SugiyamaM, IkomaS, FunauchiM, KanamaruA. Blockade of IL-18 receptor signaling delays the onset of autoimmune disease in MRL-Faslpr mice. J Immunol, 2004; 173:5312–5318.
8.
DegenWG, van ZuilekomHI, ScholtesNC, van DaalN, SchijnsVE. Potentiation of humoral immune responses to vaccine antigens by recombinant chicken IL-18 (rChIL-18)Vaccine, 2005; 23:4212–4218.
9.
MunetaY, ShimojiY, YokomizoYet al. Molecular cloning of porcine Interleukin-beta converting enzyme and differential gene expression of IL-beta converting enzyme, IL-beta, and IL-18 in porcine alveolar macrophases. J Interferon Cytokine Res, 1999; 19:1289–1296.
10.
LiuB, LiG, SuiX, YinJ, WangH, RenX. Expression and functional analysis of porcine aminopeptidase N produced in prokaryotic expression system. J Biotechnol, 2009; 141:91–96.
11.
RenX, WangM, YinJ, RenY, LiG. Heterologous expression of fused genes encoding the glycoprotein 5 from PRRSV: a way for producing functional protein in prokaryotic microorganism. J Biotechnol, 2010; 147:130–135.
12.
LiG, ZengY, YinJ, LillehojHS, RenX. Cloning, prokaryotic expression, and biological analysis of recombinant chicken IFN-gamma. Hybridoma, 2010; 29:1–6.
13.
RenX, GlendeJ, Al-FalahM, de VriesV, Schwegmann-WesselsC, QuX, TanL, TschernigT, DengH, NaimHY, HerrlerG. Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus. J Gen Virol, 2006; 87:1691–1695.
14.
YinJ, LiG, RenX, HerrlerG. Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol, 2007; 127:335–347.
15.
MengF, YinJ, LiX, YangW, LiG, RenX. Production and characterization of a monoclonal antibody against spike protein of transmissible gastroenteritis virus. Hybridoma, 2010; 29:345–350.
16.
WangC, RenX. Preparation, characterization of polyclonal antibody against severe acute respiratory syndrome-associated coronavirus spike protein. Hybridoma, 2010; 29,6.
17.
RenX, LiG, LiuB. Binding characterization of determinants in porcine aminopeptidase N, the cellular receptor for transmissible gastroenteritis virus. J Biotechnol, 2010; 150:202–206.
18.
LiG, HongJ, RenX, YinJ, FengS, HuoG. Prokaryotic expression of Stx 1B subunit of Escherichia coli O157:H7 and its monoclonal antibody preparation and characterization. Hybridoma, 2010; 29:283–289.
19.
RenX, WangM, YinJ, LiG. Phages harboring specific peptides to N protein of PRRSV distinguish the virus from other viruses. J Clin Microbiol, 2010; 48:1875–1881.
