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Expression and biological activity assay of bovine interleukin-18 fusion protein

Published online by Cambridge University Press:  27 June 2008

Tian Zhao-Ju ,
Zheng Yu-Shu ,
Liu Cui-Yan ,
Hu Jing-Dong  and
Zhao Hong-Kun
Tian Zhao-Ju
Affiliation:
The Faculty of Laboratory Medicine, Taishan Medical University, Tai'an 271016, China College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China
Zheng Yu-Shu
Affiliation:
College of Animal Science, Henan Science College, Xinxiang 453003, China
Liu Cui-Yan
Affiliation:
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China
Hu Jing-Dong
Affiliation:
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China
Zhao Hong-Kun*
Affiliation:
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

The cDNA of bovine interleukin-18 (BoIL-18) was subcloned into pGEX6P-1 vector and transformed into Escherichia coli BL21(DE3). The recombinant protein was successfully expressed in E. coli by induction of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 0.3 mmol/l for 8 h. SDS-PAGE indicated that the BoIL-18 fusion protein, 44 kDa, was highly expressed. Densitometric scanning showed that the fusion protein expression was about 31.8% of the total bacterial protein. The biological activity of the chromatographically purified protein was assayed. The peripheral blood mononuclear cells (PBMC) proliferation test indicated that the BoIL-18 fusion protein could enhance PBMC proliferation when its concentration was more than 0.10 mg/l. Enzyme-linked immunosorbent assay (ELISA) showed that the BoIL-18 fusion protein could induce interferon (IFN)-γ production from spleen lymphocytes when it was at a concentration of more than 0.20 mg/l, and that the inducing effect of BoIL-18 fusion protein on IFN-γ was directly proportional to its concentration. This verified that the purified BoIL-18 fusion protein possessed a functional activity and could be applied in further studies.

Keywords

biological activity assaybovine interleukin-18prokaryotic expressionpurification
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 5 , Issue 1 , April 2008 , pp. 7 - 11
Copyright
Copyright © China Agricultural University 2008

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Footnotes

First published in Journal of Agricultural Biotechnology 2007, 15(4): 579–583

References

Chen, KS (2004) The research evolve of biological activity of IL-18. Jiujiang Medical Journal (Natural Sciences) 4: 81–84.Google Scholar
Goto, H, Osaki, T, Nishino, K, et al. (2002) Construction and analysis of new vector systems with improved interleukin-18 secretion in axenogeneic human tumor model. Journal of Immunotherapy 25(1): 535–541.CrossRefGoogle ScholarPubMed
Huang, X, Zhou, J, Zhang, FK and Cai, SM (2002) Study on antitumor effect of IL-l8 on ovarian carcinoma cell line HO-8910. China Oncology 12(2): 106–109.Google Scholar
Liu, WQ, Hu, JD, Yang, SH, Zhao, HK, Gao, YD and Zhong, JF (2005) cDNA clone and expression of mature bovine interleukin-18 gene. Acta Veterinaria et Zootechnica Sinica 36(9): 873–876.Google Scholar
Nakamori, M, Iwahashi, M, Nakamura, M, Ueda, K, Zhang, X and Yamaue, H (2003) Intensification of antitumor effect by T helper 1-dominant adoptive immunogene therapy for advanced orthotopic colon cancer. Clinical Cancer Research 9(6): 2357–2365.Google Scholar
Okamura, H, Tsutsui, H, Komatsu, T, et al. (1995) Cloning of a new cytokine that induces IFN-γ production by T cells. Nature 378(11): 88–91.CrossRefGoogle ScholarPubMed
Shoda, LK, Zarlenga, DS and Hirano, A (1999) Cloning of a cDNA encoding bovine interleukin-18 and analysis of IL-18 expression in macrophages and its IFN-gamma-inducing activity. Journal of Interferon and Cytokine Research 19(10): 1169–1177.CrossRefGoogle ScholarPubMed
Yoshimura, K, Hazama, S, lizuka, N, et al. (2001) Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igκ leader sequence. Cancer Gene Therapy 8(1): 9–16.CrossRefGoogle ScholarPubMed