Hostname: page-component-cd9895bd7-gbm5v Total loading time: 0 Render date: 2024-12-28T11:23:47.306Z Has data issue: false hasContentIssue false
  • English
  • Français

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]
Get access Rights & Permissions [Opens in a new window]

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

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