猪α干扰素与白介素-18融合基因的克隆、原核表达及其抗病毒活性的初步研究

Abstract

Abstract: In order to explore the antiviral effect of porcine interferon-α and interleukin-18 fusion gene （poIFN-α-IL-18）, poIFN-α-IL-18 was amplified from pMD18-T/IFN-α and pMD18-T/IL-18 by using fusion-PCR in this study. The recombinant expression plasmid pET-28b/IFNα-IL18 was constructed by inserting poIFNα-IL18 into vector pET-28b（+） separately. SDS-PAGE was used for analyzing the protein after induced by IPTG. The protein was purified by Ni²⁺-NTA. Antiviral activities of the purified protein were tested by cytopathogenic effect inhibition assay. Results showed that the recombinant expression plasmid pET-28b/IFNα-IL18 was successfully constructed. SDS-PAGE could detect the protein bands about 37 ku. After Ni²⁺-NTA affinity chromatography purification, the activity of purified fusion protein against astrovirus was better than the single protein. Antiviral activities of the purified proteins, poIFN-α-IL-18, poIFN-α and poIL-18, were about 1.03×10⁵,1.28×10⁴ and 0.11×10⁴ U/mg.

Key words: fusion gene; cloning; expression; antiviral activity

ZHANG Pan-pan, HE Jing, KANG Yin-feng, XIANG Bin, REN Tao. Cloning and Prokaryotes Expression of Porcine Interferon-α and Interleukin-18 Fusion Gene and Detection of its Antiviral Activity[J]. .

References

1 王丽，刘金波，何涛，等. 猪干扰素-γ基因在大肠杆菌中的克隆表达及其活性测定［J］. 中国医学工程，2007，15(3)：35～37.
2 王彦彬，崔保安，陈红英，等. 猪干扰素α在昆虫细胞中分泌表达及其抗病毒活性检测［J］. 中国农业科学，2009，42(7)：1435～1441.
3 刘剑锋，王立贤，张沅，等. 猪干扰素基因研究进展及其在抗病育种中的应用展望［J］. 中国畜牧兽医，2003，30(3)：30～33.
4 林毅，奉孝书. 猪干扰素生产及应用研究进展［J］. 今日畜牧兽医，2010，5(2)：69～70.
5 高立芬. IL-18与免疫相关性疾病［J］. 国外医学—免疫学分册，2001，24(3)：165～167.
6 曹雪涛. 干扰素与白介素之间的相互关系［J］. 微生物学免疫学进展，1989，1(8）：30～38.
7 Chevaliez S, Pawlotsky J M. Interferons and their use in persistent viral infections［J］. Handb Exp Pharmacol, 2009, 189: 203～241.
8 Dinarello C A, Novick D, Puren A J, et al. Overview of interleukin 18: More than an interferon-gamma inducing factor[J］. J Leukoc Biol, 2003, 63(6): 658～664.
9 Lefevre F, Boulay V. A novel and atypical type one interferon gene expressed by trophoblast during early pregnancy［J］. J Biol Chem, 1993, 268: 19760～19768.
10 Nakamori M, Iwahashi M, Nakamura M, et al. Intensification of antitumor effect by T helper-1 dominant adoptive immunogene therapy for advanced orthotopic colon cancer［J］. Clin Cancer Res, 2003, 9(6): 2357～2365.
11 Pestka S, Krause C D, Walter M R. Interferons, interferon-like cytokines, and their receptors［J］. Immunol Rev, 2004, 202: 8～32.
12 Reed L J, Muench H. A simple method of estimating fifty per cent endpoints［J］. American Journal of Epidemiology, 1938, 27(3)： 493～497.
13 Roberts R M, Liu L, Alexenko A. New and atypical families of type Ⅰ interferons in mammals: Comparative functions, structures, and evolutionary relationships［J］. Prog Nucl Acid Res Mol Biol, 1997, 56: 287～325.
14 Vandenbroeck K, Nauwynck H, Vanderpooten A, et al. Recombinant porcine IFN-γ potentiates the secondary IgG and IgA responses to an inactied suid herpesvirus-1 vaccine and reduces postchallenge weight loss and fever in pigs［J］. J Interferon Cytokine Res, 1998, 18(9): 739～744.

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Cloning and Prokaryotes Expression of Porcine Interferon-α and Interleukin-18 Fusion Gene and Detection of its Antiviral Activity

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