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1 生物工程学报 Chin J Biotech 2009, June 25; 25(6): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 动物及兽医生物技术 I II 陈雪梅, 薛清华, 祝荣格, 付显华, 杨利敏, 孙蕾, 刘文军, : 干扰素 (IFN) 是由多种细胞受病毒感染或其他生物诱导剂刺激而产生的天然蛋白质, 主要功能为抗病毒增殖调节免疫反应和激活免疫细胞等本研究克隆并测序了猪干扰素 (PoIFN)α γ α γ 及 ω 基因构建原核表达载体 pet-his/poifn-α pet-his/poifn-γ pet-his/poifn- αγ 和 pet-his/poifn-ω, 转化大肠杆菌 Rosetta(DE3) 进行表达, 经纯化复性得到具有生物学活性的蛋白用细胞病变抑制法在 Marc-145/PRRSV Marc-145/VSV PK-15/VSV Vero/VSV MDBK/VSV 系统上进行抗病毒活性测定, 结果表明猪 α 和 αγ 融合干扰素有较为显著的抗病毒活性, 抗 PRRSV 活性高达 10 8 U/mg; 猪 γ 干扰素活性效价约为 α 干扰素的 1/2 到 1/3; 猪 ω 干扰素几乎未检测到抗病毒活性, 需进一步验证本研究对干扰素在抗病毒提高机体免疫方面的应用提供了理论依据 : 猪干扰素, 抗病毒活性 Comparison of antiviral activities of porcine interferon type I and type II Xuemei Chen, Qinghua Xue, Rongge Zhu, Xianhua Fu, Limin Yang, Lei Sun, and Wenjun Liu Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China Abstract: Interferons (IFNs) are natural proteins produced by wide variety of cells in response to viral infection or other biological inducers, and they execute diversified functions as antiviral defense, immune activation and cell growth regulation. Four genes encoding porcine interferons (PoIFN), PoIFN-α, PoIFN-γ, PoIFN-αγ or PoIFN-ω, were cloned and sequenced. The four types of porcine interferon genes were subcloned into the pet-his vector, and expressed in Escherichia coli Rosetta (DE3). The recombinant products were purified and renaturalized from inclusion bodies to obtain a native state of well biological activity. Antiviral activity assays for porcine interferons were performed and evaluated by standard procedures in following cell/virus test systems: Marc-145/PRRSV, Marc-145/VSV, PK-15/VSV, Vero/VSV or MDBK/VSV. The data showed that both PoIFN-α and PoIFN-αγ demonstrated significant antiviral activities, and the titer of them against PRRSV was up to 10 8 U/mg. PoIFN-γ had approximately half or one-thirds antiviral activity of PoIFN-α. PoIFN-ω showed inconspicuous antiviral activity. Keywords: porcine interferon, antiviral activity Received: March 18, 2009; Accepted: April 21, 2009 Supported by: Ministry of Science and Technology of China Program (No. 2006BAD06A04), Hundreds of Talents Program of Chinese Academy of Sciences. Corresponding author: Wenjun Liu. Tel: ; Fax: ; liuwj@im.ac.cn 国家科技支撑计划 (No. 2006BAD06A04), 中国科学院百人计划资助

2 : I II 807,, 3 : I II I, IFN-α β ω σ τ ω κ ξ, ; II IFN- γ, ;, IFN-λ1 IFN-λ2 IFN-λ3 [1 4] 1985, Hauptmann Swetly cdna ω [5], 116~132 α [6] ω, [2,7], [8] 1991, Mege 5 ω, 2, 3 [9],,,,,,, [10,11] PoIFN-α PoIFN-γ PoIFN-αγ PoIFN-ω, 4, 1 材料和方法 1.1 材料 DH5α Rosetta(DE3) ; pmd18-t TaKaRa ; pet-his (MDBK) (Vero) (Marc-145) (PK-15); (VSV) (PRRSV) TaKaRa ; Trizol superscript TM III first-strand synthesis system for RT-PCR Invitrogen ; Ni-NTA QIAGEN ; DMEM Gibco ; Promega ; 1.2 方法 RNA RT-PCR cdna Trizol VSV 12 h PK-15 RNA RNA, Invitrogen superscript TM first-strand synthesis system for RT-PCR cdna PCR T GenBank α (Accession No. M28623) γ (Accession No. EU249804), cdna, PCR PoIFN-α PoIFN-γ ;, PoIFN-α PoIFN-γ, PCR, PoIFN-αγ,, 2 ggcgggggtggcagcggtggcgggggatca, GGGGSGGGGS ; ω (GenBank S. scrofa domestica PoIFN-alpha -5 gene, Accession No. X57196), PK-15 cdna, PCR PoIFN-ω BamH I Hind III ( 1) PoIFN-α PoIFN-γ PoIFN-αγ PoIFN-ω PCR pmd18-t, DH5α, BamH I Hind III, PoIFN-ω MegAlign PoIFN-ω GenBank Clustal W,

3 808 ISSN CN /Q Chin J Biotech June 25, 2009 Vol.25 No.6 表 1 克隆目的基因 PoIFN-α PoIFN-γ PoIFN-αγ 和 PoIFN-ω 的引物 Table 1 Primers for cloning of target genes Genes Primers Primer sequences (5 3 ) PoIFN-α PoIFN-γ PoIFN-ω PoIFN-αγ Fusion -reverse Fusion -forward cgcggatccatgtgtgacctgcctcagaccc cccaagcttactccttcttcctgagtctgtct cgcggatccatgtcttactgccaggcgcccttt cccaagcttattttgatgctctctggcctt cgcggatccatgtgtgacctgtctcagaa cccaagcttaggatgaccccaggtctac cgcggatccatgtgtgacctgcctcagaccc ccgccaccgctgccacccccgccctccttcttcc tgag tggcagcggtggcgggggatcatcttactgccag gcgc cccaagcttattttgatgctctctggcctt pet-his/poifn-α pet-his/ PoIFN-γ pet-his/poifn-αγ pet-his/poifn-ω pmd18/poifn-α pmd18/ PoIFN- γ pmd18/poifn- αγ pmd18/poifn-ω BamH I Hind III, pet-his, pet-his/poifn- α pet-his/poifn-γ pet-his/ PoIFN-αγ pet-his/poifn-ω Rosetta(DE3), 37 o C OD ~0.6, IPTG 4 h SDS-PAGE α γ αγ ω, SDS-PAGE, PBS 2 mol/l 1 mol/l NaCl 2, 6 mol/l 5 h 4 o C, r/min 15 min,, Buffer B (8 mol/l, 0.1 mol/l NaH 2 PO 4, 0.01 mol/l Tris Cl, ph 8.0), Buffer B Ni-NTA Agarose, 30 min ph,, SDS-PAGE (4 o C), 1: 30,, 4 o C 48 h : 0.5 mol/l 2.5 mmol/l EDTA 10%~20%, 0.1 mol/l Tris 1 mmol/l PoIFN-α PoIFN-γ PoIFN-αγ PoIFN-ω 0.22 μm, 4 o C Bradford [12] α γ αγ ω PoIFN-α PoIFN-γ PoIFN-αγ PoIFN-ω / : Marc-145/PRRSV Marc-145/VSV PK-15/VSV Vero/VSV MDBK/VSV (CPE) [13 15] ~ /ml, 100 μl/ 96, 37 o C 5% CO 2 6~8 h, ( ) 4 6, 10% FBS DMEM, 2~4 18 h, DMEM (300~500 TCID 50 ), 30 h(vsv) 72 h(prrsv), 90%,, (Tekan, Grödig, Austria) 570 nm, Reed-Muench [15] 2~3 2 结果 2.1 PoIFN-α PoIFN-γ 和 PoIFN-αγ 基因鉴定 PoIFN-α PoIFN-γ PoIFN-αγ 1%, 501 bp 441 bp 969 bp, 2.2 PoIFN-ω 基因序列分析 VSV PK-15 RNA N 23, 504 bp, 168 GenBank, X57196, α,, ω, : 1) ω α ( 2); 2) (Cys) (Pro) ω, ; 3) 109~113 5

4 : I II 809, C 6, ω, α ( 1) [2] ; 4) MegAlign Clustal W,, ω ( 2) 2.3 重组猪干扰素 α γ αγ 和 ω 的表达纯化与复性 Rosetta(DE3), 37 o C IPTG 4 h, SDS-PAGE, PoIFN-α PoIFN-γ PoIFN-αγ PoIFN-ω 19 kd 17 kd 36 kd 19 kd, 90% ( 3) N Bradford mg/ml mg/ml mg/ml mg/ml 表 2 猪人猫和牛干扰素在核苷酸和氨基酸水平上的全序列 ( 含信号肽 ) 相似性 Table 2 Similarities (%) of nucleotides (top line) and amino acids (bottom line) full-length sequences among porcine, human, feline and bovine interferon * PoIFN alpha * HuIFN alpha * FeIFN alpha * BoIFN alpha * PoIFN gamma * HuIFN gamma * FeIFN gamma * BoIFN gamma * PoIFN omega * HuIFN omega * 76.3 FeIFN omega * BoIFN omega * same sequences. 图 1 干扰素成熟区氨基酸序列比较 Fig. 1 Alignment of mature amino acid sequences of interferons. Conserved positions of cysteine and proline residues present in real lined box, and an extension of six amino acids at the C-terminal presents in dotted lined box.

810 ISSN1000-3061 CN11-1998/Q Chin J Biotech June 25, 2009 Vol.25 No.6 图 2 干扰素核苷酸和氨基酸序列进化树 Fig. 2 Phylogenetic trees of interferons. (A) Phylogenetic trees based on nucleotide sequences.

5 810 ISSN CN /Q Chin J Biotech June 25, 2009 Vol.25 No.6 图 2 干扰素核苷酸和氨基酸序列进化树 Fig. 2 Phylogenetic trees of interferons. (A) Phylogenetic trees based on nucleotide sequences. (B) Phylogenetic trees based on amino acid sequences. α γ αγ PRRSV 10 7 ~10 8 U/mg, 表 3 猪干扰素在不同细胞系上对不同病毒的抗病毒活性比较 Table 3 Antiviral activities of porcine interferons against different virus on various cell line 图 3 SDS-PAGE 检测纯化的 PoIFN-α PoIFN- γ PoIFN-αγ 和 PoIFN-ω 蛋白 Fig. 3 SDS-PAGE analysis of purified PoIFN-α, PoIFN-γ, PoIFN-αγ and PoIFN-ω protein. M: protein marker; 1: purified PoIFN-α protein; 2: purified PoIFN-γ protein; 3: purified PoIFN-αγ protein; 4: purified PoIFN-ω protein. 2.4 重组猪干扰素 α γ αγ 和 ω 的抗病毒活性比较 Marc-145 PK-15 Vero MDBK VSV PRRS, Reed-Muench, α αγ, γ α 1/2 1/3( 3, 4), ω ( ),, Marc-145 / PRRSV Antiviral activities ( 10 5 U/mg) Marc-145 / VSV PK-15 / VSV Vero / VSV MDBK /VSV pifn-α pifn-γ pifn-αγ 讨论 I 4, PoIFN-α PoIFN-γ PoIFN-αγ PoIFN- ω, PoIFN-α PoIFN-γ GenBank [16], [17] Xia [18] Cencic [19] 2, PoIFN-αγ Ⅱ PoIFN-ω 1991 [9], α, Cys

6 : I II 811 图 4 Fig. 4 猪干扰素在不同细胞系上对不同病毒的抗病毒活性比较 Antiviral activities of porcine interferons against different virus on various cell line. Pro, α, 4, PoIFN-α PoIFN-γ PoIFN-αγ VSV PRRSV Marc-145 PK-15 Vero MDBK,,,, : Marc-145/PRRSV MDBK/VSV,, 10 7 ~10 8 U/mg,, 10 5 U/mg, ω, :, ;, PoIFN-αγ, I,,,,,,,,, REFERENCES [1] Lefevre F, Boulay V. A novel and atypical type one interferon gene expressed by trophoblast during early pregnancy. J Biol Chem, 1993, 268: [2] Roberts RM, Liu L, Alexenko A. New and atypical families of type I interferons in mammals: Comparative functions, structures, and evolutionary relationships. Prog Nucl Acid Res Mol Biol, 1997, 56: [3] Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors. Immunol Rev, 2004, 202: [4] Chevaliez S, Pawlotsky JM. Interferons and their use in persistent viral infections. Handb Exp Pharmacol, 2009, 189: [5] Hauptmann R, Swetly P. A novel class of human type I interferons. Nucl Acids Res, 1985, 13(13): [6] Hughes AL. The evolution of the type I interferon gene family in mammals. J Mol Evol, 1995, 41: [7] Yang LM, Xue QH, Liu WJ, et al. Cloning and characterization of a novel feline IFN-omega. J Interferon Cytokine Res, 2007, 27(2): [8] Himmler A, Hauptmann R, Adolf GR, at al. Structure and expression in Escherichia coli of canine interferon-alpha genes. J Interferon Res, 1987, 7(2): [9] Mege D, Lefevre F, Labonnardiere C. The porcine family of interferon-omega: Cloning, structural analysis, and functional studies of five related genes. J Interferon Res, 1991, 11(6): [10] Overend C, Mitchell R, He D, et al. Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with porcine reproductive and respiratory syndrome virus. J Gener Virol, 2007, 88: [11] Moraes MP, de Los Santos T, Koster M, et al. Enhanced antiviral activity against foot-and-mouth disease virus by a combination of type I and II porcine interferons. J Virol,

812 ISSN1000-3061 CN11-1998/Q Chin J Biotech June 25, 2009 Vol.25 No.6 2007, 81(13): 7124 35. [12] Zhang LX, Zhang TF, Wang JZ. The Protein Protocols Handbook. Beijing: Science Press, 2000: 42 46.,,.

7 812 ISSN CN /Q Chin J Biotech June 25, 2009 Vol.25 No , 81(13): [12] Zhang LX, Zhang TF, Wang JZ. The Protein Protocols Handbook. Beijing: Science Press, 2000: ,,.. :, 2000: [13] John A, Armstrong. Semi-micro dye-binding assay for rabbit interferon. Appl Microbiol, 1971, 21(4): [14] Sara Rubinstein, Philip C, Familletti, et al. Convenient assay for interferons. J Virol, 1981, 37(2): [15] Chinese Pharmacopoeia Commission. The Pharmacopoeia of the People s Republic of China. Beijing: Chemical Industry Press, 2005, appendix XC: :, 2005, X C: [16] Lefevre F, La Bonnardiere C. Molecular cloning and sequencing of a gene encoding biologically active porcine alpha-interferon. J Interferon Res, 1986, 6(4): [17] Cao RB, Xu XQ, Zhou B, et al. Gene modification and high prokaryotic expression of porcine interferon alpha-1. Chin J Biotech, 2004, 20(2): ,,,. α1-., 2004, 20(2): [18] Xia C, Dan W, Wu WX, et al. Cloning and expression of interferon-α/γ from a domestic porcine breed and its effect on classical swine fever virus. Vet Immunol Immunopathol, 2005, 104(1 2): [19] Cencic A, Lefevre F, Koren S, et al, Tetracycline-controlled expression of glycosylated porcine interferon-gamma in mammalian cells. Anim Biotechnol, 1999, 10(1 2): 科学出版社科学出版中心生命科学分社新书推介宋安东等编著年 5 月出版,,,, 美国国家可再生能源实验室编著鲍杰译叶勤金浩校年 5 月出版, (NREL) 2002,, (PNNL) 2003,, ,,,, ( ) ( )

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