猫干扰素FeIFN-ω和FeIFN-α的克隆、表达和体外生物学活性研究
摘要
猫干扰素omega(FeIFN-ω)属Ⅰ型干扰素,具有抗病毒,抗增殖和免疫调节功能。干扰素的生物学活性具有种属限制性,但也有报道,人IFN-ω对其他种属也有作用,而且较之IFN-α具有更为广谱的生物学活性和更高的抗病毒能力。干扰素的研究已有几十年的历史,但对猫源干扰素的研究,目前国内尚未见报道。
1.本试验从感染猫传染性腹膜炎病毒(FIPV)的普通缅甸猫脾脏中克隆猫ω型干扰素基因(FeIFN-ω);以实验室保存的含猫α型干扰素基因(FeIFN-α)的质粒Pet-28a/FeIFN-α为模板,PCR扩增FeIFN-α成熟区域。利用分子生物学技术,分别构建原核表达载体pET-His/FeIFN-ω和pET-His/FeIFN-α,转化大肠杆菌Rosetta(DE3),得到高效稳定的包涵体表达。通过包涵体洗涤和Ni-NTA亲和层析两步纯化,蛋白纯度能达到95%以上。
2.本研究采用以细胞致病效应(CPE)抑制为基础的抑制微量测定法,根据结晶紫染色,利用酶标仪测定干扰素的抗病毒活性结果。试验从两个方面比较了干扰素FeIFN-ω、FeIFN-α的体外生物学活性,同时以日本干扰素产品Intercat~(?)作为参照。第一,从干扰素广谱抗病毒活性方面,比较了三种干扰素亚型抵抗四种不同种属病毒的活性;第二,从干扰素生物学活性的种属限制性方面,比较了三种干扰素亚型在五种不同种属细胞上,抵抗水泡性口炎病毒的活性。结果显示,第一,重组猫ω型干扰素(FeIFN-ω)抵抗四种病毒的活性明显高于重组猫α型干扰素(FeIFN-α),尤其对H_9N_2亚型禽流感病毒(AIV),FeIFN-ω的活性是FeIFN-α的160倍,对犬瘟热病毒(CDV),FeIFN-ω的活性是FeIFN-α的4倍,而日本同类产品Intercat~(?)对CDV和AIV均未表现活性。第二,FeIFN-ω在五种不同种属的细胞上均显示高效的抗病毒活性,在非洲绿猿猴肾细胞(VERO)和牛肾细胞(MDBK)上,FeIFN-ω抗VSV的活性能达到0.13×10~6U/mg,是FeIFN-α活性的15～30倍。在人宫颈癌细胞(HeLa)和猪肾细胞(PK-15)上,FeIFN-ω的抗病毒活性达到5×10~4U/mg,是FeIFN-α的100倍。而Intercat~(?)只在猫肾细胞(CRFK)上表现抗病毒活性,在其他种属细胞系上均无活性。
本研究这为下一步的临床研究提供了重要的理论依据,为寻求新的有效的动物抗病毒药物,构建高效的基因工程干扰素制品提供了理论依据和研究手段。
IFN-ωand IFN-αboth belong to typeⅠinterferon which have antiviral, antiproliferative,immunomodulatory activities.The bio-activity of IFNs are limited by their species-specific,however,some reports indicated that the IFN-ωderived from human also has the same effects on other species,even could be higher effective than IFN-αon aspect of antiviral activity.
1.The FeIFN-ωgene from a Burmese cat infected with feline infectious peritonitis virus(FIPV),FeIFN-αgene was cloned based on the Recombinent plasmid Pet-28a/FeIFNα.Then the two types of feline interferon genes were subcloned into the pET-His vector,and expressed in Escherichia coli Rosetta(DE3).Recombinant interferons were induced by IPTG and inclusion body was purified by affinity chromatography with immobilized nickel chelating NTA(Ni-NTA).In this study,we successfully construct the higher expression systems of FeIFN-ωand FeIFN-αin Escherichia coli Rosetta and seeked a effective method to purify the inclusion body. The purity of INFs reached to 95%.
2.The comparison of the antiviral activity of two interferon:FeIFN-ω、FeIFN-αfocus on two aspects based on the control of Intercat(?).First,we compared antiviral activity of three subtypes of interferons against different viruses.Second,we compared the antiviral activity of them on different animal cells against VSV.The antiviral activity was assayed by the ability of FeIFNs to inhibit the cytopathic effects (CPE) of vesicular stomatitis virus.When the CPE reached 75～100%in viral control, the cells were strained with crystal violet absorbance was measured at 570 nm on a Sunrise microtiter plate reader.Through the research of bio-activity of FeIFNs,the results showed that:the antiviral activities against four viruses of FeIFN-ωwere higher than those of FeIFN-α.Against H_9N_2 subtype avian influenza virus(AIV) and canine distemper virus(CDV),the antiviral activities of FeIFN-ωwere 160 folds and 4 folds higher than those of FeIFN-α,and the Japanese product Intercat(?) showed no antiviral activity.The antiviral activities against VSV on five different mammal cells of FeIFN-ωwere higher than those of FeIFN-α.On VERO and MDBK cells,the antiviral activities of FeIFN-ωwere15 folds and 30 folds higher than those of FeIFN-α,on HeLa and PK-15,the antiviral activities of FelFN-ωwere100 folds higher than those of FelFN-α,and the Japanese product Intercat(?) showed no antiviral activity on these cells except CRFK cell.Meanwhile.
All these results can provide strong support to antiviral therapy in clinic and new drug development of pets.
1.本试验从感染猫传染性腹膜炎病毒(FIPV)的普通缅甸猫脾脏中克隆猫ω型干扰素基因(FeIFN-ω);以实验室保存的含猫α型干扰素基因(FeIFN-α)的质粒Pet-28a/FeIFN-α为模板,PCR扩增FeIFN-α成熟区域。利用分子生物学技术,分别构建原核表达载体pET-His/FeIFN-ω和pET-His/FeIFN-α,转化大肠杆菌Rosetta(DE3),得到高效稳定的包涵体表达。通过包涵体洗涤和Ni-NTA亲和层析两步纯化,蛋白纯度能达到95%以上。
2.本研究采用以细胞致病效应(CPE)抑制为基础的抑制微量测定法,根据结晶紫染色,利用酶标仪测定干扰素的抗病毒活性结果。试验从两个方面比较了干扰素FeIFN-ω、FeIFN-α的体外生物学活性,同时以日本干扰素产品Intercat~(?)作为参照。第一,从干扰素广谱抗病毒活性方面,比较了三种干扰素亚型抵抗四种不同种属病毒的活性;第二,从干扰素生物学活性的种属限制性方面,比较了三种干扰素亚型在五种不同种属细胞上,抵抗水泡性口炎病毒的活性。结果显示,第一,重组猫ω型干扰素(FeIFN-ω)抵抗四种病毒的活性明显高于重组猫α型干扰素(FeIFN-α),尤其对H_9N_2亚型禽流感病毒(AIV),FeIFN-ω的活性是FeIFN-α的160倍,对犬瘟热病毒(CDV),FeIFN-ω的活性是FeIFN-α的4倍,而日本同类产品Intercat~(?)对CDV和AIV均未表现活性。第二,FeIFN-ω在五种不同种属的细胞上均显示高效的抗病毒活性,在非洲绿猿猴肾细胞(VERO)和牛肾细胞(MDBK)上,FeIFN-ω抗VSV的活性能达到0.13×10~6U/mg,是FeIFN-α活性的15～30倍。在人宫颈癌细胞(HeLa)和猪肾细胞(PK-15)上,FeIFN-ω的抗病毒活性达到5×10~4U/mg,是FeIFN-α的100倍。而Intercat~(?)只在猫肾细胞(CRFK)上表现抗病毒活性,在其他种属细胞系上均无活性。
本研究这为下一步的临床研究提供了重要的理论依据,为寻求新的有效的动物抗病毒药物,构建高效的基因工程干扰素制品提供了理论依据和研究手段。
IFN-ωand IFN-αboth belong to typeⅠinterferon which have antiviral, antiproliferative,immunomodulatory activities.The bio-activity of IFNs are limited by their species-specific,however,some reports indicated that the IFN-ωderived from human also has the same effects on other species,even could be higher effective than IFN-αon aspect of antiviral activity.
1.The FeIFN-ωgene from a Burmese cat infected with feline infectious peritonitis virus(FIPV),FeIFN-αgene was cloned based on the Recombinent plasmid Pet-28a/FeIFNα.Then the two types of feline interferon genes were subcloned into the pET-His vector,and expressed in Escherichia coli Rosetta(DE3).Recombinant interferons were induced by IPTG and inclusion body was purified by affinity chromatography with immobilized nickel chelating NTA(Ni-NTA).In this study,we successfully construct the higher expression systems of FeIFN-ωand FeIFN-αin Escherichia coli Rosetta and seeked a effective method to purify the inclusion body. The purity of INFs reached to 95%.
2.The comparison of the antiviral activity of two interferon:FeIFN-ω、FeIFN-αfocus on two aspects based on the control of Intercat(?).First,we compared antiviral activity of three subtypes of interferons against different viruses.Second,we compared the antiviral activity of them on different animal cells against VSV.The antiviral activity was assayed by the ability of FeIFNs to inhibit the cytopathic effects (CPE) of vesicular stomatitis virus.When the CPE reached 75～100%in viral control, the cells were strained with crystal violet absorbance was measured at 570 nm on a Sunrise microtiter plate reader.Through the research of bio-activity of FeIFNs,the results showed that:the antiviral activities against four viruses of FeIFN-ωwere higher than those of FeIFN-α.Against H_9N_2 subtype avian influenza virus(AIV) and canine distemper virus(CDV),the antiviral activities of FeIFN-ωwere 160 folds and 4 folds higher than those of FeIFN-α,and the Japanese product Intercat(?) showed no antiviral activity.The antiviral activities against VSV on five different mammal cells of FeIFN-ωwere higher than those of FeIFN-α.On VERO and MDBK cells,the antiviral activities of FeIFN-ωwere15 folds and 30 folds higher than those of FeIFN-α,on HeLa and PK-15,the antiviral activities of FelFN-ωwere100 folds higher than those of FelFN-α,and the Japanese product Intercat(?) showed no antiviral activity on these cells except CRFK cell.Meanwhile.
All these results can provide strong support to antiviral therapy in clinic and new drug development of pets.
引文
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[2]XU DZ,HUANG K L,ZHAO K,et al.Vaccination with recombinant HBSAG-HBIG complex in healthy adults[J].Vaccine,2005,23(20);2658-2664.
[3]王子轼,王涛.干扰素研究进展[J].江苏畜牧兽医职业技术学院学报,2003(1):41-46。
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[5]张学文,章怀云。干扰素诱导细胞抗病毒的分子机制。湖南农业大学学报(自然科学版),2001,27(1):79-83。
[6]Roberts MR,Cross JC,Leaman DW et al.Unique features of the tropHoblast interferons.PHarm Ther,1991,51:329-345.
[7]Farin C.E.,Imakawa K.,Hansen T.R.,McDoonell,et al.,Expression of trophoblastic interferon genes in sheep and cattle.Biological Reproduction,1990,43:210-218.
[8]Kenji Oritani,Paul W,Kincade,Cai Zhang et al.Type Ⅰ interferons and Limitin:a comparison of structures,receptors,and functions.Cytokine and Growth Factor Rev,2001,12:337-348.
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[12]Oritani K,Kincade PW,Tomiyama Y.Limitin:an interferonlike cytokine without myelo-erythroid suppressive properties.J.Mol.Med.(in press).
[13]Sekellick MJ,Marcus PI.Interferon induction by viruses.Virology 1982;117:280-5.
[14]史喜菊。牛IFN-α/γ基因的克隆、表达及重组蛋白的应用研究:[博士学位论文]。北京:中国农业大学,2004。
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[2]XU DZ,HUANG K L,ZHAO K,et al.Vaccination with recombinant HBSAG-HBIG complex in healthy adults[J].Vaccine,2005,23(20);2658-2664.
[3]王子轼,王涛.干扰素研究进展[J].江苏畜牧兽医职业技术学院学报,2003(1):41-46。
[4]候云德。分子病毒学。北京:学苑出版社,1990,598-642。
[5]张学文,章怀云。干扰素诱导细胞抗病毒的分子机制。湖南农业大学学报(自然科学版),2001,27(1):79-83。
[6]Roberts MR,Cross JC,Leaman DW et al.Unique features of the tropHoblast interferons.PHarm Ther,1991,51:329-345.
[7]Farin C.E.,Imakawa K.,Hansen T.R.,McDoonell,et al.,Expression of trophoblastic interferon genes in sheep and cattle.Biological Reproduction,1990,43:210-218.
[8]Kenji Oritani,Paul W,Kincade,Cai Zhang et al.Type Ⅰ interferons and Limitin:a comparison of structures,receptors,and functions.Cytokine and Growth Factor Rev,2001,12:337-348.
[9]Leung D.W.,Daniel J.C.,Goeddel D.V.,The structure and bacterial expression of thee distinct bovine interferon-βgenes.Journal of Biotechnology,1984,May,58-63.
[10]杨汉春.动物免疫学[M].2版:北京:中国农业出版社,2004:98
[11]龚国忠,郑宣鹤,赖力英,等.Ⅰb型丙肝病毒在外周血单核细胞感染状态及其对IFN 应答的影响[J].中华微生物和免疫,1998,18(6):4522-4524。
[12]Oritani K,Kincade PW,Tomiyama Y.Limitin:an interferonlike cytokine without myelo-erythroid suppressive properties.J.Mol.Med.(in press).
[13]Sekellick MJ,Marcus PI.Interferon induction by viruses.Virology 1982;117:280-5.
[14]史喜菊。牛IFN-α/γ基因的克隆、表达及重组蛋白的应用研究:[博士学位论文]。北京:中国农业大学,2004。
[15]Roberts R.,Liu L.,Quingtao Guo et al.,The evolution of the type Ⅰ interferons.Journal of Interferon and Cytokine Research,1998,18:805-816.
[16]Kenji O.,Kincade P.W.,Cai Zhang,Yoshiaki T.,Type Ⅰ interferons and limitin:a comparison of structures,receptors,and functions.Cytokine and Growth Factor Reviews,2000,12:337-347.
[17]Renqiu Hu,Joseph B.,Schmeisser H.,et at.,Human IFN-α protein engineering:The amino acid residues at positions 86 and 90 are important for antiproliferative activity.The Journal of Immunology,2001,167:1482-1489.
[18]Radhakrishnan R.,Walter LJ,Huza A,et al.,Zinc mediated dimmer of human interferon-α2b revealed by X-ray crystallography.Structure,1996,4:1453-1463.
[19]Klaus W.,Gsell B.,Labhardt AM,et al.,The thee-dimensional high resolution structure of human interferon-α2a determined by heteronuclear NMR spectroscopy in solution.J Mol Biol,1997,274:661-675..
[20]enda T,Saitoh S,Mitsui Y.Refined crystal structure of recombinant murine interferon-βat 2.15 A resolution.J Mol Biol,1995,253:187-207.
[21]arpusas M,Nolte M,Benton CB,et al.,The crystal structure of human interferon-β at 2.2 A resolution.Proc Natl Acad Sci USA,1997,94:1813-1181.
[22]Kile BT,Schulman BA,Alexander WS,et al.,The SOCS box:a tale of destruction and degradation.Trends Biochem Sci,2002,27:235-241.
[23]Walter MR,Narula SK.Crystal structure of a complex between interferon-γ and its soluble high-affinity receptor.Nature,1995,376:230-235.
[24]Thiel DJ,Observation of an unexpected third receptor molecule in the crystal structure of human interferon-γ receptor complex.Structure Fold Des,2000,8:927-936.
[25]Krause CD,Seeing the light:preassembly and ligand-induced changes of the interferon-γreceptor complex in cells.Mol Cell Proteomics,2002,1:805-815.
[26]Jonson H.M.,Bazer F.W.,Szente B.E.,et al.,How interferons fight disease.Sci.Am.,1994,68:270-275.
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