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以刀豆素A(ConA)诱导的梅山猪外周血淋巴细胞中提取的总RNA为模板,采用RT-PCR方法克隆扩增出全长猪γ干扰素基因(PoIFNγ,501bp)。测序结果证实扩增得到的PoIFNγ与GenBank上所报道的猪γ干扰素基因同源性为100%。将PoIFNγ插入腺病毒穿梭载体pShuttle-CMV,与腺病毒骨架载体pAdEasy-1同源重组。在293A包装细胞系中成功包装成完整的病毒粒子。将此重组后的腺病毒连续接种传至第10代,每代提取病毒基因组,PCR均能扩增出目的片段,以细胞病变抑制法(CPE50)测定干扰素生物活性达1.3×106U/mL,证明该种表达猪γ-干扰素的重组腺病毒能稳定传代。
Abstract：The total RNA was extracted from peripheral blood mononuclear cells(PBMC) which was isolated from Meishan porcine and induced with concanavaline A(ConA),then the porcine interferon gamma gene(PoIFNγ,501bp) was amplified by RT-PCR.The result of sequencing demonstrated that the amplified PoIFNγ had 100% nucleotide homology with the other porcine IFNγ sequence published on GenBank.The objective gene(PoIFNγ) was inserted into adenoviral shuttle vector,pShuttleCMV,to construct recombinant plasmid pSh-PoIFNγ.And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37℃ for 24h on kanamycin resistance plate and selected for smaller colonies.Then,the extracted recombinant plasmid was named pAd-Sh-PoIFNγ,which was confirmed by PacⅠ digestion,and transformed into XL10-Glod(r) for copious preparation.pAd-Sh-PoIFNγ linearized with PacⅠwas co-transfected with liposome into 293 package cellline.After 7d-10d,the typical cytopathic effect indicated that recombinant adenoviral genome(deleted with E1 and E3 genes) carrying PoIFNγ was successfully packaged into intact virion.The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR.The antiviral activity of PoIFNγ was tested by CPE50 method.The results showed that the PoIFNγ expressed by adenovirus had high antiviral activity,which was 1.3×106 U/mL against VSV in MDBK cells.The results demonstrated that the recombinant adenovirus carrying PoIFNγ could be stably passaged.
[1]Muller U,Steinhoff U,Reis L F,et al.Functional roleof typeⅠand typeⅡinterferons in antiviral defense[J].Science.1994,264(5167):1918-1921.
[2]王晓鸣,李玉英,金奇,等.人γ干扰素突变体(rIFN-γ132-PGIL)的构建及其生物学活性的测定[J].病毒学报,1989,5(4):340-345.
[3]Xia C,Dan W,Wen-Xue W,et al.Cloning and expres-sion of interferon-α/γfrom a domestic porcine breed andits effect on classical swine fever virus[J].Vet ImmunolImmunopathol,2005,81-89.
[4]Rubin B Y,Gupta S L.Differential efficacies of humantype I and type II interferons as antiviral and antiprolifer-ative agents[J].Proc Natl Acad Sci USA,1980,77(10):5928-5932.
[5]Sonnenfeld G,Mandel A D,Merigan T C.The immu-nosuppressive effect of type II mouse interferon prepara-tions on antibody production[J].Cell Immunol,1977,34(2):193-206.
[6]Sonnenfeld G,Mandel A D,Merigan T C.Time anddosage dependence of immunoenhancement by murinetype II interferon preparations[J].Cell Immunol,1978,40(2):285-293.
[7]Crane J L Jr,Glasgow L A,Kern E R,et al.Inhibitionof murine osteogenic sarcomas by treatment with type Ior type II interferon[J].J Natl Cancer Inst,1978,61(3):871-874.
[8]Bautista E M,Molitor T W.IFN gamma inhibits por-cine reproductive and respiratory syndrome virus replica-tion in macrophages[J].Arch Virol,1999,144(6):1191-1200.
[9]Suradhat S,Intrakamhaeng M,Damrongwatanapokin S.The correlation of virus-specific interferon gamma pro-duction and protection against classical swine fever virusinfection[J].Vet Immunol Immunopathol,2001,83(3-4):177-189.
[10]Heath A W,Playfair H L.Conjugation of interferon-gamma to antigen enhances its adjuvanticity[J].Immu-nology,1990,71(3):454-456.
[11]Dijikmans R,Vandenbroeck K,Beaken E,et a1.Se-quence of the porcine interferon-gamma gene[J].NuclAcids Res,1990,18(14):42-59.
[12]郭瀛军,吴丹,陈蕊雯,等.猪γ干扰素基因的克隆、表达及其纯化[J].生物工程学报,2001,17(2):183-186.
[13]黄文林.分子病毒学[M].北京:人民卫生出版社,2002:247-248.
[14]Russell W C.Update on adenovirus and its vectors[J].J Gen Virol,2000,81(Part11):2573-2604.
[15]Mittal S K,Bett A J,Prevec L,et al.Foreign geneexpression by human adenovirus type 5-based vectorsstudied using firefly luciferase and bacterial beta-galac-tosidase gene as reporters[J].Virology,1995,210(1):226-230.
[16]Lefevre F,Lharidon R,Borras-Cuesta F,et a1.Pro-duction,purification and biological propertities of allEscherichia coli-derived recombinant porcine alpha in-terferon[J].J Gen Virol,1990,71:1057-1063.
[17]Weingartl H M,Derbyshire J B.Antiviral activity a-gainst transmissible gastroenteritis virus,and cytotox-icity of natural porcine interferons alpha and beta[J].Can J Vet Res,1991,55(2):143-149.
[18]Kishko I H,Vasylenko M I.The use of gamma-inter-feron for the prevention and treatment of infectious dis-eases in piglets and calves[J].Mikrobiol Z,1999,61(5):28-32.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.001836
中图分类号:S852.5
引用信息:
[1]姚清侠,徐卓菲,何雁南,等.表达猪γ干扰素的重组腺病毒的构建和活性鉴定[J].病毒学报,2007(05):394-398.DOI:10.13242/j.cnki.bingduxuebao.001836 .
基金信息:
国家863计划基金(2002AA245071); 湖北省科技攻关(2004AA202B01)
2007-09-15
2007-09-15