绵羊痘病毒甘肃古浪株RING finger基因的克隆表达及序列分析
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摘要
【目的】对绵羊痘病毒甘肃古浪株RING finger蛋白的基因进行克隆,表达以及序列分析,初步探究绵羊痘病毒RING finger蛋白是否具有E3泛素连接酶活性,为阐明其在绵羊痘病毒感染过程中对泛素蛋白酶体系统的调控作用奠定基础.【方法】以绵羊痘病毒甘肃古浪株DNA为模板,通过PCR扩增RING finger基因.利用Pfam数据库、DNAstar等软件进行序列及遗传进化分析;将RING finger基因片段插入原核表达载体pGEX-4T-1中,构建pGEX-SPPVRFP重组表达载体,在大肠杆菌BL21(DE3)中诱导表达,并进行SDS-PAGA和Western-blot分析.【结果】绵羊痘病毒RING finger基因由723个核苷酸组成,编码的240个氨基酸的分子量约为28.5 ku,具有RING finger结构域.不同羊痘病毒株间RING finger基因核苷酸序列同源性高达99.2%,氨基酸序列同源性高达98.3%.不同的RING finger蛋白都含有8个保守的半胱氨酸和组氨酸.SDS-PAGA分析显示,重组SPPVRFP大小约为55 ku,Western-blot分析显示,重组SPPVRFP不能与绵羊痘病毒阳性血清反应.【结论】成功克隆、表达并纯化了绵羊痘病毒RING finger基因,对SPPV GS-GL株RING-finger蛋白进行序列分析,推测其可能具有E3泛素连接酶活性.
【Objective】 The gene of RING finger protein of sheeppox virus was cloned,expressed and sequenced,aiming to analyze whether sheeppox virus RING finger protein has E3 ubiquitin ligase activity in order to provide a foundation for further studies on the regulatory functions of SPPVRFP in the ubiquitin protease system during sheeppox virus infection.【Method】 The RING finger gene was amplified with PCR by using DNA of sheeppox virus Gansu Gulang strain as a template.The sequence and genetic evolution analysis were carried out by using Pfam database and DNAstar software,respectively.The RING finger gene fragment was ligated into the prokaryotic expression vector pGEX-4 T-1,then the recombinant prokaryotic expression vector pGEX-SPPVRFP was constructed to induce the expression in E.coli BL21(DE3),and analyzed by SDS-PAGA and Western-blot.【Result】 The sheeppox virus RING finger gene consisted of 723 nucleotides,encoding 240 amino acids with a molecular weight mass of 28.5 ku,and it had a RING finger domain.The nucleotide sequence homology of RING finger genes among different sheeppox virus strains was as high as 99.2%,and the amino acid sequence homology was as high as 98.3%.It was found that the RING finger of SPPV had 8 conserved cysteines and histidines.The SDS-PAGA analysis showed that the relative molecular weight of recombinant SPPVRFP was about 55 ku,and Western-blot analysis showed that recombinant SPPVRFP did not reacted with the sheeppox virus positive sera.【Conclusion】 The sheeppox virus RING finger gene is successfully cloned,expressed and purified.It is speculated that it may have ubiquitin ligase acticity.
【Objective】 The gene of RING finger protein of sheeppox virus was cloned,expressed and sequenced,aiming to analyze whether sheeppox virus RING finger protein has E3 ubiquitin ligase activity in order to provide a foundation for further studies on the regulatory functions of SPPVRFP in the ubiquitin protease system during sheeppox virus infection.【Method】 The RING finger gene was amplified with PCR by using DNA of sheeppox virus Gansu Gulang strain as a template.The sequence and genetic evolution analysis were carried out by using Pfam database and DNAstar software,respectively.The RING finger gene fragment was ligated into the prokaryotic expression vector pGEX-4 T-1,then the recombinant prokaryotic expression vector pGEX-SPPVRFP was constructed to induce the expression in E.coli BL21(DE3),and analyzed by SDS-PAGA and Western-blot.【Result】 The sheeppox virus RING finger gene consisted of 723 nucleotides,encoding 240 amino acids with a molecular weight mass of 28.5 ku,and it had a RING finger domain.The nucleotide sequence homology of RING finger genes among different sheeppox virus strains was as high as 99.2%,and the amino acid sequence homology was as high as 98.3%.It was found that the RING finger of SPPV had 8 conserved cysteines and histidines.The SDS-PAGA analysis showed that the relative molecular weight of recombinant SPPVRFP was about 55 ku,and Western-blot analysis showed that recombinant SPPVRFP did not reacted with the sheeppox virus positive sera.【Conclusion】 The sheeppox virus RING finger gene is successfully cloned,expressed and purified.It is speculated that it may have ubiquitin ligase acticity.
引文
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[14] Nerenberg B T H,Taylor J,Bartee E,et al.The poxviral RING protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories[J].Journal of Virology,2005,79(1):597-601.
[15] Mottet K,Bareiss B,Milne C D,et al.The poxvirus encoded ubiquitin ligase,p28,is regulated by proteasomal degradation and autoubiquitination[J].Virology,2014(468/470):363-378.
[16] Bareiss B,Barry M.Fowlpox virus encodes two p28-like ubiquitin ligases that are expressed early and late during infection[J].Virology,2014(462/463):60-70.
[17] Brick D J,Burke R D,Minkley A A,et al.Ectromelia virus virulence factor p28 acts upstream of caspase-3 in response to UV light-induced apoptosis[J].Journal of General Virology,2000,81(4):1087-1097.
[18] Huang J,Huang Q,Zhou X,et al.The Poxvirus p28 virulence factor is an E3 ubiquitin ligase[J].Journal of Biological Chemistry,2004,279(52):54110-54116.
[19] Tulman E R,Afonso C L,Lu Z,et al.The genomes of sheeppox and goatpox viruses[J].Journal of Virology,2002,76(12):6054-6061.
[20] Mansur D S,Carlos M D M,Unterholzner L,et al.Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry:a mechanism to inhibit NF-κB activation and promote immune evasion and virulence[J].PLoS Pathogens,2013,9(2):e1003183.
[21] Mohamed M R,Mcfadden G.NFκB inhibitors:strategies from poxviruses[J].Cell Cycle,2009,8(19):3125-3132.
[2] 赵文华,杨仕标,姚俊,等.山羊痘病毒及口疮病毒ITR-059基因双重PCR检测方法的建立[J].中国兽医科学,2017,47(5):569-576.
[3] 王晓霞,郑亚东,骆学农,等.绵羊痘病毒甘肃流行株糖蛋白基因ORF117的克隆及其原核表达[J].中国预防兽医学报,2009,31(5):337-340.
[4] Mehlhorn H.World Organisation for Animal Health (OIE)[M]//Encyclopedia of Parasitology:Springer Berlin Heidelberg,2015:115-117.
[5] 农业部兽医局.一三类动物疫病释义[M].北京:中国农业出版社,2011.
[6] 刘辉志,韩世平.泛素-蛋白连接酶(E3)—RING-finger蛋白的研究进展[C]//河南省植物生理学会三十周年庆典暨学术研讨会论文集.河南:河南省科学技术协会,2010.
[7] Stone S L,Hauksdottir H,Troy A,et al.Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis[J].Plant Physiol,2005,137(1):13-30.
[8] Lim S D,Yim W C,Moon J C,et al.A gene family encoding ring finger proteins in rice:their expansion,expression diversity,and co-expressed genes[J].Plant Molecular Biology,2010,72(4/5),369-380.
[9] Lee H K,Cho S K,Son O,et al.Drought stress-induced Rma1H1,a RING membrane-anchor E3 ubiquitin ligase homolog,regulates aquaporin levels via ubiquitination in transgenic Arabidopsis plants[J].Plant Cell,2009,21(2):622-641.
[10] Freemont P S.The RING finger.A novel protein sequence motif related to the zinc finger[J].Ann N Y Acad Sci,1993,684(1):174-192.
[11] 田丽,包满珠,张蔚.植物逆境相关RING finger蛋白的研究进展[J].湖北农业科学,2018,57(6):5-11.
[12] Kosarev P,Mayer K F,Hardtke C S.Evaluation and classification of RING-finger domains encoded by the Arabidopsis genome[J].Genome Biology,2002,3(4):1-12.
[13] Randow F,Lehner P J.Viral avoidance and exploitation of the ubiquitin system[J].Nature Cell Biology,2009,11(5):527-534.
[14] Nerenberg B T H,Taylor J,Bartee E,et al.The poxviral RING protein p28 is a ubiquitin ligase that targets ubiquitin to viral replication factories[J].Journal of Virology,2005,79(1):597-601.
[15] Mottet K,Bareiss B,Milne C D,et al.The poxvirus encoded ubiquitin ligase,p28,is regulated by proteasomal degradation and autoubiquitination[J].Virology,2014(468/470):363-378.
[16] Bareiss B,Barry M.Fowlpox virus encodes two p28-like ubiquitin ligases that are expressed early and late during infection[J].Virology,2014(462/463):60-70.
[17] Brick D J,Burke R D,Minkley A A,et al.Ectromelia virus virulence factor p28 acts upstream of caspase-3 in response to UV light-induced apoptosis[J].Journal of General Virology,2000,81(4):1087-1097.
[18] Huang J,Huang Q,Zhou X,et al.The Poxvirus p28 virulence factor is an E3 ubiquitin ligase[J].Journal of Biological Chemistry,2004,279(52):54110-54116.
[19] Tulman E R,Afonso C L,Lu Z,et al.The genomes of sheeppox and goatpox viruses[J].Journal of Virology,2002,76(12):6054-6061.
[20] Mansur D S,Carlos M D M,Unterholzner L,et al.Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry:a mechanism to inhibit NF-κB activation and promote immune evasion and virulence[J].PLoS Pathogens,2013,9(2):e1003183.
[21] Mohamed M R,Mcfadden G.NFκB inhibitors:strategies from poxviruses[J].Cell Cycle,2009,8(19):3125-3132.