全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

一种新的海洋微藻病毒丝氨酸蛋白酶基因的克隆表达及其活性分析

DOI: 10.3969/j.issn.0253-4193.2014.08.011, PP. 101-110

Keywords: 海洋球石藻病毒,丝氨酸蛋白酶,基因克隆,重组表达,活性分析

Full-Text   Cite this paper   Add to My Lib

Abstract:

从海洋球石藻Emilianiahuxleyi病毒EhV99B1的基因组中克隆了丝氨酸蛋白酶(Sp)基因(GenBank登录号:KC161207),对该基因的开放阅读框(ORF)进行系统的生物信息学分析,并在大肠杆菌中融合表达,通过亲和层析法获得了纯化的重组Sp。结果表明:EhV99B1-Sp基因的ORF为1110bp,编码368个氨基酸,蛋白相对分子质量为39.5kDa;该基因片段与GenBank中EhV86-Sp的同源性很高,核苷酸及其对应的氨基酸序列同源性分别为95%和97%,而与其他物种Sp序列的同源性仅为28%~32%,说明其可能是丝氨酸蛋白酶家族中的一个新成员;预测的二级结构特征显示EhV99B1-Sp的蛋白结构域中具有典型的LTAGHC(组氨酸活性位点区域)和AICNGDSGGPLF(丝氨酸活性位点区域)两个丝氨酸蛋白酶催化活性位点的氨基酸基序,是一个两次跨膜蛋白;将该基因在大肠杆菌中进行低温诱导表达,得到分子量为60kDa的重组蛋白,经鲤鱼肌肉丝氨酸蛋白酶(MBSP)抗体检测证实为Sp,且重组蛋白在大肠杆菌细胞中具有明显的生物学活性。本研究结果为进一步探讨EhV99B1-Sp在病毒与宿主相互作用过程中的调节作用及其功能与应用奠定基础。

References

[1]  Rohwer F,Thurber R V. Viruses manipulate the marine environment[J]. Nature,2009,459(7244): 207-212.
[2]  Bidle K D,Varid A. A chemical arms race at sea mediates algal host-virus interactions[J]. Curr Opin Microbiol,2011,14(4): 449-457.
[3]  Vardi A,Van Mooy B A S,Fredricks H F,et al. Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton[J]. Science,2009,326(5954): 861-865.
[4]  Castberg T,Thyrhaug R,Larsen A,et al. Isolation and characterization of a virus that infects Emiliania huxleyi (Haptophyta)[J]. J Phycol,2002,38(4): 767-774.
[5]  Simpson A A,Nandhagopal N,Van Etten J L,et al. Structural analyses of Phycodnaviridae and Iridovirdae[J]. Acta Crystallogr,Sect D: Biol Crystallogr,2003,59(12): 2053-2059.
[6]  Solioz M,Yen H C,Marris B. Release and uptake of gene transfer agent by Rhodopseudomonas capsulata[J]. J Bacteriol,1975,123(2): 651-657.
[7]  Biers E J,Wang K,Pennington C,et al. Occurrence and expression of gene transfer agent genes in marine bacterioplankton[J]. Appl Environ Microbiol,2008,74(10): 2933-2939.
[8]  Monier A,Pagarete A,Vargas C,et al. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus[J]. Genome Res,2009,19(8): 1441-1449.
[9]  Bidle K D,Haramaty L,Ramos J B,et al. Viral activation and recruitment of metacaspases in the unicellular coccolithophore,Emiliania huxleyi[J]. Proc Natl Acad Sci USA,2007,104(14): 6049-6054.
[10]  Pagarete A,Lanzen A,Puntervoll P,et al. Genomic sequence and analysis of EhV-99B1,a new Coccolithovirus from the Norwegain Fjords[J]. Intervirology,2013,56(1): 60-66.
[11]  Gaines P J,Sampson C M,Rushlow K E,et al. Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis[J]. Insect Mol Biol,1999,8(1): 11-22.
[12]  Lindell D,Jaffe J D,Coleman M L,et al. Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution[J]. Nature,2007,449(7158): 83-86.
[13]  Kegel J U,Blaxter M,Allen M J,et al. Transcriptional host-virus interaction of Emiliania huxleyi (Haptophyceae) and EhV-86 deduced from combined analysis of expressed sequence tags and microarrays[J]. Eur J Phycol,2010,45(1): 1-12.
[14]  Wilson W H,Schroeder D C,Allen M J,et al. Complete genome sequence and lytic phase transcription profile of a Coccolithovirus[J]. Science,2005,309(5737): 1090-1092.
[15]  汪世华,王文勇,黄益洲,等. 丝氨酸蛋白酶的研究进展[J]. 福建农业学报,2007,22(4): 453-456.
[16]  Suzuki C K,Rep M,Van Dijl J M,et al. ATP-dependent proteases that also chaperone protein biogenesis[J]. Trends Biochem Sci,1997,22(4): 118-123.
[17]  Kalisz H M. Microbial proteinases[J]. Adv Biochem Eng/Biotechnol,1988,36: 1-65.
[18]  Gottesman S,Maurizi M R. Regulation by proteolysis: energy-dependent proteases and their targets[J]. Microbiol Rev,1992,56(4): 592-621.
[19]  Nierman W C,Pain A,Anderson M J,et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus[J]. Nature,2005,438(7071): 1151-1156.
[20]  崔士超,谢利娟,吴红芝. 丝氨酸蛋白酶与植物器官衰老关联的研究进展[J]. 安微农业科学,2010,38(15): 7742-7743,7745.
[21]  张彦锋,刘静雯,张稚兰,等. 海洋球石藻(Emiliania huxleyi)病毒硫氧还蛋白(TRX)基因的克隆及生物信息学分析[J]. 海洋与湖沼,2010,41(2): 293-300.
[22]  Sambrook J, Russell D W.分子克隆实验指南[M]. 3版. 黄培堂,译. 北京:科学出版社,2002.
[23]  季星来,孙之荣. 基于结构的丝氨酸蛋白酶超家族进化分析[J]. 电子学报,2001,29(12A): 1756-1758.
[24]  黄娟,杨维青,赵祖国. 细菌生物被膜菌群间遗传物质的转移[J]. 中国抗生素杂志,2013,38(2): 98-101.
[25]  Lopez J F,Shi Y,Tyson G W,et al. Microbial community gene expression in ocean surface waters[J]. Proc Natl Acad Sci USA,2008,105(10): 3805-3810.
[26]  La Scola B,Desnues C,Pagnier I,et al. The virophage as a unique parasite of the giant mimivirus[J]. Nature,2008,455(7209): 100-104.
[27]  Gonzlez J M,Esteban M. A poxvirus Bcl-2-like gene family involved in regulation of host immune response: sequence similarity and evolutionary history[J]. Virol J,2010,7: 59-70.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133