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...

少孢节丛孢菌胞外丝氨酸蛋白酶P186基因的克隆、序列分析及表达

DOI: 10.7668/hbnxb.2014.04.015, PP. 93-97

Keywords: 少孢节丛孢菌,丝氨酸蛋白酶P186,克隆,表达

Full-Text   Cite this paper   Add to My Lib

Abstract:

根据GenBank中少孢节丛孢菌丝氨酸蛋白酶P186基因序列设计特异性引物,运用RT-PCR扩增获得目的基因片段,构建重组表达质粒pET32a-P186,转化到大肠杆菌BL21(DE3),以IPTG进行了诱导表达。结果表明,P186基因cDNA全长为1224bp,编码407个氨基酸,其中第1~21位为信号肽序列,氨基酸序列的第156~167位、192~202位、343~353位分别是天冬氨酸(Asp160)、组氨酸(His192)、丝氨酸(Ser345)活性位点所在区域,属于Subtilase家族,包括2个潜在的N-联糖基化位点(Asn249、Asn342)及与底物结合的S1区(SBP)Ser251Ile252Gly253和Ala277Ala278Gly279。SDS-PAGE分析结果显示,表达产物的分子质量约为62kDa。WesternBlot分析结果表明,P186重组蛋白可以与抗蛋白粗提液多克隆抗体发生反应。为了揭示少孢节丛孢菌捕食线虫的分子机制,首次克隆并表达了少孢节丛孢菌丝氨酸蛋白酶P186基因,为进一步研究丝氨酸蛋白酶P186的生物学功能奠定了基础。

References

[1]  Paraud C,Chartier C.Biological control of infective larvae of a gastro-intestinal nematode(Teladorsagia circumcincta)and a small lungworm(Muellerius capillaris)by Duddingtonia flagrans in goat faeces [J].Parasitology Research,2003,89(2):102-106.
[2]  Terrill T H,Larsen M,Samples O,et al.Capability of the nematode-trapping fungus Duddingtonia flagrans to reduce infective larvae of gastrointestinal nematodes in goat feces in the southeastern United States:dose titration and dose time interval studies [J].Veterinary Parasitology,2004,120(4):285-296.
[3]  Niu X M,Zhang K Q.Arthrobotrys oligospora:a model organism for understanding the interaction between fungi and nematodes [J].Mycology,2011,2(2):59-78.
[4]  Liang L M,Wu H,Liu Z H,et al.Proteomic and transcriptional analyses of Arthrobotrys oligospora cell wall related proteins reveal complexity of fungal virulence against nematodes [J].Applied Microbiology and Biotechnology,2013,97(19):8683-8692.
[5]  Tunlid A,Rosen S,Ek B O, et al.Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora [J].Microbiology,1994,140(7):1687-1695.
[6]  Andersson K M,Meerupati T,Levander F,et al.Proteome of the nematode-trapping cells of the fungus Monacrosporium haptotylum [J].Applied and Environmental Microbiology,2013,79(16):4993-5004.
[7]  孟庆玲,王为升,王俊伟,等.少孢节丛孢菌的分离鉴定及捕食线虫活性研究[J].中国预防兽医学报,2011,33(3):189-193.
[8]  Dijksterhuis J,Veenhuis M,Harder W,et al.Nematophagous fungi:physiological aspects and structure-function relationships [J].Advances in Microbial Physiology,1994,36:111-144.
[9]  Yang J,Huang X,Tian B,et al.Isolation and characterization of a serine protease from the nematophagous fungus, Lecanicillium psalliotae,displaying nematicidal activity [J].Biotechnology Letters,2005,27(15):1123-1128.
[10]  Minglian Z,Minghe M,Keqin Z.Characterization of a neutral serine protease and its full-length cDNA from the nematode-trapping fungus Arthrobotrys oligospora [J].Mycologia,2004,96(1):16-22.
[11]  Yang J K,Tian B Y,Liang L M,et al.Extracellular enzymes and the pathogenesis of nematophagous fungi [J].Applied Microbiology and Biotechnology,2007,75(1):21-31.
[12]  Huang X W,Zhao N H,Zhang K Q.Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host [J].Research in Microbiology,2004,155(10):811-816.
[13]  Yang J K,Wang L,Ji X L,et al.Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into Nematode-Trap formation [J].PLOS Pathogens,2011,7(9):e1002179.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133