水产学报2011，Vol.35Issue(9)：1320-1326,7.DOI:10.3724/SP.J.1231.2011.17463
急性病毒性坏死病毒dUTPase基因的克隆、表达及其产物的酶学活性分析
Cloning, expression of Chlamys farreri acute viral necrosis virus dUTPase gene and the determination of its enzymatic activity
摘要
Abstract
Acute viral necrosis virus(AVNV) was reported as one causative agent responsible for summer mass mortality of adult Zhikong scallop(Chlamys farreri), which is widely cultured along northern China coast. To explore its pathogenesis at the molecular level, we cloned a gene which was predicted to encode AVNV dUTPase based on the genomic sequence of C. Farreri A VNV completed by our laboratory. The gene encodes a protein of 248aa with a predicted molecular mass of 26.4 ku. To obtain the C. Farreri AVNV Open Reading Frame 074, which probably encodes AVNV dUTPase, a pair of specific primers was designed based on the genomic sequence of C. Farreri AVNV. Then this paper amplified the expected DNA by PCR, using the total genomic DNA extracted from infected C. Farreri tissues as template. Amplified PCR fragments were subcloned into the prokaryotic expression vector pET-32a( + ). After that,we transformed the recombinant plasmid pET32a-dut into E. Coli BL21 (DE3) strain and expressed it under IPTG induction. SDS-PAGE analysis showed that the molecular mass of the induced recombinant protein was about 46 ku. The Western-blotting and mass spectrometry analysis proved that the expressed protein is the target protein. Then we purified recombinant protein with Co2 + purification column and got the recombinant protein with a purity of more than 90%. The analysis of the enzymatic activity indicated that the recombinant dUTPase could specifically catalyse the hydrolysis of dUTP and the activity of enzyme was enhanced by Mg2+ while inhibited by EDTA.关键词
栉孔扇贝/dUTP焦磷酸酶/急性病毒性坏死病毒/原核表达/酶学活性Key words
Chlamys farreri/dUTP pyrophosphatase/acute viral necrosis virus (AVNV)/prokaryotic expression/enzymatic activity分类
生物科学引用本文复制引用
贾志磊,王崇明,任伟成,梁彦韬,曲朋,李赟..急性病毒性坏死病毒dUTPase基因的克隆、表达及其产物的酶学活性分析[J].水产学报,2011,35(9):1320-1326,7.基金项目
国家“八六三”高技术研究发展计划(2006AA100307) （2006AA100307）
现代农业产业技术体系建设专项资助(nycytx-47) （nycytx-47）
