文章摘要
钱 璟,王崇明,潘鲁青,黄 倢.急性病毒性坏死病毒引物酶表达及酶学活性分析[J].水产学报,2013,37(9):1401~1408
急性病毒性坏死病毒引物酶表达及酶学活性分析
Expression and enzymatic activity analysis of primase from acute viral necrosis virus
投稿时间:2012-12-26  修订日期:2013-04-22
DOI:DOI:10.3724/SP.J.1231.2013.38510
中文关键词: 引物酶  原核表达  Pico-Green核酸染料  酶学活性  多聚胞嘧啶寡核苷酸
英文关键词: primase  prokaryotic expression  Pico-Green fluorescent dye  enzymatic activity  poly(d C)
基金项目:现代农业产业技术体系建设专项(CARS-48)
作者单位
钱 璟 中国海洋大学水产学院,海水养殖教育部重点实验室 
王崇明 中国水产科学研究院黄海水产研究所 
潘鲁青 中国海洋大学水产学院,海水养殖教育部重点实验室 
黄 倢 中国水产科学研究院黄海水产研究所 
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中文摘要:
      根据急性病毒性坏死病毒(acute viral necrosis virus,AVNV)ORF 024序列设计引物,以AVNV的DNA为模板进行扩增AVNV引物酶(primase)基因,同时构建表达质粒pET32a-prim,并转化至E.coli BL21(DE3)进行诱导表达。SDS-PAGE检测显示诱导表达两条蛋白条带,其中分子量为60 ku的蛋白条带与预期表达蛋白条带大小一致,另一蛋白条带分子量约为55 ku,经Western-blotting及质谱分析鉴定分子量60 ku的蛋白条带为AVNV-引物酶,而表达出的55 ku蛋白条带存在部分引物酶多肽片段。RNA结合荧光染料Pico-Green在30 min内荧光强度比较稳定,从而确定30 min为进行AVNV引物酶活性分析的最佳终止反应时间,并在引物合成实验中观察到30 min内引物酶活性较高。当使用多聚胞嘧啶寡核苷酸poly(d C)为模板时,重组引物酶能特异性催化底物GTP。0.1 mmol/L Zn2+可显著增强引物酶活性,而1 mmol/L Mn2+和EDTA能抑制引物酶活性。
英文摘要:
      Acute viral necrosis virus(AVNV)was reported as one causative agent responsible for mass mortality of adult Chlamys farreri,which is widely cultured along northern China coast.To explore its pathogenesis at the molecular level,a gene was cloned which was predicted to encode AVNV primase based on the genomic sequence of AVNV completed by our laboratory.The gene encodes a protein of 350 aa with a predicted molecular mass of 60 ku.To obtain AVNV ORF 024,which probably encodes AVNV primase,a pair of specific primers was designed based on the genomic sequence of AVNV.Then this paper amplified the expected DNA by PCR,and used the total genomic DNA extracted from infected C.farreri tissues as template.Amplified PCR fragments were cloned into the prokaryotic expression vector pET-32a(+).After that,the plasmid pET32a-prim was transformed into E.coli BL21(DE3)stain,and AVNV primase was expressed under IPTG induction.SDS-PAGE analysis showed that the two induced recombinant proteins’ molecular mass was about 60 and 55 ku,the Western-blotting and mass spectrometry analysis proved that the expressed protein(60 ku)was the primase,while another expressed protein(55 ku)had some of the primase peptide fragments.Meanwhile,the experiments showed that Pico-Green(it was able to specifically bind to a random short segments of RNA)as the fluorescent dye which had a stable fluorescence intensity in 30 min.So the 30 min was the termination time for the enzymatic activity analysis of the recombined primase.In the presence of template of the poly(d C),the analysis of the enzymatic activity indicated that the recombinant primase could specifically catalyze the hydrolysis of GTP.The enzymatic activity of primase was enhanced by 0.1 mmol/L Zn 2+ but inhibited by 1 mmol/L Mn2+ and EDTA.
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