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高溳, 金春英, 王蔚. 对虾白斑综合症病毒IE1基因表达与鉴定[J]. 中国公共卫生, 2011, 27(5): 535-536. DOI: 10.11847/zgggws-2011-27-05-04
引用本文: 高溳, 金春英, 王蔚. 对虾白斑综合症病毒IE1基因表达与鉴定[J]. 中国公共卫生, 2011, 27(5): 535-536. DOI: 10.11847/zgggws-2011-27-05-04
GAO Yun, JIN Chun-ying, WANG Wei. Prokaryotic cloning, expression and identification of IE1 gene from white spot syndrome virus[J]. Chinese Journal of Public Health, 2011, 27(5): 535-536. DOI: 10.11847/zgggws-2011-27-05-04
Citation: GAO Yun, JIN Chun-ying, WANG Wei. Prokaryotic cloning, expression and identification of IE1 gene from white spot syndrome virus[J]. Chinese Journal of Public Health, 2011, 27(5): 535-536. DOI: 10.11847/zgggws-2011-27-05-04

对虾白斑综合症病毒IE1基因表达与鉴定

Prokaryotic cloning, expression and identification of IE1 gene from white spot syndrome virus

  • 摘要: 目的 探讨对虾白斑综合症病毒极早期基因IE1原核表达载体的构建与表达。方法 将IE1编码序列克隆入pGEX-4T-2原核表达载体,并转染大肠埃希菌BL21,异丙基-β-D硫代半乳糖苷(IPTG)诱导谷胱甘肽硫转移酶(GST-IE1)融合蛋白表达,亲和层析法纯化,并用聚丙烯酰胺变性凝胶电泳(SDS-PAGE)和蛋白印迹(western b lot)对融合蛋白进行鉴定。结果 克隆到IE1基因序列长675 bp,编码224个氨基酸,理论分子量为25 kDa,等电点为4.87,所构建的重组体经PCR、双酶切及测序鉴定与目标基因相符,SDS-PAGE结果表明,目的基因在大肠埃希氏菌BL21中表达成功。结论 克隆构建了对虾白斑综合症病毒极早期基因IE1,并在原核细胞中获得表达。

     

    Abstract: Objective To express the white spot syndromevirus(WSSV)mimediate early prote in(IE1)in prokaryotic expression system,and to purify and identify the prote in expressed.Methods The encoding fragment of IE1 was inserted into plasmid pGEX-4T-2 and the recom binant plasmid pGEX-4T-2-IE1 was transformed into E.coli BL21 and induced by isopropy lD thiog alacto side(IPTG).The expressed glutathione Stran sferase(GST)IE1 was purified by affinity chromatog raphy column and identified by sodium dodecyl sulfate poly acrylamide gelelectropho resis(SDS-PAGE)and western blot.Results Anovel cDNA sequence encoding IE1 with amolecular mass of 25015.2,pI 4.87,which was 675bp and encoding 224 aminaacids,was obtained.The recom binant plasmid pGEX-4T-2-IE1 was constructed and expressed in fusion protein.Conclusion Recom binant GST-IE1 prote in was obtained and could be used in the functional study of IE1.

     

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