Prokaryotic Expression of Anguillae herpesvirus ORF95 Protein
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摘要: 鳗鲡疱疹病毒(Anguillae herpesvirus, AngHV)是淡水鳗鲡的主要病毒性疾病病原之一。根据鳗鲡疱疹病毒(AngHV-1)的基因组序列(GenBank:NC-013668)设计引物,扩增鳗鲡疱疹病毒福建株(AngHV-FJ)ORF95基因的开放阅读框序列,克隆至pMD19-T载体;经限制性内切酶酶切和测序鉴定,进一步将其克隆至原核表达载体pET-32a,构建了表达质粒32a-ORF95,将其转化表达菌株BL21(DE3),经IPTG诱导,实现了ORF95在大肠杆菌中的高效表达。在0.1 mmol·L-1 IPTG、23℃诱导14 h的条件下,可溶性ORF95蛋白的表达量较高,经镍柱纯化获得了高纯度的融合蛋白。Abstract: Anguillae herpesvirus(AngHV)is one of the major viral pathogen of freshwater eels. In this study, the open reading frame of AngHV-FJ ORF95 gene was synthesized according to the gene sequence of AngHV-1 provided by GenBank:(NC-013668),and then cloned into the pMD19-T vector. After restriction digestion and sequence verification,the ORF95 gene was inserted into the prokaryotic expression vector pET-32a to construct the recombinant expression plasmid 32a-ORF95. The plasmid was then transformed into E. coli BL21 (DE3) strain to induce expression of ORF95 protein. The soluble ORF95 protein can be highly expressed under the induction of IPTG (0.1 mmol·L-1) at 23℃ for 14h,and then purified by Ni-NTA His.Binding affinity purification. The protein can be used further to understand the characteristic of ORF95 protein and the AngHV prevention.
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Key words:
- Anguillae herpesvirus /
- ORF95 /
- gene cloning /
- prokaryotic expression
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[1] UENO Y,KITAO T,CHEN S N,et al. Characterization of a herpes-like virus isolated from cultured Japanease eels in Taiwan[J]. Fish Pathology, 1992, 27: 7-17. [2] JAKOB E,NEUHAUS H,STEINHAGEN D, et al. Monitoring of Herpesvirus anguillae (HVA) infections in European eel,Anguilla anguilla(L.), in northern Germany[J]. J Fish Dis, 2009,32(6): 557-561. -
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