具有酶活性的兔出血症病毒RNA依赖的RNA聚合酶在大肠杆菌中的表达。
Expression of enzymatically active rabbit hemorrhagic disease virus RNA-dependent RNA polymerase in Escherichia coli.
作者信息
Vázquez A L, Martín Alonso J M, Casais R, Boga J A, Parra F
机构信息
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, Spain.
出版信息
J Virol. 1998 Apr;72(4):2999-3004. doi: 10.1128/JVI.72.4.2999-3004.1998.
Abstract
The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3Dpol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.
摘要
兔出血症病毒（RHDV）（分离株AST/89）的RNA依赖性RNA聚合酶（3Dpol）编码区通过基于谷胱甘肽S-转移酶的载体在大肠杆菌中表达，这使得能够以毫克量纯化出预期分子量约为58 kDa的均一酶。重组多肽表现出对利福平和放线菌素D耐药的、依赖于聚腺苷酸的聚尿苷酸聚合酶活性。该酶在有或无寡聚尿苷酸引物存在的情况下，与合成的RHDV亚基因组RNA进行体外反应时也显示出RNA聚合酶活性。在寡聚尿苷酸引发的反应中合成了模板大小的产物，而在不添加引物的情况下，合成的RNA产物长度可达模板的两倍。双倍长度的RNA产物是双链的，并且能与正链和负链探针杂交。
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