CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI OF THE COAT PROTEIN GENE OF A NEW POTYVIRUS INFECTING SOUTH-AFRICAN PASSIFLORA
CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI OF THE COAT PROTEIN GENE OF A NEW POTYVIRUS INFECTING SOUTH-AFRICAN PASSIFLORA
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DOI：
10.1007/bf01309786
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发表时间：
1993-01-01
影响因子：
2.7
通讯作者：
RYBICKI, EP
中科院分区：
文献类型：
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作者：
BRAND, RJ;BURGER, JT;RYBICKI, EP
对引起南非西番莲木质病的马铃薯Y病毒基因组RNA的3‘-聚(A)-近端1418个核苷酸(NT)的互补DNA进行了克隆和测序。该序列含有一个1188个核苷酸的长开放阅读框(ORF)，没有起始密码子，以及一个230个核苷酸的3‘-非编码区(3’-NCR)，其后是一个多聚腺苷酸区。与其他马铃薯Y病毒外壳蛋白(CP)序列的比较表明，分子量为31722的279个CP由开放阅读框架3‘端的836nt编码。这种病毒不仅仅是西番莲木质化病毒(PWV)的南非株：推断的CP序列与其他PWV序列株的CP只有遥远的亲缘关系，尽管它是与PWV相关的一个不同的马铃薯Y病毒亚群的一部分。因此，该病毒被命名为南非西番莲病毒(SAPV)。将含CP基因的DNA克隆到pUEX-2细菌表达载体中，并在大肠杆菌中以β-Gal-CP融合蛋白的形式表达。融合蛋白与天然病毒多克隆抗血清反应，部分纯化的融合蛋白抗血清与病毒CP在Western blotting中反应。
Complementary DNA representing 1418 nucleotides (nt) of the 3'-poly(A)-proximal tract of the genomic RNA of a potyvirus causing woodiness disease in South African passionfruit, was cloned and sequenced. The sequence contained a single long open reading frame (ORF) of 1188 nt with no initiation codon, and a 3'-non-coding region (3'-NCR) of 230 nt followed by a polyadenylate tract. Comparison of the ORF with other potyviral coat protein (CP) sequences led to the prediction that a 279 residue CP of MW 31722 is encoded by 836 nt at the 3'-terminus of the ORF. This virus is not merely a South African strain of passionfruit woodiness virus (PWV): the deduced CP sequence is only distantly related to CPs of other sequenced strains of PWV, although it is part of a distinct subgroup of potyviruses related to PWV. The virus was therefore designated as South African passiflora virus (SAPV). The DNA containing the putative CP was cloned into the pUEX 2 bacterial expression vector and expressed in Escherichia coli as a beta-gal-CP fusion protein. The fusion protein reacted with polyclonal antisera raised against the native virus, and antisera raised against partially purified fusion protein reacted with viral CP in Western blots.