Woloshuk S L, Xiong Z, Hellmann G M, Wernsman E A, Weissinger A K, Lommel S A
Department of Crop Science, North Carolina State University, Raleigh.
Arch Virol. 1993;132(1-2):161-70. doi: 10.1007/BF01309850.
Complementary DNA libraries representing the capsid protein cistron of the potato virus Y (PVY) isolate 'Chilean', 'Hungarian', MsNr, NsNr, O, and 'Potato US' were synthesized and used as template for polymerase chain reaction (PCR) amplification. An AUG codon for initiating a discrete capsid protein (CP) open reading frame was embedded upstream of the first codon of the CP cistrons. PCR-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pBS(+). The fidelity of each PCR-amplified PVY CP cistron was tested by transcribing recombinant plasmids in vitro and translating the transcripts in two cell free translation systems. Translation analysis of in vitro transcribed PVY CP cistrons consistently yielded a polypeptide co-migrating with authentic CP that was immunoprecipitated by anti PVY 'Chilean' antibodies. The nucleotide sequence of each capsid protein gene was determined by dideoxy sequence analysis. Each capsid protein gene was determined to be 801 nucleotides in length, encoding a deduced protein of 267 amino acids with calculated M(r) ranging from 29,799 to 29,980. The nucleic acid sequence similarity between the six isolates ranged between 89 to 97% and the amino acid similarity between 91 to 99%. The high level of amino acid sequence similarity confirms the classification of these viruses as isolates of PVY.
合成了代表马铃薯Y病毒（PVY）“智利”株系、“匈牙利”株系、MsNr、NsNr、O株系和“美国马铃薯”株系衣壳蛋白顺反子的互补DNA文库，并将其用作聚合酶链反应（PCR）扩增的模板。在衣壳蛋白（CP）顺反子的第一个密码子上游嵌入了一个用于启动离散衣壳蛋白开放阅读框的AUG密码子。将预期大小为0.8千碱基的PCR扩增产物克隆到转录载体pBS(+)中。通过体外转录重组质粒并在两个无细胞翻译系统中翻译转录本，测试了每个PCR扩增的PVY CP顺反子的保真度。对体外转录的PVY CP顺反子的翻译分析始终产生一种与天然CP共迁移的多肽，该多肽被抗PVY“智利”抗体免疫沉淀。通过双脱氧序列分析确定了每个衣壳蛋白基因的核苷酸序列。每个衣壳蛋白基因的长度为801个核苷酸，编码一个推导的267个氨基酸的蛋白质，计算的分子量在29,799至29,980之间。六个分离株之间的核酸序列相似性在89%至97%之间，氨基酸相似性在91%至99%之间。氨基酸序列的高度相似性证实了这些病毒作为PVY分离株的分类。
