López L, Muller R, Balmori E, de la Riva G, Ramírez N, Doreste V, López M, Peréz S, Oramas P, Selman-Housein G
Division of Plant Biotechnology, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Virus Genes. 1994 Sep;9(1):77-83. doi: 10.1007/BF01703437.
Total RNA from infected Physalis floridana was isolated to generate complementary DNA corresponding to the coat protein (GP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1-3) for fusion protein expression in E. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRVcub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.
从受感染的弗罗里达酸浆中分离出总RNA，以生成与马铃薯卷叶病毒（PLRV）古巴分离株的外壳蛋白（CP）基因相对应的互补DNA。该cDNA通过聚合酶链反应（PCR）进行扩增，并克隆到细菌表达载体pEX(1-3)中，以便在大肠杆菌中表达融合蛋白。通过针对PLRV CP的特异性抗体检测该产物。确定了CP基因的编码序列，预测的CP长度为208个氨基酸（23 kD）。将核苷酸序列和推导的氨基酸序列与其他PLRV分离株进行比较，发现在其他分离株的核苷酸和氨基酸序列水平上，其同一性为97 - 99.5%。PLRVcub CP推导的氨基酸序列与其他黄症病毒具有相当的同源性。我们认为，所描述的方案可能适用于其他低丰度或分离繁琐的植物病毒，因为该方法比克隆已知序列的植物病毒外壳蛋白基因的传统方法耗时更少。
