论文部分内容阅读
以甜菜坏死黄脉病毒(Beet Necrotic Yellow Vein Virus,简称BNYVV)内蒙分离物(NM)RNA为模板,通过反转录和PCR扩增得到了BNYVV RNA4基因组的cDNA克隆pGBF6。序列分析结果表明,pGBF6含有全长RNA4 cDNA插入片段,大小为1465个核苷酸,含有一个849个核苷酸的开放阅读框架,编码产生由282个氨基酸组成的分子量为31kDa的蛋白。与法国F2分离物RNA4相比,其核苷酸序列和由此推导的氨基酸序列同源性分别为97.1％和96.4％,并在5’端非编码区比F2分离物缺失了3个核苷酸。将RNA4编码区cDNA克隆到原核表达载体pFLAG·MAC上,获得融合蛋白表达质粒pFMBF87。所构建的融合蛋白由载体序列编码的14个氨基酸和31kDa蛋白C端的233个氨基酸组成。经IPTG诱导,Westem blotting分析表明,该融合蛋白在大肠杆菌中得到高效表达。本文还对内蒙分离物的株系划分进行了讨论。
The cDNA clone pGBF6 of BNYVV RNA4 genome was obtained by reverse transcription and PCR amplification using Inner Mongolia isolate (NM) RNA of Beet Necrotic Yellow Vein Virus (BNYVV) as a template. Sequence analysis showed that pGBF6 contained a full-length RNA4 cDNA insert of 1465 nucleotides in size and contained an open reading frame of 849 nucleotides encoding a protein of 282 kDa in molecular weight of 31 kDa. Compared with the French F2 isolate RNA4, the nucleotide sequences and deduced amino acid sequence identities were 97.1% and 96.4% respectively, and 3 nucleotides were deleted from the F2 isolate in the 5 ’non-coding region acid. The RNA4 coding region cDNA was cloned into the prokaryotic expression vector pFLAG · MAC to obtain the fusion protein expression plasmid pFMBF87. The constructed fusion protein consists of 14 amino acids encoded by the vector sequence and 233 amino acids at the C-terminal of the 31 kDa protein. After induced by IPTG, Westem blotting analysis showed that the fusion protein was highly expressed in E.coli. This article also discussed the division of isolates in Inner Mongolia.