甜菜坏死黄脉病毒RNA4 cDNA克隆、序列分析及其编码蛋白基因在大肠杆菌中的表达

cDNA CLONING, SEQUENCING AND EXPRESSION OF RNA4 FROM BEET NECROTIC YELLOW VEIN VIRUS

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。

With beet necrotic yellow vein virus (BNYVV) NM isolate from Inner Mongolia, the full length RNA4 cDNA was amplified by reverse transcription and polymerase chain reaction (RT-PCR), and cloned into pGEM7zf(+) to obtain the recombinant plasmid pGBF6. The sequence analysis showed that the RNA genome has 1465 nucleotides and contains a single open reading frame of 849 nucleotides, which encoded a 31kDa protein consisted of 282 amino acids. Comparing with the French F2 isolate, it shows 97.1% identity in terms of nucleotide with 3 nucleotides deletion at its 5' noncoding region and 96.4% homology of deduced amino acids respectively. The expression vector pFMBF87 was constructed by plasmid pFLAG . MAC and part of the coding region. The IPTG-inducible fusion protein is consist of C-terminal 233 amino acids of the 31kDa protein encoded by BNYW RNA4 besides a flag peptide of 8 amino acids at the N terminal. With induction of IPTG, a 27kDa protein was specifically expressed in E. coli in high level and its affinity to BNYW RNA4 antibodies was identified by Western blot. The relationship of BNYW NM to other strain groups was discussed in this paper as well.