克隆草莓轻型黄边病毒CP基因,并构建其原核表达载体,在大肠杆菌中诱导表达CP蛋白。利用SMYEV的检测引物,通过RT-PCR技术筛选带SMYEV的草莓植株;根据NCBI中SMYEV序列设计扩增CP基因全长引物,通过RT-PCR获得SMYEV沈阳分离物CP基因全长序列;将CP基因克隆到原核表达pGEX-6P-1上,利用IPTG诱导CP基因在大肠杆菌BL21(DE3) 中表达蛋白。利用RT-PCR扩增获得SMYEV分离物SY05的CP基因全长序列,由729个核苷酸组成,编码242个氨基酸残基。SY05与其他SMYEV分离物的核苷酸同源性为80.1%～97.4%,氨基酸同源性为92.1%～99.6%,其中,与SMYEV分离物SY03的氨基酸一致性为98.3%。将SY05的CP基因成功克隆到原核表达载体pGEX-6P-1上,构建了重组表达载体p6P-EV-CP,并将其导入大肠杆菌BL21(DE3) 中。SDS-PAGE分析表明,IPTG诱导SMYEV分离物SY05的CP基因在大肠杆菌中表达出分子量为52.0 kDa的融合蛋白。克隆出SMYEV分离物SY05的CP基因,实现了其在原核细胞中的表达,为SMYEV抗血清的制备奠定了重要基础。
The objective of this study is to clone and analyze the sequence characterization of strawberry mild yellow edge virus CP gene, then construct its prokaryotic expression vector and express its protein in Escherichia coli Rosetta-gami B(DE3). Virus detection primers was used to detect the strawberry plantlets infected with SMYEV by RT-PCR, and a pair of primers were designed according to the uploaded sequence of SMYEV CP gene in NCBI to isolate the gene of SMYEV isolated in Shenyang region(SY05). The CP gene of SY05 was subcloned into expression vector pGEX-6p-1 and then transferred into Escherichia coli Rosetta-gami B(DE3) pLysS, finally this CP protein was induced by IPTG to express. Sequence analysis indicates that the SMYEV CP gene from SY05 is 729 bp in length and encodes 242 amino acid residues. The nucleotide sequence of this CP gene shares 80. 1%-97. 4% homology with other CP genes of SMYEV isolated from different plants. The deduced protein shares 92. 1%-99. 6% with the other homologous genes, including a 98. 3% homology with that isolated from SY03. The CP gene was subcloned into the expression vector pGEX-6P-1 and transferred into prokaryotic cells. Induced with IPTG, a 52. 0 kDa induction expression band of fusion protein was found in SDS-PAGE detection. All the results showed that we have isolated the CP gene of SMYEV in SY05 and prokaryotic expression of this gene was realized. This research provides a foundation for the serology research of SMYEV detection in strawberry.
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