车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备

为制备车前草花叶病毒(Plantago asiatica mosaic virus,PlAMV)衣壳蛋白(capsid protein,CP)及其多克隆抗体,采用RT-PCR方法从甘肃省种植的切花百合上克隆了PlAMV cp基因的部分序列,连接表达载体pET-28a(+),转化Escherichia coli BL21...

Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of its polyclonal antibody

The cp gene of Plantago asiatica mosaic virus (PlAMV) was cloned from an isolate of PlAMV, obtained from lily growing in Gansu Province, China. The cp gene fragment of 621 bp was constructed into the prokaryotic expression vector pET-28a (+), and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to express the fusion CP protein. After IPTG induction and Ni-NTA gravity column chromatography, purified CP fusion protein was obtained and used as immunogen to prepare a rabbit-derived anti-CP polyclonal antibody. BLAST result showed that the similarity of nucleotide and amino acid sequences of cp gene was up to 74.7%-100% and 83.6%-100% between the cloned PlAMV isolate and the known PlAMV isolates, respectively. We noted that cp sequences from different hosts were significantly different, which suggested the distribution of PlAMV population was influenced by species of host. SDS-PAGE indicated that CP protein was highly expressed in E. coli BL21 (DE3), and had a relative protein molecular weight of 24 ku. Western blot result demonstrated that the prepared polyclonal antibody could successfully capture the natural PlAMV capsid proteins, which was used to check the expression level of PlAMV protein in susceptible plant tissue. This work provided a reference for the study of the pathogenic mechanism of PlAMV and the development of serological detection techniques for the virus.