关键词: 薯蓣褪绿坏死花叶病毒, 外壳蛋白, 原核表达, 抗血清

Abstract: Yam chlorotic necrotic mosaic virus（YCNMV）is a new tentative species of genus Macluravirus in the family Potyviridae. Referring to the 3'-terminal sequence amplified with degenerate primers Sprimer/M4 by RT-PCR, we designed the specific primer pair of CP gene of YCNMV. The PCR product of CP gene was inserted into the pET30a prokaryotic expression vector, which was subsequently expressed in BL21（DE）strain by induction. Fusion protein was purified with Ni⁺ -NTA affinity column and was used to immunize New Zealand white rabbits to produce the antiserum. Results showed that the CP gene ligated in the prokaryotic expression vector shared 99.65% homology with the template CP gene in both nucleotide and protein sequence. SDS-PAGE results indicated that the CP fusion protein was about 44 kD in size, which was in accord with the prediction. The antiserum was tested by both ID-ELISA method and Western blotting, which showed that the polyclonal antibody had a relatively high level of titer, and reliably, sensitively and specifically bound with the YCNMV virus particles. These results indicate that the antiserum is suitable for YCNMV detection.

Key words: yam chlorotic necrotic mosaic virus, coat protein, prokaryotic expression, antiserum