摘要
薯蓣褪绿坏死花叶病毒(yam chlorotic necrotic mosaic virus,YCNMV)是马铃薯Y病毒科柘橙病毒属暂定成员。采用RT-PCR方法,以Sprimer/M4简并引物对扩增获得的YCNMV分离物3'端基因组序列为参考序列,根据该序列设计CP基因特异引物获得YCNMV CP基因并插入p ET-30a表达载体中,在BL21(DE3)菌株中诱导表达。获得的融合蛋白经Ni+-NTA柱纯化后免疫新西兰大白兔,制备抗血清。结果表明,连接在表达载体中的CP基因序列及表达的蛋白氨基酸序列与预期的CP基因核苷酸和氨基酸序列同源性均为99.65%。聚丙烯酰胺凝胶电泳结果表明,获得的融合蛋白大小约为44 k D,与预期蛋白大小相符。制备的抗血清经Western blotting和ID-ELISA检测表明,获得的多克隆抗体效价较高,能可靠、灵敏、特异地与YCNMV病毒颗粒结合,可应用到该病毒的检测中。
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.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第8期206-212,共7页
Biotechnology Bulletin
基金
国家自然科学基金项目(31101416)
云南大学生命科学学院科学研究与人才培养开放基金项目(2013S205)
云南省烟草公司项目(2011YN60
2012YN10
2012YN36
2012YN13)
关键词
薯蓣褪绿坏死花叶病毒
外壳蛋白
原核表达
抗血清
yam chlorotic necrotic mosaic virus
coat protein
prokaryotic expression
antiserum