薯蓣褪绿坏死花叶病毒CP基因的原核表达及抗血清的制备

Prokaryotic Expression of CP Gene of Yam Chlorotic Necrotic Mosaic Virus and Preparation of Its Antiserum

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.