摘要
根据 GenBank 上 WSSV 囊膜蛋白基因 vp19 和 vp28 的序列,设计并合成两对引物,PCR 扩增得到 vp19和vp28两基因,大小分别为370bp和630bp。通过EcoRI位点连接两基因,再按正确的阅读框插入表达载体pET-22b(+)中,构建出重组表达载体 pET-vp(19+28)并转化大肠杆菌 BL21(DE3)。基因工程菌株 35℃IPTG 诱导,表达产物经 SDS-PAGE 检测显示有与预期大小 41kDa 相吻合的融合蛋白带。用 Ni2+-柱纯化的基因工程蛋白免疫新西兰大白兔制备抗血清,进行螯虾活体中和病毒实验,结果表明抗血清对 WSSV 的中和效率达到了 100%。
Two pairs of primers were designed according to the sequences of envelope genes, vp19 and vp28 of White spot syndrome virus (WSSV) in the GenBank. The two DNA fragments about 370bp and 630bp amplified by PCR were linked to the EcoR I restriction endonuclease site and cloned into E.coli expression vector pET- 22b(+) in the proper Open Reading Frame(ORF). With IPTG induction at 35℃, the molecular weight of the engineered protein was about 41kDa, which was identified by SDS-PAGE analysis. Antiserum of the fusion envelope protein VP (19+28) purified by Ni2+- column chromatography was prepared by immunizing rabbit. The VP (19+28) antiserum was used to neutralize the WSSV infection on crayfish by intramuscular injection. The result showed that the antiserum was effective in the neutralization of WSSV on the crayfish which were reared with artificial food at 15-22℃.
出处
《中国病毒学》
CSCD
2004年第6期587-590,共4页
Virologica Sinica
