摘要
利用PCR技术从虎源猫泛白细胞减少症病毒(FPV)HLJ株细胞培养物中扩增VP2基因,测序后插入原核表达载体pGEX-6p-1构建pGEX-6p-VP2重组质粒,转化BL21(DE3)中进行诱导表达。表达蛋白经切胶法纯化,所得产物用Western blot法检测其抗原活性。以纯化的重组VP2蛋白作为抗原,通过建立检测家猫FPV自然感染抗体的间接ELISA方法来研究该蛋白作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。测序结果显示:该基因全长1755bp,编码584个氨基酸。SDS-PAGE和Western blot显示表达产物以包涵体形式存在,大小约为84000(其中目的蛋白58000,GST标签26000),并具有抗原性。间接ELISA抗体检测法的应用结果显示表达的重组VP2蛋白可以作为诊断抗原用于家猫FPV自然感染抗体的间接ELISA检测。并初步证明该蛋白具有作为猫科动物猫泛白细胞减少症通用诊断抗原的特性。
The amplified VP2 gene was subcloned into the prokaryotic expressing vector pGEX-6p-1,and then transferred into E.coli BL21（DE3） for expression under IPTG treatment.The purified recombinant protein was analysed by Western blot.Based on the purified recombinant protein,an indirect enzyme-linked immunosorbent assay （ELISA） for detecting antibodise was developed to study characteristics of common diagnostic antigen of VP2 protein.Sequencing result showed that the full length of the VP2 gene was 1755 bps and encoded 584 amino acids.SDS-PAGE and Western blotting assays revealed that the fusion protein with GST-tag,which was approximately 84 000,was highly effectively expressed in E.coli and had good immunoreactivity.The indirect ELISA indicated a good antigenic specificity of the expressed VP2 protein and the feasibility to be used as diagnostic antigen for the diagnosis of FPV infection in cats.Recombinant VP2 protein showed an ability to be used as common diagnostic antigen for the diagnosis of FPV infection in felids
出处
《中国兽医学报》
CAS
CSCD
北大核心
2010年第5期602-606,共5页
Chinese Journal of Veterinary Science
基金
国家林业局野生动植物保护管理项目基金资助项目(2007)
