鹅细小病毒Rep1蛋白的原核表达及抗体制备

Prokaryotic expression of the Rep1 protein of goose parvovirus and preparation of specific antibody

为深入研究鹅细小病毒(GPV)Rep蛋白的功能,采用PCR方法扩增出完整的Rep1基因,并克隆入原核表达载体pET-28a(+)。将重组质粒pET28-Rep1导入到大肠埃希菌BL21(DE3)中表达。SDS-PAGE分析表明:37℃下经1mmol·L^(-1) IPTG诱导4h,Rep1蛋白可获得高效表达。重组蛋白经切胶免疫BALB/c小鼠制备针对Rep1蛋白的多克隆抗体。经间接免疫荧光检测,1∶1 000稀释的多抗能特异性识别在鹅胚成纤维细胞上增殖的GPV抗原,说明所制备的多抗具有良好反应原性,可用于后续针对Rep蛋白的相关功能研究。

In order to explore the function of the Rep protein of Goose parvovirus （GPV）, the Repl gene was cloned into the pET-28a prokaryotic expression vector and the recombinant plasmids were transformed into BL21 （DE3） E. coll. The Repl protein was expressed successfully following 1 mmol · L^-1 of IPTG induction. The approximate molecular weight of Repl protein analyzed by SDS-PAGE was 80 ku. The antisera against Repl protein was produced in BALB/c mouse immunized with the recombinant protein. The prepared antisera reacted specifically with the proteins of GPV which replicated in goose embryo fibroblast （GEF） cells by indirect immunofluorescence assay （IFA）. The antisera prepared can be used for elucidating the role of the Rep protein in course of GPV infection.