摘要
为了制备抗鸭肠炎病毒(DEV)VP22蛋白的单克隆抗体(MAb),本研究以DEV Clone-03基因组DNA为模板,通过PCR方法扩增UL49基因并克隆至pMD-18载体。重组质粒经测序鉴定后,将UL49基因亚克隆于原核表达载体pET-30a,构建重组表达质粒pET-30a-UL49。重组质粒转化大肠杆菌BL21(DE3)后,经IPTG诱导,在大肠杆菌中获得表达,并对表达的重组蛋白VP22进行western blot鉴定。结果显示,表达的重组蛋白分子量约为36ku,与预期的大小一致。重组蛋白能够被鼠抗DEV阳性血清所识别,表明该重组蛋白具有良好的免疫原性。将重组蛋白纯化、复性后免疫BALB/c小鼠,制备MAb,经间接ELISA、western blot和间接免疫荧光试验进行筛选及鉴定,获得4株能够稳定分泌抗DEVVP22蛋白MAb的杂交瘤细胞株,分别命名为2B10、2G1、3F10和3G2。其MAb亚类分别属于IgG2b、IgG2b、IgA和IgM。4株MAbs都能够与DEV Clone-03发生特异性反应,表明能够识别天然构象的VP22蛋白。这4株MAb可用于DEV VP22蛋白功能研究及抗原表位的研究。
In this study, the UL49 gene was amplified from the genome of duck enteritis virus （DEV） Clone-03 strain by PCR method and was cloned into pMD-18 vector. UL49 gene was subcloned into the prokaryotic expression vector pET-30a. The E. coli BL21 （DE3） contained the recombinant plasmid was induced by IPTG and the recombinant protein was analyzed by SDS-PAGE. The recombinant protein with a molecular weight of 36 ku could be detected by western blot using the antiserum against DEV. Monoclonal antibodies （MAbs） against VP22 protein of DEV were developed using recombinant protein VP22 as antigen. Four MAbs against VP22 were obtained following screening by indirect enzyme-linked immunosorbent assay （ELISA）, western blot and indirect immunofluorescence assay. Four MAbs, designated as 2B10, 2G1, 3F10 and 3G2 respectively, could recognize the VP22 protein in DEV-infected chicken embryo fibroblasts. This study paved the way for future study of biological function and antigenic epitope of VP22 protein.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第9期721-725,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
"十一五"国家科技支撑计划(2006BAD6A03)
