摘要
采用RT-PCR方法从新城疫病毒(NDV)ClassⅠ强毒株9a5b中扩增出了L基因C末端的基因片段,将其克隆到表达载体pGEX-6P-1中,测序验证后转化入表达宿主菌BL21进行IPTG诱导表达,表达的蛋白接种8周龄BALB/c小鼠,制备L蛋白的单克隆抗体,用间接免疫荧光试验(IFA)、酶联免疫吸附试验(ELISA)和Western-bolt方法共同检测所获得的抗体。结果显示,重组菌可表达出分子质量约为39ku的重组融合蛋白。SDS-PAGE分析显示,表达的蛋白以包涵体的形式存在于菌体中。多种方法筛选显示成功制备了4株单克隆抗体。免疫特异性鉴定结果表明,在所获得的单抗中,4株单抗均具有ELISA和IFA效价,且4株单抗与ClassⅠ毒株的反应均强于与ClassⅡ毒株的反应。
L gene of ClassⅠ9a5b virulent strain was amplified by RT-PCR and cloned to pGEX-6P-1 expression vector.After sequencing,the recombinant plasmid was transformed into BL21 which were then induced by IPTG to produce a recombinant protein of 39 ku.BALB/c mice were immunized with the purified expression product of L protein.The ELISA,indirect immunofluorescence assay IFA and Western-blot analysis were used to characterize and identify antibodies.Results showed that a panel of 4 hybridoma cell lines were successfully prepared.All the 4 McAbs had the ability of ELISA and IFA,and all the 4 McAbs had stronger reaction with ClassⅠ than ClassⅡ.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2012年第4期347-351,共5页
Chinese Veterinary Science
基金
国家自然科学基金青年基金项目(31101822)
公益性行业(农业)专项(201003012)
关键词
新城疫病毒
原核表达
单克隆抗体
L蛋白
杂交瘤细胞
Newcastle disease virus
prokaryotic expression
monoclonal antibody
L protein
hybridoma cell
