禽网状内皮组织增生症病毒单抗制备及在外源病毒检测中的应用

关键词:  禽网状内皮组织增生症病毒  外源病毒检验  gp90  单克隆抗体  间接免疫荧光染色法

Key words:  avian reticuloendotheliosis virus  exogenous viruses detection  gp90  monoclonal antibody  indirect immunofluorescence staining method.

摘要:

为了建立外源病毒检验用间接免疫荧光染色方法,本研究将禽网状内皮组织增生症病毒囊膜蛋白gp90(SU)的基因合成后连接至原核表达载体中进行表达,获得REV-SU蛋白,通过Ni柱亲和层析获得纯化的重组蛋白,免疫BALB/C小鼠,应用杂交瘤技术获得1株分泌特异性抗REV SU蛋白的单克隆抗体杂交瘤细胞系(2A2株),该细胞株制备的腹水与不同REV毒株具有良好的反应性,荧光效价可达1：51200,与不同ALV毒株和其他常见禽的病原均没有交叉反应,特异性良好。采用该细胞株分泌的单克隆抗体建立的间接免疫荧光染色法能检出至少5个TCID50REV病毒感染量,与多克隆抗体作为检测一抗具有同等的效力,且检测背景更加纯净,适用于外源性REV病毒检验。

Abstract:

To establish an indirect immunofluorescence staining method for detecting exogenous viruses, the gene of the envelope protein gp90 (SU) of avian reticuloendotheliosis virus (REV) was synthesized and ligated into prokaryotic expression vector for expression, and REV-SU protein was obtained in this study. Then, the purified recombinant protein was obtained by Ni affinity chromatography. After immunizing BALB/C mice, a hybridoma cell line (strain 2A2) secreting specific monoclonal antibody against REV-SU protein was obtained with hybridoma technique. The ascites prepared using this cell line had high reactivity with different REV strains, with the immunofluorescence titer reaching 1 : 51,200, but no cross-reaction with different ALV strains or other conventional avian viruses, showing high specificity. The indirect immunofluorescence staining method established with the monoclonal antibody secreted by this cell line could detect more than 5 TCID50 REV,as the same effect as the detection based on polyclonal antibody, and the detection background was more pure. The results confirm that this method is suitable for the detection of exogenous REV.