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【目的】制备高效价鼠和兔抗Ⅰ型马立克氏病毒(Marek’s disease virus,MDV)sorf2蛋白的多克隆抗体并对其特异性进行鉴定。【方法】以Ⅰ型MDV GX0101为模板,利用PCR扩增sorf2基因,分别克隆进原核表达载体pET-28a(+)和pET-32a(+)中,转化大肠杆菌BL21(Rosetta),经异丙基硫代-β-D半乳糖苷(IPTG)诱导后进行融合蛋白的表达和纯化。用纯化的融合目的蛋白常规免疫6-8周龄Balb/c小鼠和成年新西兰大白兔,制备抗sorf2蛋白的多克隆抗体。用Western blot和间接免疫荧光试验鉴定多克隆抗体的特异性。【结果】Ⅰ型MDV的sorf2基因在原核表达载体中能够正确表达,免疫小鼠和兔后能获得与sorf2蛋白发生特异性反应的高效价的多克隆抗体。【结论】制备的抗sorf2蛋白的多克隆抗体能够特异的鉴定MDV sorf2基因缺失株,有效的区分MDV疫苗株HVT(FC-126)与Ⅰ型MDV毒株,用于临床MDV野毒的分离鉴定。
【Objective】 Polyclonal antibodies against Marek’s disease virus (MDV) sorf2 were prepared and their specificity was evaluated. 【Method】 The sorf2 gene was amplified by PCR using type Ⅰ MDV GX0101 as a template and cloned into prokaryotic expression vector pET-28a (+) and pET-32a (+) respectively. The recombinant was transformed into E. coli BL21 (Rosetta) Expression and purification of fusion protein after induction with thio-β-D-galactoside (IPTG). Anti-sorf2 protein polyclonal antibodies were prepared by routine immunization of 6-8 week old Balb / c mice and adult New Zealand white rabbits with purified fusion protein. Western blot and indirect immunofluorescence assay were used to identify the specificity of the polyclonal antibody. 【Results】 sorf2 gene of type Ⅰ MDV was correctly expressed in prokaryotic expression vector, and high titer polyclonal antibodies specific for sorf2 protein could be obtained after immunization of mice and rabbits. 【Conclusion】 The prepared polyclonal antibody against sorf2 could specifically identify the MDF sorf2 gene deletion strain and effectively discriminate between the MDV vaccine strain HVT (FC-126) and the type Ⅰ MDV strain for the isolation and identification of the clinical MDV field virus .