抗Ⅰ型马立克氏病毒sorf2蛋白的多克隆抗体制备及其特异性鉴定

Preparation and identification of polyclonal antibody to serotype I Marek＇s disease virus sorf2 protein

摘要:

摘要:【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。

关键词:关键词:Ⅰ型马立克氏病毒，sorf2 基因，多克隆抗体，Western blot，间接免疫荧光

Abstract:

Abstract:［Objective］To obtain mice and rabbit polyclonal antibody of serotype I Marek＇s disease virus (MDV) sorf2 protein with higher titer and to identify the specificity．［Methods］ Using serotype I MDV GX0101 as template，we amplified sorf2 gene and then cloned it into pET-28a (+) and pET-32a (+) respectively．The recombinant plasmid pET-28a-sorf2 and pET-32a-sorf2 was separately transformed into Escherichia coli BL21 (Rosetta) competent cell which was induced with isopropylthio-β-D-galactoside (IPTG)．After purification，immuned 6－8 Balb/c mice and adult New Zealand white rabbit with the purified fusion protein and the anti-sorf2 polyclonal antibody were prepared．The specificity of the serum was detected by Western blot and the indirect immunofluorescence assay (IFA) method．［Results］Serotype I MDV sorf2 protein was expressed successfully in the recombinant E coli． Mice and rabbit anti-sorf2 polyclonal antibody with higher titer could react with sorf2 protein specifically．［Conclusion］The prepared anti-sorf2 polyclonal antibody could identify MDV sorf2 gene deletion strain specifically．In addition，it could be used for differential of MDV vaccine poison HVT and serotype I MDV，which was useful for the separation and identification of clinical MDV.

Key words:Keywords: serotype I Marek＇s disease virus，sorf2 gene，polyclonal antibody，Western blot，immunofluorescence assay