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

【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] 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 （1PTG）. 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 coll. 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.