抗羊口疮病毒囊膜蛋白B2L小鼠多克隆抗体的制备和应用

Preparation and application of anti-envelope protein B2L mouse polyclonal antibody of ORF virus

目的获取羊口疮病毒(ORFV)囊膜蛋白B2L并制备鼠抗B2L蛋白多克隆抗体。方法采用PCR扩增获得B2L基因,并将其分别克隆至原核表达载体p ET-32a和真核表达载体p3×FLAG-CMV-14。两个重组表达载体均经过鉴定,重组原核表达载体使用BL21大肠杆菌表达,异丙基β-D-硫代半乳糖苷(IPTG)诱导表达目的蛋白。通过Tris-尿素溶液洗去非目的蛋白,Western blot法鉴定。将定量的纯化蛋白免疫BALB/c小鼠,制备鼠抗ORFV B2L多克隆抗体。将真核表达重组质粒p3×FLAG-B2L转染Vero细胞、BHK-21细胞,使用间接免疫荧光法(IFA)对抗B2L蛋白的多克隆抗体鉴定,并应用于免疫组织化学染色检测患病羊唇部组织ORFV的表达。结果 Western blot法确定了制备的B2L蛋白的特异性; IFA结果表明制备的小鼠抗B2L多克隆抗体有特异性和反应原性,免疫组织化学染色法结果表明该抗体可以检测出患病羊唇部组织的ORFV。结论成功制备了特异性的小鼠抗B2L多克隆抗体。

Objective To obtain the envelope protein B2 L of orf virus( ORFV) and prepare highly specific polyclonal antibody against B2 L. Methods The B2 L gene was amplified by PCR,and subcloned into the vectors p ET-32 a andp3 × FLAG-CMV-14,p ET-32 a-B2 L followed by expression of B2 L protein in E. coli,and induction of the target protein byIPTG,purification by urea solution and identification via Western blot analysis. Furthermore,the BALB/c mice wereimmunized with B2 L protein to prepare highly-specific anti-B2 L polyclonal antibody. In addition,plasmid p3 × FLAG-B2 L wastransfected into Vero cells and BHK-21 cells which was used to confirm its specificity and antigenicity by indirectimmunofluorescence assay( IFA). Results Western blotting demonstrated that the B2 L protein had high-level specificity.The results of IFA indicated that the anti-B2 L specific polyclonal antibody had specificity and reactivity. Then Immunohisto-chemistry showed that it could neutralize natural ORFV. Conclusion Highly specific and purified polyclonal antibody againstB2 L protein of the ORFV was successfully prepared.