鸡CD40L基因的原核表达及多克隆抗体的制备

Prokaryotic expression of chicken CD40L gene in E.coli and the preparation of its polyclonal antibody

为了研究鸡CD40L作为分子免疫佐剂的免疫原性,试验采用鸡的脾脏细胞克隆鸡CD40L基因,构建表达载体pET28a-cCD40L进行原核表达,以纯化的重组蛋白免疫长耳白兔,获得兔抗鸡CD40L多克隆抗体,用间接ELISA检测多克隆抗体效价,并进行Western-blot检测。结果表明:获得分子质量为26 ku的重组融合蛋白,多克隆抗体效价为1∶12 800;表达的融合蛋白不仅能与鼠抗His克隆抗体发生特异性反应,而且与获得的兔抗鸡CD40L多克隆抗体发生特异性反应。说明鸡CD40L融合蛋白具有较高的反应活性,所获得的兔抗鸡CD40L多克隆抗体具有良好的特异性。

To study the immunogenicity of chicken CD40L gene as a molecular immunological adjuvant, chicken CD40L （cCIMOL） gene was cloned using chicken splenocytes, and then was used to construct expression vector pET28a -cCD40L for prokaryotic expression. The recombinant protein was purified and immunized the long - eared rabbit to prepare rabbit anti - chicken CD40L polyclonal antibody. The polyclonal antibody was used fnr the detection of its titer using indirect ELISA, and then was used for Western - blot analysis. The results showed that the obtained recombinant fusion protein had a molecular weight of about 26 kD, and the titer of anti - serum was 1：12 800. The expressed fusion protein could occur specific reactions with both mouse - anti His monoclonal antibody and rabbit anti - chicken CD40L polyclonal antibody. The results indicate that the chicken CD40L fusion protein has high reactive activity, and the obtained rabbit anti - chicken CD40L polyclonal antibody has good specificity.