BLCAP蛋白多克隆抗体制备及其免疫学特性鉴定

Preparation and Identification of Immunological Traits of Polyclonal Antibody Against BLCAP

目的:利用大肠杆菌BL21(DE3)表达BLCAP融合蛋白并制备和鉴定其多克隆抗体。方法:构建原核表达重组质粒pET32a(+)-BLCAP并转化到宿主菌BL21中,诱导表达带有His标签的目的融合蛋白,经Ni2+亲和层析纯化回收后免疫日本大耳白兔制备多克隆抗体,用ELISA测定抗体的效价,Western blot检测抗体的特异性和亲和性。结果:成功获得分子质量约为28 kU纯化的融合蛋白,免疫日本大耳白兔后得到抗BLCAP的多克隆抗体。ELISA测定显示抗体效价可达到1∶10 000。通过Western blot检测证明该抗体有较好的针对BLCAP蛋白的专一性。结论:重组质粒表达的BLCAP融合蛋白具有良好的抗原性。制备的特异性和效价良好的抗BLCAP的多克隆抗体,能够满足针对BLCAP免疫印迹和细胞免疫组化检测等实验要求,为今后深入研究BLCAP蛋白性质与功能提供了有用的实验工具。

Objective： To express BLCAP fusion protein in E. coli BL21（DE3） and prepare and identify polyclonal antibody. Methods： The prokaryotic expression plasmid pET32a（＋）-BLCAP was constructed and the positive recombinant plasmid was transformed into BL21（DE3）. The expressed His-tagged protein was purified by Ni^2＋ affinity chromatography column and purified fusion protein was used to immune the rabbit for preparing polyclonal antibody. The titer and specificity of the rabbit＇s antiserum was measured by ELISA and Western blotting. Results： The Mr 28 kU fusion protein was successfully expressed. The polyclonal antibody was also successfully prepared after immunizing the rabbit. The titer of the antiserum measured by ELISA could achieve 1 ： 10 000. The specificity of antibody was proved by Western blotting analysis of prokaryotic expression product of BLCAP. Conclusion： The BLCAP fusion protein has the good antigenicity. The BLCAP polyclonal antibody with high titer and specificity can satisfy Western blot and immunohistochemistry experiment request, which was the foundation to study the characters and function of BLCAP protein.