1种改良的高效单特异性兔多克隆抗体的制备方法

A modified and effective method of obtaining monospecific polyclonal antibody from rabbits

目的建立1种改良的高效单特异性兔多克隆抗体的制备方法。方法用RT-PCR方法获得bax保守N端1～123位氨基酸基因片段,并将其插入pET42a原核表达载体,诱导表达的Bax融合蛋白组合应用GST、His亲和层析技术获得免疫原(pET42a/Bax融合蛋白),HPLC鉴定纯度达95%。利用改良快速免疫法获得人Bax兔多克隆抗体,并经蛋白A柱亲和层析技术,抗原亲和纯化技术获得高效价高特异性的抗体。间接ELISA检测抗体滴度、Western blot和免疫组化试验检测抗体特异性,并与商业化抗体进行对比。结果通过快速免疫法得到人Bax兔多克隆抗体,经过Protein A柱纯化,再经抗原亲和纯化后,间接ELISA证明,抗体效价均达1:51 200;Western blot显示,只有经过抗原亲和纯化后的抗体特异性高,无其他杂带;免疫组化证明,在原发性肝癌组织中,人Bax兔多克隆抗体能特异地和内源性Bax结合,其高效高特异性已达国外Santa Cruse公司Bax商业化抗体水平。结论快速免疫法与抗原亲和纯化相结合,获得人Bax高效单特异性兔多克隆抗体,建立了1种改良的高效单特异性兔多克隆抗体的制备方法。

Objective To search for a modified and effective method of obtaining monospecific polyclonal antibody from rabbits and obtain the separately high-titer and specific antibodies of bax,a member in bcl-2 family,and β-actin.Methods Obtain Bax gene of conservative 1 to 123 amino acids from N pole by RT-PCR.Subclone the RT-PCR gene product into the Protokaryon expression vectors pET42a.The recombinant Bax were purified by affinity chromatogram with His and GST columns.The purity identified by HPLC approaches 95%.Bax Rabbit polyclonal antibody serum obtained by a modified rapid immune procedure was purified by protein A column and antigen affinity chromatography.The titre and specificity of the Bax antibody were determined by ELISA,Western blot and immunohistochemistry assay.Results Anti-Bax antibody was obtained by modified rapid immune procedure,after protein A column and antigen affinity chromatography,ELISA illustrated that the titre of the two antigens was 51 200;Western blot suggests that only after antigen affinity chromatography,the antigen was more specific without other mixed band;The immunohistochemistry revealed that anti-Bax antibody could bind the human endogenous bax protein in the primary hepatoma tissue and its high titre and high specificity had catch up with the foreign commercial anti-Bax antibody from Santa Cruse.Conclusion The high-titre and specific anti-Bax（Bax fusion protein） antibody were obtained,which illustrated that the combination of rapid immunization method and antigen affinity chromatography is a modified and effective method of obtaining monospecific polyclonal antibody from rabbits.