CAJ | 학술논문

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.
목적 건립1충개량적고효단특이성토다극륭항체적제비방법.방법 용RT-PCR방법획득bax보수N단1～123위안기산기인편단,병장기삽입pET42a원핵표체재체,유도표체적Bax융합단백조합응용GST、His친화층석기술획득면역원(pET42a/Bax융합단백),HPLC감정순도체95%.이용개량쾌속면역법획득인Bax토다극륭항체,병경단백A주친화층석기술,항원친화순화기술획득고효개고특이성적항체.간접ELISA검측항체적도、Western blot화면역조화시험검측항체특이성,병여상업화항체진행대비.결과 통과쾌속면역법득도인Bax토다극륭항체,경과Protein A주순화,재경항원친화순화후,간접ELISA증명,항체효개균체1∶51 200;Western blot현시,지유경과항원친화순화후적항체특이성고,무기타잡대;면역조화증명,재원발성간암조직중,인Bax토다극륭항체능특이지화내원성Bax결합,기고효고특이성이체국외Santa Cruse공사 Bax상업화항체수평.결론 쾌속면역법여항원친화순화상결합,획득인Bax고효단특이성토다극륭항체,건립료1충개량적고효단특이성토다극륭항체적제비방법.
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.