CAJ | 학술논문

目的构建血管紧张素Ⅱ相关新基因BC097361的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗BC097361蛋白多克隆抗体并进行鉴定.方法应用RT-PCR技术,以LX02细胞总RNA为模板,扩增BC097361目的基因片段,构建原核表达载体pET-32a(+)-BC097361.转化大肠埃希菌BL21(DE3),异丙基-β-半乳糖苷诱导并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,Western blot分析证实融合蛋白表达的特异性.大量表达后利用Ni+亲和柱对表达蛋白进行纯化及柱上复性.纯化蛋白免疫新西兰兔,获得抗BC097361蛋白的多克隆抗体.以纯化的BC097361蛋白为抗原,分别以免疫前后的新西兰兔血清作为第一抗体,利用Western blot和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测.结果扩增获得BC097361基因片段,成功表达了BC097361相关蛋白,经十二烷基硫酸钠一聚丙烯酰胺凝胶电泳和Western blot分析得到证实.成功获得融合蛋白及兔抗BC097361多克隆抗体.酶联免疫吸附法检测证实多克隆抗体效价>1:320 000,Western blot、免疫组织化学检测证明多克隆抗体的特异性良好.结论利用大肠埃希菌BL21(DE3)能够成功表达BC097361蛋白,获得高特异性、高效价兔抗BC097361蛋白的多克隆抗体,为今后研究BC097361蛋白的生物学特性奠定了基础.
목적 구건혈관긴장소Ⅱ상관신기인BC097361적원핵표체재체,유도융합단백적표체,병대기진행순화;제비토항BC097361단백다극륭항체병진행감정.방법 응용RT-PCR기술,이LX02세포총RNA위모판,확증BC097361목적 기인편단,구건원핵표체재체pET-32a(+)-BC097361.전화대장애희균BL21(DE3),이병기-β-반유당감유도병통과십이완기류산납-취병희선알응효전영분석,Western blot분석증실융합단백표체적특이성.대량표체후이용Ni+친화주대표체단백진행순화급주상복성.순화단백면역신서란토,획득항BC097361단백적다극륭항체.이순화적BC097361단백위항원,분별이면역전후적신서란토혈청작위제일항체,이용Western blot화매련면역흡부법대다극륭항체진행특이성분석급효개검측.결과 확증획득BC097361기인편단,성공표체료BC097361상관단백,경십이완기류산납일취병희선알응효전영화Western blot분석득도증실.성공획득융합단백급토항BC097361다극륭항체.매련면역흡부법검측증실다극륭항체효개>1:320 000,Western blot、면역조직화학검측증명다극륭항체적특이성량호.결론 이용대장애희균BL21(DE3)능구성공표체BC097361단백,획득고특이성、고효개토항BC097361단백적다극륭항체,위금후연구BC097361단백적생물학특성전정료기출.
Objective To express and purify of the BC097361 recombinant protein,and to prepare the BC097361 specific rabbit polyclonal antibody.Methods BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+),and the resulting plasmid was transformed into E.coli BL21 (DE3).The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting.The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody.The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA.Results The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body.ELISA indicated the titer of polyclonal antibody > 1:320 000.The high specificity was comfirmed with Western blot.Conclusions The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.