重组人胱抑素C重链单域抗体的制备

发布时间：2019-03-24 13:56

【摘要】：研究在大肠杆菌中表达重组人胱抑素C(Cystatin C,Cys C)重链单域抗体(Variable domain of the heavy chain of heavy-chain antibody,VHH),经纯化和复性后,建立测定人血清Cys C的胶乳增强免疫比浊法(Particle enhanced turbidimetric immunoassay,PETIA)。根据大肠杆菌编码蛋白的特性设计Cys C重链单域抗体编码基因序列,人工合成目的基因克隆至PET32a(+)载体中,构建重组人Cys C重链单域抗体原核表达质粒PET32a(+)-Cys C重链单域抗体,测序鉴定正确后转入大肠杆菌BL21中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,所获得的目的抗体经Ni-NTA亲和层析法进行纯化,通过稀释复性,SDS-PAGE鉴定其纯度。Cys C重链单域抗体化学偶联胶乳颗粒,建立PETIA法测定人血清Cys C。SDS-PAGE电泳分析显示,表达的Cys C重链单域抗体相对分子质量约为53 k,经Ni-NTA亲和层析法纯化后抗体纯度达90%以上,复性回收率为60%左右。用Cys C重链单域抗体建立的测定人血清Cys C的PETIA,能检测到血清中的Cys C含量。成功构建Cys C重链单域抗体原核表达系统并获得了有活性的Cys C重链单域抗体,该抗体可用于建立测定Cys C的PETIA法,为下一步开发Cys C的免疫检测试剂盒奠定了基础。
[Abstract]:To study the expression of recombinant human cystatin C (Cystatin C (Cys C) heavy chain single domain antibody (Variable domain of the heavy chain of heavy-chain antibody,VHH) in E. coli, after purification and renaturation, To establish a latex enhanced immunoturbidimetric assay (Particle enhanced turbidimetric immunoassay,PETIA) for the determination of Cys C in human serum. The Cys C heavy chain single domain antibody encoding gene sequence was designed according to the characteristic of E. coli protein, and the target gene was synthesized and cloned into PET32a () vector. The recombinant human Cys C heavy chain single domain antibody prokaryotic expression plasmid PET32a ()-Cys C heavy chain single domain antibody was constructed, identified by sequencing and transferred into E. coli BL21. Isopropyl-尾-D-galactogalactoside (IPTG) was induced and expressed. The purified antibody was purified by Ni-NTA affinity chromatography, and its purity was identified by dilution and renaturation by SDS-PAGE. Cys C heavy chain single domain antibody was chemically coupled with latex particles. PETIA method was established for the determination of human serum Cys C.SDS-PAGE electrophoresis. The relative molecular weight of Cys-C heavy chain single domain antibody was about 53 kg. the purity of the antibody purified by Ni-NTA affinity chromatography was more than 90%, and the recovery rate of renaturation was about 60%. The content of Cys C in human serum Cys C can be detected by PETIA, established by using heavy chain single domain antibody against Cys C. The prokaryotic expression system of Cys-C heavy chain single domain antibody was successfully constructed and the active Cys-C heavy chain single domain antibody was obtained. The antibody can be used to establish PETIA assay for the determination of Cys-C and lay a foundation for the further development of Cys-C immune detection kit.
【作者单位】：南京理工大学环境与生物工程学院;中国人民解放军第八一医院检验科;南京大学生命科学学院;
【基金】：973项目资助(No.2014CB744501)
【分类号】：R943

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