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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。
The recombinant domain of the heavy chain of heavy-chain antibody (VHH) was expressed in Escherichia coli. After purification and renaturation, Serum Cys C latex-enhanced turbidimetric immunoassay (Particle enhanced turbidimetric immunoassay, PETIA). According to the characteristics of the encoded protein of Escherichia coli, the coding sequence of Cys C heavy chain single chain antibody was designed and the target gene was cloned into PET32a (+) vector to construct recombinant prokaryotic expression plasmid PET32a (+) - Cys C heavy chain single domain antibody was sequenced and then transformed into E. coli BL21 for expression under the induction of isopropyl-β-D-thiogalactoside (IPTG). The obtained antibody was purified by Ni-NTA affinity chromatography Analytical method was purified, purified by dilution, SDS-PAGE to identify its purity. Cys C heavy chain single-domain antibody conjugated latex particles, the establishment of PETIA method for the determination of human serum Cys C. SDS-PAGE electrophoresis analysis showed that the relative molecular weight of the expressed Cys C heavy chain single-domain antibody was about 53 kDa. After purification by Ni-NTA affinity chromatography, the purity of the antibody was over 90%, and the recovery rate was about 60% . The PETIA for human serum Cys C, established using the Cys C heavy chain single domain antibody, was able to detect Cys C levels in the serum. The prokaryotic expression system of Cys C heavy chain single domain antibody was successfully constructed and an active Cys C heavy chain single domain antibody was obtained. This antibody can be used to establish the PETIA assay for measuring Cys C and lay the foundation for further development of Cys C immunoassay kit The foundation.