重组人胱抑素C重链单域抗体的制备

Preparation of Recombinant Human Cystatin C Variable Domain of the Heavy Chain of Heavy-Chain Antibody

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。

To study the expression of recombinant human cystatin C（Cys C） variable domain of the heavy chain of heavy-chain antibody（VHH） in Escherichia coli BL21,after purified through Ni-NTA affinity chromatography and refolded by means of dilution,the particle enhanced turbidimetric immunoassay（PETIA） for determining concentration of human serum Cys C was established.The gene of recombinant human Cys C VHH was obtained by means of synthesis.The c DNA fragment was cloned into p ET-32a（＋）expression vector to construct prokaryotic expression vector p ET-32a（＋）-Cys C VHH,which was analyzed by means of DNA sequencing.The prokaryotic expression vector p ET-32a（＋）-Cys C VHH was transformed into Escherichia coli BL21,then Escherichia coli BL21 was induced to express recombinant human Cys C VHH by the inducer of IPTG.Recombinant human Cys C VHH was purified through Ni-NTA affinity chromatography and refolded by means of dilution.The prepared recombinant human Cys C VHH was identified by means of SDS-PAGE.Then the recombinant human Cys C VHH chemical coupled latex particles,PETIA method for determination concentration of human serum Cys C was established.The result of SDS-PAGE showed that recombinant human Cys C VHH was expressed successfully and the molecular weight of the expressed recombinant human Cys C VHH was 53 k,the purity of recombinant human Cys C VHH reached to 90% by means of Ni-NTA affinity chromatography.The refolding rate was about 60% by means of dilution refolding.With the recombinant human Cys C VHH,PETIA for determining concentration of human serum Cys C was established.This method can be detected Cys C content in the serum.The recombinant human Cys C VHH prokaryotic expression system was constructed successfully,biological activity of the recombinant human Cys C VHH was obtained,which could be used to establish PETIA for determining concentration of human serum Cys C.These have laid a foundation of development of immunological method for detection of human serum Cys C.