重组人IL-1β分子的克隆、表达及其单克隆抗体的制备

The Molecular Cloning,Expression of Recombinant Human IL-1β and the Preparation of Its Monoclonal Antibody

目的克隆人IL-1β基因,纯化其原核表达的产物,并制备其单克隆抗体(mAb)。方法重组原核表达质粒pET-32a(+)-IL-1β在宿主菌BL21(DE3)表达后,用割胶回收、电洗脱的方法纯化原核表达的蛋白,并用其免疫BALB/c小鼠,采用常规杂交瘤技术制备相应的mAb。用ELISA法检测mAb的效价,并进行Ig亚类、特异度的检测。结果本研究利用HL-60细胞系cDNA为模板经PCR克隆了人IL-1β基因,纯化的重组人IL-1β蛋白纯度和含量分别为95%和0.75μg/μl。共筛选出能稳定分泌抗人IL-1β的杂交瘤细胞2株,分别为5G5和8H8。抗体亚型均为IgG2a,轻链均为κ链。结论克隆了人IL-1β基因,并成功纯化了其原核表达的蛋白,以纯化的蛋白为免疫原制备的鼠抗人IL-1β的单克隆抗体(mAb),为实验室及临床研究奠定了一定的基础。

Objective To clone recombinant human IL-1B gene and express with a prokaryotic expression system and to prepare the monoclonal antibody against it.Methods The cDNA of HL-60 cell line was used as a template,the human IL-1β gene was detected by PCR.IL-1β expressed by a prokaryotic expression system was purified.Mice were immunized with purified fusion protein IL-1β for three times.The specificity and sensitivity of anti-human IL-1β monoclonal antibody were characterized by Western blot and indirect ELISA.Results Fusion protein IL-1β was highly expressed in E.coli with a molecular weight of about 51kd,its purity was 95%.Western blot and FACS results showed that the monoclonal antibodies could specifically recognize the target protein expressed in the E.coli expression system and HL-60 cell line.Conclusions The IL-1βsene is successfully cloned,the fusion protein of which and the mAb against recombinant human IL-1β are prepared,which provids a useful tool for laboratory and clinical research.