论文部分内容阅读
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 human IL-1β gene and purify its prokaryotic expression product and prepare its monoclonal antibody (mAb). Methods The recombinant prokaryotic expression plasmid pET-32a (+) - IL-1β was expressed in E. coli BL21 (DE3) and purified by tapping and electroelution. The expressed protein was purified and used to immunize BALB / c mice The corresponding mAbs are prepared by conventional hybridoma techniques. The titer of the mAb was measured by ELISA and the Ig subclass was tested for its specificity. Results The human IL-1β gene was cloned by PCR using HL-60 cell line cDNA as a template. The purity and content of the purified recombinant human IL-1β protein were 95% and 0.75 μg / μl, respectively. A total of 2 hybridomas secreting anti-human IL-1β were screened for 5G5 and 8H8, respectively. Antibody subtypes were IgG2a, light chain are kappa chain. Conclusions The human IL-1β gene was cloned and the prokaryotic expression protein was successfully purified. The purified mAb of human anti-human IL-1β was purified with the purified protein, which lays the foundation for laboratory and clinical research A certain basis.