人降钙素原的原核表达及其单克隆抗体的制备

目的: 原核表达人降钙素原（procalcitonin，PCT）重组蛋白，制备高纯度的PCT单克隆抗体。方法: 将构建的PCT-pET22b表达载体转入大肠埃希菌BL21中诱导表达PCT蛋白，经Ni-NTA亲和层析法纯化，用蛋白质印迹法和胶体金法对其进行鉴定。获得的PCT重组蛋白采用长程免疫法皮下多点注射Balb/c小鼠，取小鼠脾细胞和骨髓瘤细胞（SP2/0）融合，筛选阳性杂交瘤细胞株并进行亚克隆4次。将细胞株注入小鼠腹腔收集腹腔积液获得单克隆抗体，经辛酸硫酸铵法和Protein G亲和层析法进行纯化。结果： SDS-PAGE电泳结果显示在相对分子质量约14 kDa处有一明显条带，与目的蛋白大小相符。蛋白质印迹法和胶体金法证明该可溶性蛋白为PCT。采用细胞融合技术成功筛选出7株阳性杂交瘤细胞，将其命名为B3，C4，C7，E9，F8，F10，G2。选B3和C4细胞株通过体内诱生法制备单克隆抗体，经纯化后纯度可达95%以上。间接ELISA法检测B3、C4抗体效价均可达10⁷，亲和常数分别为5.06×10⁸，7.3×10⁸。结论： 获得高纯度的PCT重组蛋白及其单克隆抗体，为PCT检测试剂盒的进一步研究提供了支持。

Prokaryotic expression of human procalcitonin andpreparation of its monoclonal antibody

Objective: To express human procalcitonin(PCT) recombinant protein in prokaryocytes and prepare high purity PCT monoclonal antibody. Methods: The constructed PCT-pET22b expression vector was transfected into Escherichia coli BL21 to induce expression of PCT protein, which was purified by Ni-NTA affinity chromatography and identified by Western blotting and colloidal gold method. The obtained PCT recombinant protein was subcutaneously injected into Balb/c mice by long-term immunization, and then spleen cells and myeloma cells(SP2/0) were fused, the positive hybridoma cell lines were screened and subcloned 4 times. The cell strain was injected into the peritoneal cavity of the mouse to collect the peritoneal effusion to obtain monoclonal antibody, which was purified by ammonium octoate ammonium sulfate and Protein G affinity chromatography. Results: SDS-PAGE analysis showed a significant band at a relative molecular weight of approximately 14 kDa, which was consistent with the size of the target protein. Western blotting and colloidal gold methods demonstrated that the soluble protein was PCT. Seven positive hybridoma cells were successfully screened by cell fusion technique and named as B3, C4, C7, E9, F8, F10, G2. B3 and C4 cell strains were selected to prepare monoclonal antibodies by in vivo induction. After purification, the purity of antibody was over 95%. Indirect ELISA showed that the titers of antibodies B3 and C4 were up to 10⁷, the affinity constants were 5.06×10⁸ and 7.3×10⁸, respectively. Conclusion: High purity PCT recombinant protein and its monoclonal antibody were obtained, which provided support for the further preparation of PCT reagent kit. ［Key words］procalcitonin； cell fusion； monoclonal antibody