论文部分内容阅读
目的:克隆细胞因子midkine(MK)基因,表达其融合蛋白,制备抗MK单克隆抗体(mAb)并检测MK在肿瘤细胞中的表达。方法:利用MK基因的特异性引物,通过RT-PCR从人肾癌组织mRNA中扩增人MKcDNA分子。将其定向克隆于原核表达载体中,在大肠杆菌中表达MS2-MK融合蛋白。以纯化的融合蛋白免疫BALB/c小鼠,用传统的杂交瘤技术,进行细胞融合、筛选、克隆化并制备mAb腹水。用ELISA法分析其Ig亚类,用免疫细胞化学染色法检测MK在肿瘤细胞中的表达。结果:成功地克隆出MK基因并表达了MS2-MK融合蛋白。通过免疫和筛选,获得2株分泌小鼠抗人MKmAb的杂交瘤细胞,其分泌的mAb的Ig亚类分别为IgG1和IgG2a。免疫细胞化学染色显示,2株mAb与人胃癌细胞株MGC803和胃癌组织呈强阳性反应。结论:在原核细胞中获得MS2-MK融合蛋白的表达,并制备出抗MK的mAb,为研究MK的生物学活性提供了条件。
OBJECTIVE: To clone the gene of midkine (MK), express its fusion protein, prepare anti-MK monoclonal antibody (mAb) and detect the expression of MK in tumor cells. METHODS: Human MKcDNA molecules were amplified from human renal cancer tissue mRNA by RT-PCR using specific primers for the MK gene. It was cloned into prokaryotic expression vector and expressed in Escherichia coli MS2-MK fusion protein. BALB / c mice were immunized with the purified fusion protein, and cell fusion, screening, cloning, and preparation of mAb ascites were performed using conventional hybridoma techniques. The Ig subclasses were analyzed by ELISA and the expression of MK in tumor cells was detected by immunocytochemical staining. Results: The MK gene was successfully cloned and the MS2-MK fusion protein was expressed. Two hybridoma cells secreting mouse anti-human MKmAb were obtained by immunization and screening, and the Ig subclasses of the secreted mAbs were IgG1 and IgG2a, respectively. Immunocytochemical staining showed that the two mAbs were strongly positive to human gastric cancer cell line MGC803 and gastric cancer. Conclusion: The expression of MS2-MK fusion protein in prokaryotic cells and the preparation of anti-MK mAb provided the conditions for the study of the biological activity of MK.