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目的:原核表达GRP78并制备抗GRP78的特异性单克隆抗体(mAb),并初步鉴定相应mAb的特性。方法:从肺癌患者组织中抽提总RNA,RT-PCR获得grp78全长基因,并通过原核重组表达GRP78蛋白;以纯化的GRP78蛋白免疫BALB/c小鼠,成功免疫的小鼠脾细胞与骨髓瘤Sp2/0细胞融合,筛选得到分泌特异性抗GRP78的mAb的杂交瘤细胞株;并通过Western blot及免疫组化初步鉴定其特异性。结果:成功构建重组表达质粒PET-28a-grp78并由大肠杆菌表达获得GRP78蛋白,被该蛋白免疫的BALB/c小鼠与Sp2/0细胞融合、筛选,获得3株稳定分泌抗GRP78抗体的杂交瘤细胞株,分别命名为2A1、4B8和4A12。采用其中1株4A12 mAb对3个肺癌患者组织样本进行Western blot检测以及免疫组化分析,结果均显示4A12 mAb能够特异识别肺癌组织表达的GRP78。结论:成功制备了抗GRP78的mAb,并能够特异识别肺癌组织表达的GRP78,为进一步研究GRP78在肿瘤治疗中的作用提供基础。
OBJECTIVE: To prokaryotic express GRP78 and to prepare specific anti-GRP78 monoclonal antibody (mAb), and to identify the characteristics of the corresponding mAb. Methods: Total RNA was extracted from lung cancer tissues and the full-length grp78 gene was obtained by RT-PCR. GRP78 protein was expressed by prokaryotic recombination. BALB / c mice were immunized with purified GRP78 protein. The spleen cells and bone marrow Hybridoma cell lines secreting mAbs specific to GRP78 were screened by fusion of Sp2 / 0 cells. The specificity of the hybridoma cells was identified by Western blot and immunohistochemistry. Results: The recombinant plasmid pET-28a-grp78 was successfully constructed and GRP78 protein was expressed in E. coli. The BALB / c mice immunized with the protein were fused with Sp2 / 0 cells and screened to obtain three hybrids stably secreting anti-GRP78 antibody The tumor cell lines, named 2A1, 4B8 and 4A12, respectively. Western blot and immunohistochemistry analysis of the tissue samples from one lung cancer patient with 4A12 mAb showed that 4A12 mAb could specifically recognize GRP78 expressed in lung cancer. Conclusion: The anti-GRP78 mAb was successfully prepared and can specifically recognize GRP78 expressed in lung cancer tissues, which provided a basis for further study on the role of GRP78 in tumor therapy.