Preparation of polyclonal antibody against extracellular domain protein of CD47
刘海静 1苏雨萌 1王芊艺1
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作者信息
1. 北京亦庄国际生物医药科技有限公司，北京 100176
折叠
摘要
目的 制备和鉴定抗CD47胞外区蛋白多克隆抗体。 方法 通过逆转录PCR(reverse transcription-PCR，RT-PCR)扩增CD47分子胞外区基因序列，分别重组到原核表达载体pET32a(＋)和pET31b(＋)中，利用大肠杆菌表达CD47蛋白，并优化异丙基硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside，IPTG)诱导蛋白表达工艺，表达出CD47蛋白。用纯化的CD47蛋白免疫Balb/c小鼠，获得小鼠多克隆抗体，使用亲和层析法纯化CD47多抗，并对多抗进行酶联免疫吸附试验(enzyme-linked immunosorbent assay，ELISA )效价检测和Western blot特异性鉴定。 结果 成功构建pET32a(＋)-CD47和pET31b(＋)-CD47重组质粒。按照实验设置不同条件诱导表达蛋白后，选出最佳表达载体pET32a(＋)-CD47。在大肠杆菌中获得表达最佳蛋白表达工艺。表达制备CD47蛋白，经过五次免疫后，得到CD47多抗。用ELISA法检测制备多抗的效价能达到1∶128 000。Western blot检测结果显示，自制的CD47多抗能准确检测出小鼠心脏组织样品。 结论 成功制备了CD47胞外区蛋白多抗，为进一步研究CD47生物学功能奠定了基础。 Objective To prepare and identify antibody of CD47 extracellular domain protein. Methods The extracellular domain gene fragment of CD47 was amplified by reverse transcription PCR (RT-PCR) and recombined into prokaryotic expression vector pET32a(+ ) and pET31b(+ ) respectively. The CD47 protein was expressed in Escherichia coli(E.coli) and the isopropyl-β-D-thiogalactopyranoside (IPTG) induced protein expression process was optimized to express the CD47 protein. Balb/c mice were immunized with purified CD47 protein to obtain mouse polyclonal antibodies. Anti-CD47 polyclonal antibodies were purified by affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used for potency detection and specificity identification respectively. Results Recombinant plasmids pET32a (+ )-CD47 and pET31b(+ )-CD47 were successfully constructed. After inducing the expression of proteins in different experimental conditions, the optimal expression vector PET32a(+ )-CD47 was selected. The optimal expression technology of PET32a(+ )-CD47 was obtained in E. coli. Express and prepare CD47 protein, after five immunizations, obtain CD47 polyclonal antibody. The titer of CD47-polyclonal antibody can reach 1∶128 000 using ELISA. Western blot results showed that the self-made CD47 can identify mouse heart tissue samples accurately. Conclusion The polyclonal antibody of CD47 extracellular domain protein was successfully prepared, laying the foundation for further study on the biological function of CD47.
Abstract
Objective To prepare and identify antibody of CD47 extracellular domain protein. Methods The extracellular domain gene fragment of CD47 was amplified by reverse transcription PCR (RT-PCR) and recombined into prokaryotic expression vector pET32a(+ ) and pET31b(+ ) respectively. The CD47 protein was expressed in Escherichia coli(E.coli) and the isopropyl-β-D-thiogalactopyranoside (IPTG) induced protein expression process was optimized to express the CD47 protein. Balb/c mice were immunized with purified CD47 protein to obtain mouse polyclonal antibodies. Anti-CD47 polyclonal antibodies were purified by affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used for potency detection and specificity identification respectively. Results Recombinant plasmids pET32a (+ )-CD47 and pET31b(+ )-CD47 were successfully constructed. After inducing the expression of proteins in different experimental conditions, the optimal expression vector PET32a(+ )-CD47 was selected. The optimal expression technology of PET32a(+ )-CD47 was obtained in E. coli. Express and prepare CD47 protein, after five immunizations, obtain CD47 polyclonal antibody. The titer of CD47-polyclonal antibody can reach 1∶128 000 using ELISA. Western blot results showed that the self-made CD47 can identify mouse heart tissue samples accurately. Conclusion The polyclonal antibody of CD47 extracellular domain protein was successfully prepared, laying the foundation for further study on the biological function of CD47.
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