| 王会平,郜赵伟,刘 冲,刘 丽,和 婷,董 轲.CD14蛋白表达、纯化及单克隆抗体制备[J].现代生物医学进展英文版,2020,(19):3616-3620. |
| CD14蛋白表达、纯化及单克隆抗体制备 |
| Expression and Purification of CD14 Protein and Preparation of Anti-CD14 Monoclonal Antibody |
| Received:March 27, 2020 Revised:April 23, 2020 |
| DOI：10.13241/j.cnki.pmb.2020.19.003 |
| 中文关键词: CD14 克隆 表达 纯化 |
| 英文关键词: CD14 Clone Expression Purification |
| 基金项目:国家自然科学基金面上项目（81572974） |
| Author Name | Affiliation | E-mail | | WANG Hui-ping | Department of Clinical Laboratories, The Second Affiliated Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China | huiping0419@126.com | | GAO Zhao-wei | Department of Clinical Laboratories, The Second Affiliated Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China | | | LIU Chong | Department of Clinical Laboratories, The Second Affiliated Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China | | | LIU Li | Department of Clinical Laboratories, The Second Affiliated Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China | | | HE Ting | Department of Clinical Laboratories, The Second Affiliated Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China | | | DONG Ke | Department of Clinical Laboratories, The Second Affiliated Hospital, Air Force Medical University, Xi'an, Shaanxi, 710038, China | |
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| 中文摘要: |
| 摘要 目的：制备CD14重组蛋白及抗CD14单克隆抗体。方法：从人外周血淋巴细胞中克隆CD14编码基因，将其连接至质粒pRSETC，构建表达质粒pRSETC/CD14，转染大肠杆菌表达菌株BL21(DE3)，筛选阳性克隆、用IPTG诱导表达，SDS-PAGE检测CD14蛋白表达水平，应用镍柱进行亲和纯化，SDS-PAGE及Western blot进行纯化产物鉴定。纯化后的CD14蛋白免疫BALB/c小鼠，利用杂交瘤技术筛选分泌单克隆抗体细胞株，制备单克隆抗体，利用Western blot鉴定抗体活性。结果：获得CD14编码基因，并成功进行了原核表达，SDS-PAGE显示CD14以包涵体形式表达，纯化产物的纯度超过95%。利用杂交瘤技术筛选出稳定分泌抗CD14抗体的细胞株，并制备了高纯度的单克隆抗体，抗体具有CD14蛋白结合活性。结论：成功表达纯化了CD14蛋白，并制备了抗CD14单克隆抗体，为后续开发CD14检测技术提供抗体。 |
| 英文摘要: |
| ABSTRACT Objective: To express CD14 protein and prepare anti-CD14 monoclonal antibody. Methods: The CD14 encoding sequence was cloned from human peripheral blood lymphocytes, which was connected to the pRSETC vector, the expression plasmid pRSETC/CD14 was transfected into BL21(DE3). Expression of recombinant protein was induced by using IPTG. SDS-PAGE and western-blot were used to detected the purified recombinant protein. Hybridoma cells stably secreting anti-CD14 monoclonal antibodies were prepared by using hybridoma technology, the activity of purified antibody was identified by Western blot. Results: The coding sequence of CD14 was obtained. SDS-PAGE showed that recombinant protein expressed in the forms of inclusion body. And moreover, SDS-PAGE showed the purification rate of the purified product was higher than 95%. The positive hybridoma clone which secreted the mAb against CD14 protein were obtained, high purity monoclonal antibody which had the binding activity to CD14 protein was prepared. Conclusion: CD14 protein and anti-CD14 monoclonal antibody were successfully prepared, which provided the experimental basis for the development of CD14 detection technology. |
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