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人CD7胞外区慢病毒载体的构建及其稳转细胞系的构建、表达和纯化
发表时间：2016-02-15 浏览量：3251 下载量：1227
| 全部作者： | 李甲璐，汤金乐，于远，杨林 |
| 作者单位： | 苏州大学唐仲英血液学研究中心 & 血液学协同创新中心 |
| 摘 要： | 为筛选能够识别具有天然构象的CD7单克隆抗体或者纳米抗体，有必要建立CD7分子的分泌型表达哺乳动物稳定细胞株来纯化糖基化CD7抗原蛋白，以此作为抗体筛选用抗原。研究通过提取Jurkat细胞总RNA，经RT-PCR扩增获得目的片段CD7-EX，并直接构建到慢病毒载体上，从而获得了带有His标签的慢病毒重组表达载体pCDH-CMV-copGFP-CD7-EX. 通过包装慢病毒，转染293T细胞系，命名为293T-GFP-CD7-EX. 经Western blotting鉴定，该His-CD7-EX融合蛋白可与His标签抗体特异性结合，表明融合蛋白可在构建的细胞系中表达。然后用无血清培养基培养293T-GFP-CD7-EX细胞，收集培养上清，对上清进行亲和层析纯化，纯化产物经考马斯亮蓝染色鉴定后得到证实。结果显示，成功构建了能够持续分泌表达CD7胞外区片段的稳转细胞系，并经过纯化获得了CD7胞外区重组蛋白。 |
| 关 键 词： | 蛋白质工程；CD7胞外区（CD7-EX）；稳转细胞系；真核表达；蛋白纯化 |
| Title： | Lentiviral vector construction, expression & purification from stably transfected cell line of human CD7 extracellular domain |
| Author： | LI Jialu, TANG Jinle, YU Yuan, YANG Lin |
| Organization： | Cyrus Tang Hematology Center & Collaborative Innovation Center of Hematology, Soochow University |
| Abstract： | In order to screen monoclonal antibody or nanobody targeting CD7 protein with natural conformation, it is necessary to obtain glycosylated CD7 antigen by establishing secretory stable mammalian cell line, which will accelerate the purification of CD7 antigen as well. Accordingly, total RNA was isolated from CD7 positive Jurkat cells, and human CD7 extracellular domain (CD7-EX) fragment was obtained by RT-PCR. Then, recombinant expression lentiviral vector with His taqs was constructed as pCDH-CMV-copGFP-CD7-EX, and 293T cells were transduced with the recombinant lentivirus which was named as 293T-GFP-CD7-EX. The supernatant from serum free culture medium of transduced 293T cells was performed affinity purification, by which the recombinant CD7-EX protein was successfully prepared by affinity chromatography and examined by Coomassie brilliamt blue staining. Furthermore, the His-CD7-EX fusion protein was proved by Western blotting assay with anti-His. Taken together, a secretory CD7-EX expressing stable cell line of 293T-GFP-CD7-EX was established which lays a foundation to prepared recombinant glycosylated CD7 antigen for antibody screening in the future. |
| Key words： | protein engineering; CD7 extracellular domain (CD7-EX); stable cell line; eukaryotic expression; protein purification |
| 发表期数： | 2016年2月第3期 |
| 引用格式： | 李甲璐，汤金乐，于远，等. 人CD7胞外区慢病毒载体的构建及其稳转细胞系的构建、表达和纯化[J]. 中国科技论文在线精品论文，2016，9（3）：228-234. |
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