| 摘要: |
| 目的 建立小鼠PD-1/PD-L1（mPD-1/mPD-L1）体外分子结合模型，为研究PD-1/PD-L1生物学活性及建立高通量药物筛选分子模型奠定基础。方法 重组质粒在原核细胞表达mPD-1和mPD-L1蛋白，利用亲和层析及切胶方法纯化相应蛋白；应用ELISA和GST Pull-down方法验证mPD-1与其配体mPD-L1蛋白的结合活性；以Alamar blue法检测纯化mPD-1和mPD-L1蛋白混合物对混合淋巴细胞增殖的影响。结果 SDS-PAGE和Western印迹结果显示原核表达的重组蛋白与预期基本一致，经过亲和层析和切胶、复性等方式获得了纯化的mPD-1和mPD-L1蛋白。ELISA和GST pull-down结果表明，mPD-1和mPD-L1蛋白具有体外结合的活性。淋巴细胞增殖试验进一步说明两蛋白的结合可一定程度上降低单独蛋白对淋巴细胞增殖的影响（P<0.05）。结论 利用原核细胞高效表达并纯化的mPD-1、mPD-L1蛋白成功建立体外mPD-1/mPD-L1分子结合模型，为高通量药物初筛奠定了基础。 |
| 关键词: mPD-1 mPD-L1 分子模型 淋巴细胞增殖 |
| DOI：10.3724/SP.J.1008.2010.0385 |
| 投稿时间：2009-10-12修订日期：2010-03-09 |
| 基金项目:湖北省自然科学基金(2008CDB118),三峡大学研究生科研创新基金(2009-36). |
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| Expression of mouse PD-1/PD-L1 recombinant protein in prokaryotic cells |
| QIN Xiao-lin1, LIU Chao-qi1*,YANG Fan2, ZHENG Lan-ying3 |
| (1. Institute of Molecular Biology, Three Gorges University, Yichang 443002, Hubei, China;2. Chemoradiation Department, the Second People’s Hospital of Yichang, Yichang 443002, Hubei, China;3. Department of Nursing, the People’s Hospital of Changyang Tujia Autonomous County, Changyang 443500, Hubei, China) |
| Abstract: |
| Objective To establish a molecule binding model of mouse PD-1/PD-L1 protein in vitro, so as to lay a foundation for studying the biological activities of the recombinant protein and to establish a high throughput drug screening model. Methods Prokaryotic expression plasmid pET28 a (+) /mPDL-1 and pGEX-4T-1/mPD-1 were transformed to E.coli BL21 (DE3), which was then induced with IPTG. The expression products were purified by fast affinity chromatography with FPLC Protein Purification Instrument or gel separation. ELISA and GST pull-down were used to detect the interaction between mPD-1 and mPD-L1. In addition, Alamar blue was used to test the role of protein mixtures in the mixed lymphocyte proliferation. Results SDS-PAGE and Western blotting analysis showed that the recombinant proteins were successfully expressed. Then purified mPD-1 and mPD-L1 protein was obtained by affinity chromatography, Gel seperation and refolding, etc. ELISA and GST pull-down showed that mPD-1 and mPD-L1 protein had a specific binding activity in vitro. The mPD-1/mPD-L1 protein had a significantly decreased influence on the proliferation of lymphocytes（P<0.05）. Conclusion Mouse PD-1/PD-L1 recombinant protein model has been successfully established using purified mPD-1 and mPD-L1 protein expressed prokaryotically, which lays a foundation for high-throughput drug screening. |
| Key words: mPD-1 mPD-L1 molecular models lymphocyte proliferation |
