| 引用本文： |
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张 冲,黄子珂,李思光.重组缓释白介素4的克隆表达与鉴定[J].同济大学学报（医学版）,2018,39(5):28-33. [点击复制]
- ZHANG Chong,HUANG Zi-ke,LI Si-guang.Construction and identification of recombinant slow-release Interleukin 4 gene[J].同济大学学报（医学版）,2018,39(5):28-33. [点击复制]
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| 摘要: |
| 目的 重组表达可以缓释的抗炎因子白介素4（Interleukin 4，IL-4）。方法 根据小鼠IL-4的氨基酸序列，并基于大肠杆菌密码子偏爱性合成IL-4的基因，通过聚合酶链式反应(PCR)方法将胶原蛋白结合域(collagen binding domain, CBD)的编码序列和组氨酸标签序列连接到IL-4基因上，运用无缝克隆技术将上述重组基因插入pET-30a(+)载体。重组蛋白通过组氨酸标签进行纯化，纯化的蛋白被用于诱导M0巨噬细胞分化成M2巨噬细胞，同时检测M2巨噬细胞相关基因表达以及对白介素10（Interleukin 10, IL10）的分泌。与Ⅰ型胶原蛋白的联用验证胶原蛋白对重组因子的吸附和缓释功能。结果 带有CBD序列和组氨酸纯化标签的IL-4通过镍柱纯化，具有与商业化IL-4相似的生物活性。功能实验证实其能够被胶原蛋白吸附，并能脱离胶原蛋白缓释。结论 通过大肠杆菌表达纯化获得了能够缓释的抗炎因子IL-4. |
| 关键词: 缓释 胶原蛋白结合域 白介素4 Ⅰ型胶原蛋白 |
| DOI：10.16118/j.1008-0392.2018.05.006 |
| 投稿时间：2018-02-22 |
| 基金项目:国家自然科学基金面上项目（31571405） |
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| Construction and identification of recombinant slow-release Interleukin 4 gene |
| ZHANG Chong,HUANG Zi-ke,LI Si-guang |
| (Dept. of Regenerative Medicine, Tongji University School of Medicine, Shanghai 200092, China) |
| Abstract: |
| Objective To construct and identify the slow-release anti-inflammatory factor interleukin4(IL-4) gene. Methods The mouse IL-4 gene was synthesized according to its amino acid sequence(ACCESSION: NP_067258) and the E.coli codon usage preference. Collagen binding domain(CBD) and His Tag sequence were linked with IL-4 sequence by PCR amplification with primers containing CBD and His Tag sequence. PCR products were reassembled into the cloning vector pET-30a(+) by seamless cloning technique. After NI-sepharose purification, the bioactivity of fusion protein was tested by polarizing M0 macrophage to M2 macrophage through measuring the gene expression and interleukin10(IL10) secretion. In combination with collagen I, the adsorption and release of recombinant proteins by collagen I was tested. Results IL-4-CBD-His Tag was purified successfully by Nickel column and it had the similar bioactivity with commercial IL-4. The function assay indicated that IL-4-CBD-His Tag was able to bind to collagen I and to be slowly released from collagen. Conclusion The slow-release anti-inflammatory factor IL-4 gene has been successfully constructed through E.coli expression system. |
| Key words: slow-release collagen binding domain interleukin 4 collagen I |
