| 摘要: |
| 【目的】克隆猪胸腺活化调节趋化因子CCL17基因， 研究其在原核及真核系统中的表达。【方法】提取仔猪脾脏组织总RNA,利用RT-PCR 技术扩增CCL17，并将其与已知序列的CCL17进行同源性比较；CCL17基因经测序验证后,将其分别克隆入表达载体pET-32a和pEGFP-C1，构建该基因的重组原核表达载体pET-CCL17和真核表达载体pEGFP-CCL17，分别进行双酶切和测序鉴定；前者在大肠杆菌BL21(DE3)中诱导表达目的蛋白，后者以脂质体介导法转染至猪血管内皮细胞（SUVECs），通过绿色荧光标记和Western blot检测细胞中CCL17基因的表达水平。【结果】PCR扩增得到CCL17 309 bp的DNA片段；重组表达质粒pET-CCL17和pEGFP-CCL17经双酶切鉴定和测序分析证明，载体构建成功。pET-CCL17经IPTG诱导表达后,SDS-PAGE 分析表明,目的蛋白在宿主大肠杆菌BL21(DE3)中高效表达CCL17融合蛋白，分子质量约32 ku,与其理论分子质量基本相符；pEGFP-CCL17转染SUVECs，经Western blot检测，证实导入的CCL17基因得到了表达。【结论】克隆了猪的CCL17基因,利用大肠杆菌成功表达了CCL17重组蛋白，真核重组质粒在SUVECs中也得到了表达。 |
| 关键词: 猪 胸腺活化调节趋化因子 分子克隆 原核表达 真核表达 |
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| 基金项目:国家自然科学基金项目(30270342)；西北农林科技大学引进人才基金资助项目(01140406) |
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| Cloning and expression of prokaryotic and eukaryotic of swine gene CCL17 |
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| Abstract: |
| 【Objective】The study was done to clone swine thymus activation regulating chemokine CCL17 and express it in prokaryotic and eukaryotic.【Method】The total RNA was extracted from Spleen of Piglet，the gene of CCL17 was amplified by RT-PCR,the homology was analyzed by expression vector pET-CCL17 and eukaryotic expression vector pEGFP-CCL17，which underwent restriction enzyme digestion and sequencing respectively.The recombinant protein was expressed in BL21 (DE3)，eukaryotica expression vector transfected Porcine vascular endothelial cells(SUVECs).【Result】A DNA fragment at length of 309 bp was amplified by PCR.Both restriction analysis and sequencing proved that recombinant plasmid pET-CCL17 and pEGFP-CCL17 were constructed correctly.pET-CCL17 by the challenge of IPTG,SDS-PAGE analysis showed that the target protein was overexpressed in E.coli BL21 (DE3),the fusion protein was about 32 ku,which was consistent with the prediction of CCL17recombinant protein.pEGFP-CCL17 transfected SUVECs,Western blot detection showed that CCL17gene was expressed in SUVECs.【Conclusion】We cloned swine CCL17 and expressed it by E.coli.The eukaryotic recombinant plasmid was expressed in SUVECs. |
| Key words: swine thymusand activation regulated chemokine molecular cloning prokaryotic expression eukaryotic expression |
