大鼠β-防御素-2的重组表达及抗菌活性检测

发布时间：2018-05-28 00:27

  本文选题：大鼠β-防御素- + 基因转染　； 参考：《中国现代医学杂志》2010年13期

【摘要】：目的构建大鼠β-防御素-2(rBD2)基因的真核表达载体pCAGG-rBD2,并转染小鼠纤维母细胞(L929),为解决细菌耐药性问题提供进一步的实验基础。方法以大鼠肺组织总RNA为模板,采用RT-PCR的方法获得β-防御素-2基因cDNA,将其克隆到pGEM-T Easy载体并构建含有目的片段的真核表达载体pCAGG-rBD2,经脂质体介导转染L929细胞,通过RT-PCR和Western blotting检测目的基因表达情况。结果成功克隆并构建了含有目的基因rBD2的真核表达载体pCAGG-rBD2,转染pCAGG-rBD2的细胞中检测到了rBD2基因在转录水平和蛋白水平的表达,其细胞培养的上清液对金黄色葡萄球菌具有杀灭作用。结论防御素在真核表达系统的成功表达,为进一步开发高效能杀灭耐药性致病微生物的多肽类新药提供实验基础和理论依据。
[Abstract]:Objective to construct the eukaryotic expression vector pCAGG-rBD2 of rat 尾 -defensin -2rBD2 gene and transfect it into mouse fibroblast cell line L929 to provide a further experimental basis for solving the problem of bacterial resistance. Methods using total RNA of rat lung tissue as template, 尾 -defensin-2 gene cDNAs were obtained by RT-PCR, cloned into pGEM-T Easy vector, constructed eukaryotic expression vector pCAGG-rBD2containing the target fragment, and were transfected into L929 cells by liposome-mediated transfection. The expression of target gene was detected by RT-PCR and Western blotting. Results the eukaryotic expression vector pCAGG-rBD2 containing the target gene rBD2 was cloned and constructed successfully. The expression of rBD2 gene at transcription and protein levels was detected in the transfected pCAGG-rBD2 cells. The supernatant of the cell culture could kill Staphylococcus aureus. Conclusion the successful expression of defensin in eukaryotic expression system provides experimental and theoretical basis for the further development of new polypeptide drugs with high efficacy in killing drug-resistant pathogenic microorganisms.
【作者单位】： 甘肃省新药临床前研究重点实验室(兰州大学基础医学院病理生理学研究所);
【基金】：甘肃省新药临床前研究重点实验室开放基金(No:GSKFKT-0703)
【分类号】：Q786
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本文编号：1944460