大鼠β-防御素-2的重组表达及抗菌活性检测  

Recombinant expression and antimicrobial activity of rat β-defensin-2

作　　者：李爱娟[1] 张妮[1] 陈新年[1] 

机构地区：[1]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),甘肃兰州730000

出　　处：《中国现代医学杂志》2010年第13期1982-1985,共4页China Journal of Modern Medicine

基　　金：甘肃省新药临床前研究重点实验室开放基金(No:GSKFKT-0703)

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。[ Objective ] To construct a eukaryotic expression vector of recombinant rat β-defensin-2 （rBD2）, and transfect mouse fibroblast （L929）, and detect the expression and antibacterial activity of protein of interest. [Method] The total RNA was extracted from rat lung tissue as template, and reverse transcription polymerase chain reaction （RT-PCR） was performed to amplify the full length of coding region of rBD2 gene. An eukaryotic expression plasmid pCAGG- rBD2 was constructed, and the plasmid was introduced into L929 by Lipofection. The expression of rBD2 in L929 cells was verified by RT-PCR and westem blotting. [ Results ] The eukaryotic expression vector pCAGG-rBD2 was successfully constructed, rBD2 gene was effectively expressed in L929 cell, importantly, the cells could secrete biologically active rBD2. [ Conclusion ] The recombinant plasmid pCAGG-rBD2 was expressed successfully in eukaryotic cells, which would provide an experimental and theoretical basis for addressing the problem of bacterial resistance.

关 键 词：大鼠β-防御素-2 基因转染 真核表达 生物活性 

分 类 号：Q786[生物学—分子生物学] R-332[医药卫生]