摘要
目的 克隆大鼠白细胞蛋白酶抑制因子（SLPI）基因,构建其真核表达载体,并转染大鼠真皮多能干细胞,以探讨SLPI在创伤及炎症性疾病中的意义.方法 PCR扩增大鼠皮肤SLPI基因 cDNA,酶切后和携带绿色荧光蛋白报告基因的真核表达载体pEGFP-N2重组,将SLPI基因 cDNA片段连接到pEGFP-N2载体的多克隆位点,形成重组载体pEGFP/SLPI,转化大肠杆菌DH5α菌株,构建成SLPI基因真核表达载体质粒.扩增DH5α后抽提质粒DNA,Hind Ⅲ和BamHⅠ酶切,电泳,DNA测序鉴定.鉴定正确的质粒DNA用脂质体包裹后转染大鼠真皮多能干细胞,通过荧光显微镜和RT-PCR方法 来检测基因转染及表达.结果从大鼠皮肤cDNA扩增出392 bp的DNA片段并成功重组到pEGFP-N2载体中.经酶切和DNA测序验证,插入载体的DNA片段为SLPI基因,插入方向正确.重组质粒经脂质体转染真皮多能干细胞,24 h后观察到绿色荧光蛋白报告基因和目的 基因SLPI表达.结论 大鼠SLPI基因的克隆和真核表达载体构建获得成功,为进一步研究其功能奠定了基础.
Objective To clone rat secretory leukocyte protease inhibitor （SLPI） gene and construct its eukaryotic expression vector for expression of SLPI in rat dermal muhipotent stem cells （dMSCs）. Methods Rat SLPI eDNA fragment was amplified with polymerase chain reaction （PCR） from rat skin cDNA. The purified fragment was reeombinated with an eukaryotic expression vector with enhanced green fluorescent protein, pEGFP - N2. The SLPI eDNA fragment was inserted into the vector and a new plasmid, pEGFP/SLPI was constructed. After expanding in DHSa, the new recombinant plasmid DNA was extracted, and then identified by cutting with Hind Ⅲ, BamH I nuclease and DNA sequencing. Identified plasmid DNA was transfected into rat dMSCs by liposome, and the SLPI gene expression in transfected dMSCs was detected by PCR. Results A 392 bp cDNA fragment was amplified from rat skin cDNA, and was correctly inserted into the vector pEGFP - N2. The fragment was confirmed to be SLPI gene by nuclease digestion and DNA sequencing. Rat dSMCs were transfected with the new recombinant plasmid DNA and the SLPI gene expression in transfected dMCSs was observed within 24 h. Conclusion Rat SLPI gene was cloned and its recombinant eukaryotic expression vector with green fluorescent protein was successfully constructed, which provides a useful tool for further research on the role of SLPI in wound healing.
出处
《中国急救医学》
CAS
CSCD
北大核心
2010年第7期614-617,F0003,共5页
Chinese Journal of Critical Care Medicine
基金
基金项目：国家自然科学基金项目（No.30370562）
国家重点基础研究发展规划（973）项目（No.G1999054205）
