摘要
目的研究O-胆甾醇基壳聚糖(O-CHCS)作为基因载体进行人β防御素-2(HBD2)基因转染小鼠纤维母细胞L929的可行性及检测目的基因表达产物的生物活性。方法构建重组质粒pCMV-hBD2,通过O-CHCS介导转染于L929细胞,提取转染细胞总RNA,用RT-PCR检测HBD2基因转录水平;收集转染细胞培养上清液,用Westernblotting检测HBD2基因蛋白的表达;用Kirby-Bauer纸片法检测HBD2抗菌活性,同时以Lipofect脂质体转染试剂作为阳性对照。结果经过O-CHCS介导转染的L929细胞,目的基因HBD2在转录水平和蛋白水平均有表达,转染细胞上清液对金黄色葡萄球菌有抑菌圈形成,结果同Lipofect的转染效果基本一致。结论成功构建了真核表达载体pCMV-hBD2,证实了O-CHCS可以作为基因载体用于目的基因HBD2的转染,且目的基因表达产物具有生物活性,为基因工程合成HBD2提供一条经济便捷的途径。
Objective To explore O-CHCS＇s feasibility of being a gene vector for transfecting L929 cells.MethodsWe constructed plasmid pCMV-hBD2,and the plasmid was introduced into L929 cells by O-CHCS.Total RNA was extracted from the cultured cells,RT-PCR was performed with specific primers for HBD2 and RT-PCR amplification products were identified with agarose gel electrophoresis;the expression of HBD2 protein was verified by Western blot;the antibacterial activity of purpose protein was defected by Kirby-Bauer disk diffusion method,at the same time put the Lipofect as the positive control.Results RT-PCR amplification products by agarose gel electrophoresis were the same size as the target gene by observation;HBD2 gene expression at the protein level was detected by Western blot;mediums from the cultured cells transfecting with the pCMV-hBD2 could format antibacterial circle against the Staphylococcus aureus,and these results were as same as the Lipofect.Conclusion The eukaryotic expression vector pCMV-hBD2 was successfully constructed and was introduced to the L929 cells,by doing this weverified O-CHCS could be used as a gene vector for HBD2,and provided a economic and convenient way to artificial synthesis of HBD2.
出处
《基础医学与临床》
CSCD
北大核心
2011年第6期709-712,共4页
Basic and Clinical Medicine
