摘要
目的 构建人β防御素1（ hBD1）稳定表达细胞株并检测其对多种多重耐药菌的抗菌活性.方法 将重组质粒pCMV-hBD1通过阳离子脂质体转染于非洲绿猴SV40转化的肾细胞（COS-7细胞）,经过G418压力筛选后获得单克隆细胞株；提取细胞总RNA,用RT-PCR检测目的基因在转录水平的表达；收集细胞培养上清液,用Western blot检测hBD1基因蛋白的表达；将含有表达产物hBD1的细胞培养上清液分别同各耐药菌液混合,37℃孵育不同时间后涂布于LB平板,以各实验组和对照组的菌落数的比值作为该耐药菌的存活率.结果 经过G418压力筛选所得到的稳定表达细胞株,在转录水平和蛋白水平均检测到目的基因hBD1的表达,在表达产物hBD1的作用下,多重耐药鲍氏不动杆菌、多重耐药大肠埃希菌存活率和多重耐药肺炎克雷伯杆菌的存活率可以分别降至9%、22%和50%,多重耐药嗜麦芽窄食单胞菌的存活率同对照组没有明显差异.结论 成功获得hBD1稳定表达细胞株,目的基因hBD1的表达产物对多种多重耐药菌均具有抗菌活性.
Objective To established a cell line that expresses hBD1 stably,and detected the antimicrobial activity of the hBD1 to the muhidrug resistant bacterial strains.Methods Recombinant plasmid was introduced into COS-7 cells by lipofectamine,cells were selected in culture medium containing G418 to acquired the monoclonal cell lines,total RNA were extracted from the cultured cells,expression levels of hBD1 mRNA was identified by RT-PCR,collected the supernatant solution of the cultured cell,expression levels of protein was identified by Western blot.Put the expression products and resistant organisms mixed together,after incubation in different times in 37℃,coating the mixtures in LB flat,then obtained the ratios between colonies number of experimental groups and colonies number of control groups,put those ratios as the survival rate of the drug resistance bacterias.Results The monoclonal cell lines had obtained after screened with G418,the hBD1 gene could be detected both at transcriptional and protein levels,Under the influence of expression product hBD1,survival rate of muhidrug-resistant Acinetobacter baumannii,multidrug-resistant Escherichia coli and multidrug-resistant Klebsiella pneumoniae could reduced to 9%,22% and 50%,but survival rate of multidrug-resistant Stenotrophomonas maltophilia is not have apparente difference with the control group.Conclusion The stably-transfected cell line of hBD1 was successfully constructed,and the expression products of hBD1 showed the antimicrobial activity toward multidrug resistant bacterial strains.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2011年第12期1138-1142,共5页
Chinese Journal of Microbiology and Immunology
基金
甘肃省新药临床前研究重点实验室开放基金（GSKFKT-0703）
关键词
人β防御素1
稳定表达
多重耐药
生物活性
Human beta-defensin 1
Stable transfect
Multidrug resistant
Biological activity
