稳表达人HSP72的人脐带间充质干细胞株的构建  

Construction of human umbilical cord mesenchymal stem cell lines to secret stable human HSP72 with Lenti-viral Vector

作　　者：王珍[1] 赵昌林[1] 骆敏翔 

机构地区：[1]广州中医药大学祈福医院,广东广州511495

出　　处：《中国当代医药》2016年第26期4-8,共5页China Modern Medicine

基　　金：广东省广州市番禺区科技计划项目(2013-Z03-24)

摘　　要：目的构建含人HSP72的慢病毒,筛选获得稳定表达HSP72的hUC-MSC细胞系。方法采用PCR方法从MGC fully sequenced human HSPAIA cDNA中调取HSP72基因的完整序列,利用双酶切及体外重组的方法获得pLV-HSP72-EGFP(2A)质粒,经PCR、测序方式鉴定成功后,对病毒进行包装。用病毒感染hUC-MSC,并用Puro进行抗性筛选可稳定表达HSP72的hUC-MSC细胞系。结果构建的pLV-HSP72-EGFP(2A)测序结果与GenBank收录序列一致,包装得到pLV-HSP72-EGFP(2A)病毒,筛选获得稳定表达HSP72的hUC-MSC细胞系,ELISA方法检测显示获得的细胞株可过表达HSP72。结论构建了获得稳定表达HSP72的hUC-MSC细胞系。Objective To construct Lenti-viral Vector carrying human HSP72 gene,and to screen the hUC-MSC cell line with stable expression of HSP72.Methods The human HSP72 cDNA was amplified from ＂MGC fully sequenced human HSPAIA cDNA＂ by PCR.pLV-HSP72-EGFP（2A） plasmid was obtained by double enzyme digestion and in vitro recombination,then was confirmed by PCR and DNA sequencing.The pLV-HSP72-EGFP（2A） virus was packaged.hUC-MSC was infected by the virus,and the hUC-MSC cell lines stably expressing HSP72 were screened by Puro.Results The recombinant pLV-HSP72-EGFP（2A） was constructed.The sequence was consistent with that reported in Gen Bank.The pLV-HSP72-EGFP（2A） virus was packaged and infected hUC-MSC.By ELISA,it was confirmed that the HSP72 protein could be expressed and secreted into the supernatant of hUC-MSC strain culture.Conclusion hUC-MSC cell line with stable expression of HSP72 is constructed.

关 键 词：HSP72 人脐带间充质干细胞 慢病毒载体 转染 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]