重组Bmi1基因的慢病毒的制备和鉴定  

Construction of Bmi1 lentivirus expressing vector and screening of stable transfected cells

作　　者：赵羽[1] 刘加军[1] 

机构地区：[1]中山大学附属第三医院血液科,广东广州510630

出　　处：《热带医学杂志》2013年第7期804-808,F0002,共6页Journal of Tropical Medicine

基　　金：国家自然科学基金(30772782);广东省科技计划项目(2010B030700006)

摘　　要：目的制备携带B细胞特异单克隆鼠白血病毒整合位点1基因(Bmi1基因)的慢病毒pSin-Bmi1,并在人胚胎干细胞H1中过表达Bmi1基因和BMI1蛋白。方法根据GeneBank提供的Bmi1基因序列,以H1来源的cDNA为模板扩增其cDNA序列,回收片段,Bmi1基因和pSin载体分别进行双酶切,连接后的质粒转化。经菌落PCR、双酶切鉴定及测序鉴定pSin-Bmi1阳性克隆。将阳性表达的pSin-Bmi1和包装质粒转染到293T细胞中制备病毒液。将包装好的病毒感染人胚胎干细胞H1细胞系并用嘌呤霉素筛选稳定表达BMI1的细胞株。通过Western blot验证H1细胞中外源性BMI1蛋白表达。结果经菌落PCR、限制性核酸内切酶酶切和测序证实目的基因Bmi1序列正确。Western blot检测发现感染重组Bmi1的慢病毒的细胞中有外源性BMI1蛋白的高表达,而未感染重组Bmi1的慢病毒的对照组未检测到BMI1表达。结论成功制备了携带Bmi1基因的慢病毒,并得到了稳定高表达外源Bmi1的细胞株。Objective To generate letivirus expression vector pSin-Bmil harboring Bmil gene and overexpression of Bmil gene and BMI1 protein in human H1 stem cells. Methods The recombinant positive plasmid clone was prepared using PCR amplification and endonuclease digestion. The fragments were inserted into pSin lentivirus vector and the genes were indentified by sequencing analysis. The lentivirus expression vector pSin-Bmil, together with psPAX2 and pMD2.G packaging plasmid were cotransfected into 293T cells to produce the lentivirus. H1 human embryonic stem cells were then infected by the packaging lentivirus. Puromycin was used to select BMI1 over-expressed cells. Western blotting was used to verify the expression of exogenous BMI1 proteins in H1 cell. Results The sequence of Bmil gene was confirmed by bacterial colony PCR, enzyme digestion and order-checking. The expression level of BMI1 protein was significantly increased as verified by Western blotting. Conclusion The recombinant plasmid pSin-Bmil was successfully constructed and transfected stably into H1 human embryonic stem cells.

关 键 词：Bmi1基因 慢病毒 稳定株 

分 类 号：R341[医药卫生—基础医学]