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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 prepare lentivirus pSin-Bmi1 carrying the B cell-specific monoclonal murine leukemia virus integration site 1 gene (Bmi1) and to overexpress the Bmi1 gene and BMI1 protein in human embryonic stem cell H1. Methods According to the Bmi1 gene sequence provided by GeneBank, the cDNA sequence was amplified by using the cDNAs derived from H1 as template. The fragments were recovered and the Bmi1 and pSin vectors were double digested and transformed respectively. After colony PCR, restriction enzyme digestion and sequencing, pSin-Bmi1 positive clones were identified. The positive expression of pSin-Bmi1 and packaging plasmid was transfected into 293T cells to prepare virus solution. The packaged virus was infected into human embryonic stem cell H1 cell line and puromycin was used to screen the cell line stably expressing BMI1. The expression of exogenous BMI1 protein in H1 cells was verified by Western blot. Results The colonies PCR, restriction endonuclease digestion and sequencing confirmed that the target gene Bmi1 sequence was correct. Western blot analysis showed that BMI1 protein was highly expressed in lentivirus infected Bmi1 cells but not in lentiviruses infected with recombinant Bmi1. Conclusion The lentivirus carrying Bmi1 gene was successfully prepared and the cell line stably expressing exogenous Bmi1 was obtained.