慢病毒介导的人MUC1核心肽串联重复片段修饰的树突状细胞疫苗的制备

Preparation of dentritic cell vaccine modified by lentivirus-mediated human MUC1 core tandem-repeats peptides

目的探讨制备慢病毒介导的人mucin糖粘蛋白1(MUC1)核心肽串联重复序列(TR)修饰的树突状细胞(DC)疫苗的可行性。方法人工合成编码MUC1 5个TR片段的基因序列,将其克隆入慢病毒载体pLV-GFP中,经酶切、基因测序鉴定。构建成功的慢病毒载体转染293T细胞,制备病毒。病毒液感染DC细胞。流式细胞术检测感染效率及DC表型,RT-PCR及Westernblot检测MUC1目的基因的表达。结果酶切及测序证实慢病毒载体构建成功。制备的慢病毒能有效感染DC细胞,感染效率达32.54%;流式检测证实慢病毒感染后DC成熟表型未受影响;RT-PCR及Western blot检测证实转染后的DC可有效表达MUC1。结论成功制备慢病毒介导的MUC1基因TR片段修饰的DC疫苗。

Objective To investigate the feasibility of preparation of a dentritic cell（DC） vaccine modified by lentivirus（LV）-mediated human mucin 1（MUC1） core tandem-repeats（TR） peptides.Methods Gene fragment coding five TRs of MUC1 was synthesized and cloned into the pLV-GFP vector.The construction of recombinant lentiviral vector was confirmed by restriction digestion and sequencing.LVs were produced in 293T cells by transfection of the recombinant vector plasmids.DCs were infected with LVs.The transduction efficiency and phenotypes of DCs were analyzed by flow cytometry（FCM）.The expression of MUC1 in DCs was detected by RT-PCR and Western blot.Results The recombinant lentiviral vector with five TRs of MUC1 was correctly constructed.DCs were effectively infected by LVs with a transduction efficiency of 32.54%,which was confirmed by FCM.The mature phenotypes of DCs were not altered after LVs infection as proved by FCM.The mRNA and protein expression levels of MUC1 in DCs were verified by RT-PCR and Western blot.Conclusion The DC vaccine modified by LV-mediated human MUC1 core TR peptides has been successfully prepared and identified.