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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 preparing lentivirus-mediated dendritic cell (DC) vaccine modified by human mucin glycoprotein 1 (MUC1) core peptide tandem repeats (TR). Methods The gene sequence encoding 5 TR fragments of MUC1 was synthesized and cloned into lentiviral vector pLV-GFP. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing. The constructed lentiviral vector was transfected into 293T cells to prepare the virus. Viral fluids infect DC cells. The infection efficiency and DC phenotype were detected by flow cytometry. The expression of MUC1 gene was detected by RT-PCR and Western blotting. Results Enzyme digestion and sequencing confirmed the construction of lentiviral vector. The lentivirus could effectively infect DC cells with an infection efficiency of 32.54%. Flow cytometry confirmed that the DC mature phenotype was not affected after lentivirus infection. The expression of MUC1 was confirmed by RT-PCR and Western blot. Conclusion The lentivirus-mediated MUC1 gene TR fragment modified DC vaccine was successfully prepared.