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目的:制备携带AFP基因的慢病毒,并体外感染树突状细胞(DC),制备AFP-DC疫苗。方法:以人HepG2肝癌细胞cDNA为模板PCR扩增出AFP基因片段,将该片段克隆入慢病毒载体,构建成Lenti-AFP慢病毒载体。其后,用慢病毒载体转染293T细胞,包装慢病毒表达载体。通过酶切、PCR对Lenti-AFP慢病毒载体进行鉴定。包装好的慢病毒体外感染DC,制备AFP-DC疫苗,流式细胞仪检测感染率,免疫荧光、RT-PCR法及电化学发光法检测AFP表达。结果:酶切、PCR鉴定证实,慢病毒载体插入片段为AFP基因。包装的慢病毒具有良好的感染性,可以在293T细胞中形成病毒颗粒,携带AFP基因的慢病毒感染DC,经免疫荧光、RT-PCR法及电化学发光法证实DC能够表达转染基因AFP,并且不会影响DC表型,感染率高达78.91%。结论:成功构建携带AFP基因的慢病毒载体,感染树突状细胞后表达AFP,为DC疫苗在肝癌中的临床应用提供了实验基础。
Objective: To prepare lentivirus carrying AFP gene and infect dendritic cells (DC) in vitro to prepare AFP-DC vaccine. Methods: AFP gene fragment was amplified by PCR from HepG2 hepatocarcinoma cells. The fragment was cloned into lentiviral vector and constructed into Lenti-AFP lentiviral vector. Thereafter, 293T cells were transfected with the lentiviral vector and the lentiviral expression vector was packaged. Lenti-AFP lentiviral vector was identified by restriction enzyme digestion. The packaged lentivirus infected DC in vitro, and then the AFP-DC vaccine was prepared. The infection rate was detected by flow cytometry. The expression of AFP was detected by RT-PCR and electrochemiluminescence. Results: Enzyme digestion and PCR proved that the lentiviral vector insert was AFP gene. The packaged lentivirus has good infectivity. The virus particles can be formed in 293T cells and the lentivirus carrying AFP gene can infect DC. The immunofluorescence, RT-PCR and electrochemiluminescence showed that DC could express AFP, And does not affect the DC phenotype, the infection rate as high as 78.91%. Conclusion: The successful construction of lentiviral vector carrying AFP gene and expression of AFP after infection of dendritic cells provide the experimental basis for the clinical application of DC vaccine in hepatocellular carcinoma.