摘要目的 构建甲胎蛋白(AFP)抗原肽修饰的白细胞介素-15(IL-15)基因慢病毒表达载体,包装慢病毒,体外感染人B细胞淋巴瘤细胞(BJAB)细胞,使之能够高效地表达甲胎蛋白抗原肽AFP158-166及IL-15,为体外大量扩增特异性T淋巴细胞建立实验基础.方法 通过聚合酶链反应(PCR)方法获得IL-15基因片段,定点突变方法在过渡质粒信号肽序列中插入AFP158-166表达基因,双酶切后克隆到第3代慢病毒载体pEZ-Lv201,酶切、测序鉴定正确后命名为CCS-afpIL15-Lv201.重组质粒与辅助质粒共转染293T细胞包装成慢病毒,体外感染BJAB细胞,ELISA检测IL-15的表达,液相色谱-质谱联用技术检测AFP抗原肽表达.结果 酶切、测序显示CCS-afpIL15-Lv201载体序列正确,制备成慢病毒感染BJAB细胞后,酶联免疫吸附试验(ELISA)检测IL-15的表达,24、48 h表达量分别为892.7 ng/L和1 232.6 ng/L.液相色谱-质谱联用技术检测到AFP抗原肽表达,其相对分子质量为1 204.65.结论 成功构建协同表达AFP、IL-15的慢病毒载体,并制备了具有感染性的慢病毒,实现了其在肿瘤细胞的高效表达.

abstractsObjective To construct and package alpha fetal protein (AFP) antigen epitope modified interleukin-15 (IL-15)-expression lentiviral vector,infect BJAB cells in vitro for effective expression of AFP158-166,in order to amplify specific T lymphocytes in vitro.Methods IL-15 gene was amplified by polymerase chain reaction (PCR) and part of the IL-2 signal peptide were replaced with AFP epitope using site-directed mutagenesis technique.AFP158-166 modified IL-15 gene was cloned into lentiviral plasmid pEZ-Lv201.The positive clones were verified by enzyme digestion and sequencing.The recombinant vector named CCS-afpIL15-Lv201 was then transfected with helper plasmids into 293T cells to package lentivirus.The expression of IL-15 and AFP epitope in lentiviral-infected BJAB cells was examined by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography-mass spectrometry (HPLC-MS).Results The recombinant plasmid was constructed successfully,which was demonstrated by enzyme digestion and sequencing.BJAB cells infected with AFP158-166 and IL-15-expressing lentivirus expressed IL-15 and AFP158-166 efficiently.The expression after being transfected 24h and 48h was respectively 892.7,1 232.6 ng/L.The relative molecular mass of expressed AFP158-166 was 1204.65.Conclusion The AFP158-166 and IL-15-expressing lentivirus was prepared successfully and was able to infect tumor cells,resulting efficient IL-15 and AFP158-166 expression.Resolving the difficulty of insufficient cells in adoptive cellular immunotherapy for Hepatic carcinoma can be expected.