携带免疫基因的前列腺癌特异性溶瘤腺病毒制备及功能验证

摘要

Objective To construct a prostate cancer specific oncolytic adenovirus armed with a fusion protein gene , PSA-IZ-CD40L, and to evaluate its oncolytic efficiency and immune activation ability in vitro.Methods Prostate Specific Antigen (PSA) gene, CD40L-N and CD40L-C genes were obtained from cDNA of LNCaP cells and Jurkat cells using poly-merase chain reaction (PCR) or nested-PCR, respectively.PSA,Linker,CD40L-N and CD40L-C were linked sequentially to generate fusion protein gene PSA-IZ-CD40L (PL) by overlapping PCR.Then, prostate specific oncolytic adenovirus PL-carrying gene, Ad-PL-PPT-E1A,was constructed using the oncolytic adenovirus system , which was based on Adeasy sys-tem.PC3M cells were infected by Ad-PL-PPT-E1A at serial multiplicity of infection (MOI), and the apoptosis was detec-ted by flow cytometry at several time points post-infection.For immune activation detection , PC3M cells were infected with Ad-PL-PPT-E1A at a MOI of 50, and the cell lysate was collected at 48 h post-infection.Peripheral blood mononuclear cells derived (PBMCs) from healthy donors were stimulated by the lysate from PC 3M cells or Ad-PL-PPT-E1A infected PC3M cells before proliferation was assayed using cell counting kit-8 (CCK8).Results Fusion protein gene, PSA-IZ-CD40L, was successfully constructed and cloned into the prostate cancer specific adenovirus to generate Ad -PL-PPT-PL. The expression of E1A and PL protein could be detected by reverse transcription PCR and Western-blotting.Cytopathic effect was observed in PC3M cells infected with Ad-PL-PPT-E1A.Furthermore, the apoptosis rate reached 70.67% ± 2.98%at 48 h post-infection with 200 MOI Ad-PL-PPT-E1A.Compared with the lysate of PC3M cells, that from Ad-PL-PPT-E1A infected cells could promote the proliferation of PBMCs .Conclusion We have constructed a prostate cancer spe-cific oncolytic adenovirus armed can fusion protein gene PL , Ad-PL-PPT-E1A, which could kill PC3M cells effectively and enhance the proliferation of PBMCs in vitro.%目的制备前列腺癌特异性免疫溶瘤双功能腺病毒Ad-PL-PPT-E1A，评价其体外溶瘤和免疫激活功能。方法以LNCaP细胞和Jurkat细胞的cDNA为模板，采用普通PCR和巢式PCR分别扩增获得前列腺特异性抗原（ prostate specific antigen，PSA）基因、CD40L-N 基因和 CD40L-C 基因；并采用重叠 PCR 方法将 PSA、Linker、CD40L-N 和CD40L-C基因依次连接并扩增获得融合蛋白基因PSA-IZ-CD40L（PL）。采用以Adeasy系统为基础的溶瘤腺病毒构建体系，构建并制备携带PL的溶瘤重组腺病毒Ad-PL-PPT-E1A。以不同感染复数（multiplicity of infection， MOI）的溶瘤腺病毒Ad-PL-PPT-E1A感染PC3M细胞后不同时间点，流式细胞术检测细胞凋亡情况；50 MOI的Ad-PL-PPT-E1A感染PC3M细胞，于48 h收集其裂解液，并将其作用于正常人外周血单个核细胞，CCK8检测细胞增殖能力。结果 PCR成功扩增并连接获得融合蛋白基因PL，构建并制备了携带PL基因的溶瘤腺病毒Ad-PL-PPT-E1A。 RT-PCR和Western印迹检测结果显示，Ad-PL-PPT-E1A可在PC3M细胞中高效表达E1A和PL蛋白。将Ad-PL-PPT-E1A感染PC3M细胞后，可见明显的细胞病变；同时，流式细胞检测结果显示，当感染滴度达到200 MOI时，48 h的凋亡率达到70．67％±2．98％。 CCK8结果显示，Ad-PL-PPT-E1A感染后，可恢复PC3M细胞裂解液对人外周血单个核细胞的抑制作用。结论成功制备和鉴定了前列腺癌特异性免疫溶瘤双功能腺病毒Ad-PL-PPT-E1A，并在体外证实了其具有溶瘤和免疫的双重功能。