表达人CD137配体重组腺病毒的构建及其抗肿瘤免疫作用的研究

Construction of recombinant adenovirus expressing human CD137L and its anti-tumor immunogenicity

目的构建并制备表达人CD137L蛋白的5型重组腺病毒(Ad5-CD137L),并初步研究其在小鼠中的抗肿瘤免疫效果。方法将密码子优化CD137L蛋白的表达基因插入pDC316穿梭质粒,并与pBH-GloxΔE1,3Cre腺病毒骨架质粒,共同转染HEK293细胞,构建Ad5-CD137L种子。扩增并纯化后,对所得Ad5-CD137L重组腺病毒的插入目的基因与蛋白表达情况进行鉴定。检测Ad5-CD137L在C57BL/6小鼠体内的特异性细胞免疫和抗肿瘤免疫水平。结果成功地构建了Ad5-CD137L,PCR和测序结果表明插入目的基因与构建理论值相符。免疫荧光法鉴定Ad5-CD137L能在HEK293细胞中表达目的蛋白,经Western印迹法鉴定该目的蛋白的相对分子质量约为25 000,与理论值相符。与空腺病毒相比,Ad5-CD137L能显著诱导小鼠特异性IFN-γ(t=6.315,P=0.003),抗肿瘤免疫效果更显著(t21 d=8.275,t29 d=4.492;t39 d=5.101,P均<0.001)。结论成功构建了Ad5-CD137L,该重组病毒具有特异性细胞免疫原性和抗肿瘤免疫效果。

Objective To construct and prepare a recombinant adenovirus-5 expressing human CD137L(Ad5-CD137L),and to evaluate its anti-tumor immunogenicity in mice.Methods The codon optimized gene of CD137L was inserted into pDC316 shuttle plasmid,and was co-transfected into HEK293 cells with pBHGloxΔE1,3Cre to construct the seed of Ad5-CD137L.After amplification and purification,the expression of inserted gene and protein of Ad5-CD137L were identified.The Ad5-CD137L was immunized to C57BL/6 mice to evaluate its specific cellular immunity and anti-tumor immunity.Results The Ad5-CD137L was constructed successfully.The inserted gene was confirmed by PCR and sequencing,and was consistent with the theoretical value.The target protein of Ad5-CD137L could be expressed and verified in HEK293 cells by immunofluorescence and Western-Blot,and the molecular weight of the protein is close to the theoretical value of 25000.Compare to the null adenoviruses,Ad5-CD137L could significantly induce specific IFN-γin mice(t=6.315,P=0.003),as well as had significant anti-tumor immune effect(t21 d=8.275,t29 d=4.492,t39 d=5.101,P all<0.001).Conclusions Ad5-CD137L is successful constructed.It has specific cellular immunogenicity and anti-tumor immune effect in mice.