【摘要】 目的:采用一种简便方法在复制缺陷型腺病毒载体纤突结(fibre knob)的HI loop插入精氨酸-甘氨酸-天冬氨酸(arginine-glycine-aspartate,RGD)肽,构建纤突结修饰的腺病毒载体,观察其对RD和T24肿瘤细胞的感染效率以及机体针对外源基因的抗体反应。方法:以PCR方法将RGD插入到pAdEasy-1后,通过常规方法构建可表达EGFP和人呼吸道合胞病毒(human respiratory syncytialviruse,RSV)密码子优化型融合蛋白(fusion protein,Fsyn)的腺病毒载体FGAd-RGD-EGFP和FGAd-RGD-Fsyn。用FGAd-RGD-EGFP感染RD和T24肿瘤细胞,通过荧光显微镜和流式细胞仪检测感染效率,用FGAd-RGD-Fsyn免疫BALB/c小鼠,通过ELISA检测针对外源基因的抗体反应。结果:与FGAd-EGFP相比,FGAd-RGD-EGFP对RD和T24肿瘤细胞的感染效率都有显著提高,EGFP表达效率也明显增强。与FGAd-Fsyn相比,FGAd-RGD-Fsyn所引起的机体针对外源基因的抗体反应并无差异。结论:改造后的腺病毒载体能显著提高对RD和T24肿瘤细胞的感染效率,但并不引起机体产生针对外源基因的增强的抗体反应,该载体能用于肿瘤等疾病的基因治疗研究。更多还原

【Abstract】 Objective: A simple way to construct an adenovirus vector containing a peptide-RGD in the HI loop of the fiber knob was developed and then its infection efficiency on RD and T24 cells in vitro and the humoral responses to transgenes in vivo were investigated.Methods: The pAdEasy-RGD-1 was modified by inserting RGD motif into pAdEasy-1 based on simple PCR method.Enhanced green fluorescent protein(EGFP) and codon optimized fusion protein(Fsyn) of human respiratory syncytial virus(RSV) were cloned and constucted into pAdEasy-RGD-1 to generate adenovirus vectors of FGAd-RGD-EGFP and FGAd-RGD-Fsyn,respectively.FGAd-RGD-EGFP infected RD and T24 cells to investigate its infection efficiency using fluorescent microscope and flow cytometry.FGAd-RGD-Fsyn was exploited to vaccinate BALB/c mice via intramuscular and imtranasal routes,respectively,to evaluate humoral responses to transgenes in vivo.Results: The infection efficiency on RD and T24 cells was higher with FGAd-RGD-EGFP than FGAd-EGFP,and immunization with FGAd-RGD-Fsyn generated similar serum IgG specific for RSV-F.Conclusions: PCR is a simple and practical method to construct adenovirus vector containing a peptide-RGD in the HI loop of the fiber knob.The resultant modified adenovirus vector displays improved infection efficiency on RD and T24 cells and similar humoral immune responses to transgene as compared to original adenovirus vector.Therefore,it will be an useful platform for oncotherapy.更多还原