基于甲病毒复制子的核酸疫苗的构建及实验免疫研究
发布时间：2019-02-24 12:39
【摘要】： 本研究旨在探讨表达新城疫病毒HN基因和鸡贫血病病毒Apoptin基因的“自杀性”DNA疫苗的抑瘤效应。以新城疫病毒HN基因及鸡贫血病毒Apoptin基因为效应基因,利用真核表达载体pIRESneo和西门利克(Semliki)森林脑炎病毒cDNA复制子为基础构建了pSFV载体,插入目的基因HN和Apoptin,分别构建了两种“自杀性”DNA疫苗,pSFV-HN和pSFV-Apoptin。采用限制性酶切、吖啶橙/溴化乙锭染色结合荧光显微镜观察、Western blot、RT-PCR等方法检测了外源基因的重组和在肿瘤细胞内的表达。结果显示,外源基因已经插入pSFV载体中,且可以在HCT-116细胞中有效表达。采用MTT法和DAPI染色等方法观察了重组质粒转染HCT-116细胞后的抑癌效应和死亡方式。结果显示,重组质粒能够有效抑制肿瘤细胞,并使肿瘤细胞呈现典型的凋亡特征。 本研究复制了C57BL/6小鼠荷H22肿瘤模型,探讨了“自杀性”DNA疫苗的体内抑瘤作用及其诱导的体液和细胞的免疫水平。结果显示,两种“自杀性”DNA疫苗均能有效抑制实体肿瘤的生长,并且能够有效刺激机体产生抗肿瘤免疫反应,为基因工程抗肿瘤治疗性疫苗的研发策略提供了新思路。
[Abstract]:The aim of this study was to investigate the inhibitory effect of "suicide" DNA vaccine expressing Newcastle disease virus (NDV) HN gene and chicken anaemia virus (Apoptin) gene. Based on Newcastle disease virus (NDV) HN gene and chicken anemia virus (Apoptin) gene as effector gene, pSFV vector was constructed by using eukaryotic expression vector pIRESneo and cDNA replicon of Simenlick (Semliki) Forest Encephalitis virus. The target genes HN and Apoptin, were inserted into pSFV vector. Two "suicide" DNA vaccines, pSFV-HN and pSFV-Apoptin., were constructed. Restriction enzyme digestion, acridine orange / ethidium bromide staining and fluorescence microscopy were used to detect the recombinant and expression of exogenous genes in tumor cells. The results showed that the exogenous gene had been inserted into pSFV vector and could be effectively expressed in HCT-116 cells. MTT and DAPI staining were used to observe the tumor suppressor effect and death mode of HCT-116 cells transfected with recombinant plasmid. The results showed that the recombinant plasmid could effectively inhibit tumor cells and make tumor cells show typical apoptotic characteristics. In this study, the tumor model of C57BL/6 mice bearing H22 was established, and the anti-tumor effect of "suicide" DNA vaccine in vivo and its induced humoral and cellular immunity were investigated. The results showed that the two "suicide" DNA vaccines could effectively inhibit the growth of solid tumors and stimulate the body to produce anti-tumor immune response effectively, which provided a new idea for the development of genetic engineering anti-tumor therapeutic vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2429561
[Abstract]:The aim of this study was to investigate the inhibitory effect of "suicide" DNA vaccine expressing Newcastle disease virus (NDV) HN gene and chicken anaemia virus (Apoptin) gene. Based on Newcastle disease virus (NDV) HN gene and chicken anemia virus (Apoptin) gene as effector gene, pSFV vector was constructed by using eukaryotic expression vector pIRESneo and cDNA replicon of Simenlick (Semliki) Forest Encephalitis virus. The target genes HN and Apoptin, were inserted into pSFV vector. Two "suicide" DNA vaccines, pSFV-HN and pSFV-Apoptin., were constructed. Restriction enzyme digestion, acridine orange / ethidium bromide staining and fluorescence microscopy were used to detect the recombinant and expression of exogenous genes in tumor cells. The results showed that the exogenous gene had been inserted into pSFV vector and could be effectively expressed in HCT-116 cells. MTT and DAPI staining were used to observe the tumor suppressor effect and death mode of HCT-116 cells transfected with recombinant plasmid. The results showed that the recombinant plasmid could effectively inhibit tumor cells and make tumor cells show typical apoptotic characteristics. In this study, the tumor model of C57BL/6 mice bearing H22 was established, and the anti-tumor effect of "suicide" DNA vaccine in vivo and its induced humoral and cellular immunity were investigated. The results showed that the two "suicide" DNA vaccines could effectively inhibit the growth of solid tumors and stimulate the body to produce anti-tumor immune response effectively, which provided a new idea for the development of genetic engineering anti-tumor therapeutic vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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