【摘要】： 本研究旨在探討表達新城疫病毒HN基因和雞貧血病病毒Apoptin基因的“自殺性”DNA疫苗的抑瘤效應(yīng)。以新城疫病毒HN基因及雞貧血病毒Apoptin基因為效應(yīng)基因,利用真核表達載體pIRESneo和西門利克(Semliki)森林腦炎病毒cDNA復(fù)制子為基礎(chǔ)構(gòu)建了pSFV載體,插入目的基因HN和Apoptin,分別構(gòu)建了兩種“自殺性”DNA疫苗,pSFV-HN和pSFV-Apoptin。采用限制性酶切、吖啶橙/溴化乙錠染色結(jié)合熒光顯微鏡觀察、Western blot、RT-PCR等方法檢測了外源基因的重組和在腫瘤細胞內(nèi)的表達。結(jié)果顯示,外源基因已經(jīng)插入pSFV載體中,且可以在HCT-116細胞中有效表達。采用MTT法和DAPI染色等方法觀察了重組質(zhì)粒轉(zhuǎn)染HCT-116細胞后的抑癌效應(yīng)和死亡方式。結(jié)果顯示,重組質(zhì)粒能夠有效抑制腫瘤細胞,并使腫瘤細胞呈現(xiàn)典型的凋亡特征。 本研究復(fù)制了C57BL/6小鼠荷H22腫瘤模型,探討了“自殺性”DNA疫苗的體內(nèi)抑瘤作用及其誘導的體液和細胞的免疫水平。結(jié)果顯示,兩種“自殺性”DNA疫苗均能有效抑制實體腫瘤的生長,并且能夠有效刺激機體產(chǎn)生抗腫瘤免疫反應(yīng),為基因工程抗腫瘤治療性疫苗的研發(fā)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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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