hTERT广谱抗肿瘤基因疫苗实验研究

发布时间：2018-05-20 17:46

  本文选题：广谱肿瘤抗原 + hTERT　； 参考：《中国人民解放军军医进修学院》2010年硕士论文

【摘要】： 背景与目的： 人端粒酶逆转录酶(human telomerase reversetranscrip tase, hTERT)是端粒酶复合物的核心成分,其表达状态是细胞调控端粒酶活性的重要环节。hTERT是最早发现,也是目前研究较多的广谱肿瘤相关抗原(TAA)之一。已经证实在85%以上的人类恶性肿瘤细胞中均存在hTERT的再激活,并且在肿瘤的发生发展过程中发挥关键作用。hTERT的广泛表达使其成为肿瘤治疗的理想靶点。本研究通过特异性扩增hTERT基因,成功构建了hTERT新型基因疫苗,并且建立了稳定表达hTERT的B16细胞系。为进一步研究hTERT基因疫苗在肿瘤免疫治疗中的应用奠定了研究基础。 方法： 通过RT-PCR的方法从人前列腺癌细胞系PC-3M中特异性扩增hTERT基因,连接到真核表达载体pIRES-neo中,构建真核表达质粒pIRES-neo-hTERT。脂质体法瞬时转染B16细胞,通过G418加压筛选单克隆细胞系,用western blot及免疫荧光和流式细胞术分别检测B16细胞中hTERT基因的蛋白表达。 扩增出hTERT基因片段eTERT (1609bp-2628bp),连接到真核表达载体pVAX1-IRES中,构建hTERT广谱抗肿瘤基因疫苗pVAX1-eTERTFcGB,脂质体法瞬时转染293T细胞,通过免疫荧光和流式细胞术检测其表达情况。结果： 特异性扩增得到大小与人hTERT基因相符的基因片段,序列测定证实与GenBank上登录的序列一致；成功构建真核表达质粒pIRES-neo-hTERT。质粒成功转染B16细胞,经加压筛选,得到稳定表达hTERT的B16细胞系。 成功扩增出hTERT基因片段eTERT (1609bp-2628bp),并构建hTERT广谱抗肿瘤基因疫苗pVAX1-eTERTFcGB,流式细胞仪和免疫荧光的检测结果显示,重组基因疫苗质粒pVAX1-eTERTFcGB在293T细胞中得到很好的表达。结论： 特异性扩增出hTERT基因,成功构建真核表达载体并筛选出稳定表达hTERT的B16细胞系；成功构建hTERT广谱抗肿瘤基因疫苗pVAXl-eTERTFcGB,为进一步研究hTERT基因疫苗的体内外免疫功能奠定了基础。
[Abstract]:Background and purpose: Human telomerase reverse transcriptase (telomerase reversetranscrip tase, hTERT) is the core component of telomerase complex. Its expression state is an important link of cell regulation of telomerase activity, hTERT is the earliest discovery, and it is also one of the widely studied broad-spectrum tumor-associated antigens (TAA). It has been proved that hTERT is reactivated in more than 85% of human malignant tumor cells, and plays a key role in tumorigenesis and development, making it an ideal target for tumor therapy. In this study, a novel hTERT gene vaccine was successfully constructed by specific amplification of hTERT gene, and a stable B16 cell line expressing hTERT was established. It lays a foundation for further research on the application of hTERT gene vaccine in tumor immunotherapy. Methods: The hTERT gene was specifically amplified from human prostate cancer cell line PC-3M by RT-PCR and ligated into the eukaryotic expression vector pIRES-neo to construct the eukaryotic expression plasmid pIRES-neo-hTERT. After transient transfection of B16 cells by liposome method, monoclonal cell lines were screened under G418 pressure. Western blot, immunofluorescence and flow cytometry were used to detect the protein expression of hTERT gene in B16 cells. HTERT gene fragment eTERT 1609bp-2628bpN was amplified and ligated into eukaryotic expression vector pVAX1-IRES to construct hTERT broad-spectrum anti-tumor gene vaccine pVAX1-eTERTFcGB. the 293T cells were transiently transfected with liposome method. The expression of pVAX1-eTERTFcGBwas detected by immunofluorescence and flow cytometry. Results: The gene fragment with the size of human hTERT gene was obtained by specific amplification, which was confirmed by sequence determination to be identical with the sequence entered on GenBank, and the eukaryotic expression plasmid pIRES-neo-hTERT was successfully constructed. The plasmid was successfully transfected into B16 cells, and B16 cell lines stably expressing hTERT were obtained by pressurized screening. The hTERT gene fragment eTERT 1609bp-2628bpN was successfully amplified, and the hTERT broad spectrum antitumor gene vaccine pVAX1-eTERTFcGBwas constructed. The results of flow cytometry and immunofluorescence analysis showed that the recombinant gene vaccine plasmid pVAX1-eTERTFcGB was well expressed in 293T cells. Conclusion: The hTERT gene was amplified specifically, the eukaryotic expression vector was successfully constructed and the B16 cell line stably expressed hTERT was screened, and the hTERT broad-spectrum anti-tumor gene vaccine pVAXl-eTERTFcGBwas successfully constructed, which laid a foundation for further study of the immune function of hTERT gene vaccine in vitro and in vivo.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392;R73-362

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