本文選題：廣譜腫瘤抗原 + hTERT��； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2010年碩士論文

【摘要】： 背景與目的： 人端粒酶逆轉(zhuǎn)錄酶(human telomerase reversetranscrip tase, hTERT)是端粒酶復(fù)合物的核心成分,其表達(dá)狀態(tài)是細(xì)胞調(diào)控端粒酶活性的重要環(huán)節(jié)。hTERT是最早發(fā)現(xiàn),也是目前研究較多的廣譜腫瘤相關(guān)抗原(TAA)之一。已經(jīng)證實(shí)在85%以上的人類惡性腫瘤細(xì)胞中均存在hTERT的再激活,并且在腫瘤的發(fā)生發(fā)展過程中發(fā)揮關(guān)鍵作用。hTERT的廣泛表達(dá)使其成為腫瘤治療的理想靶點(diǎn)。本研究通過特異性擴(kuò)增hTERT基因,成功構(gòu)建了hTERT新型基因疫苗,并且建立了穩(wěn)定表達(dá)hTERT的B16細(xì)胞系。為進(jìn)一步研究hTERT基因疫苗在腫瘤免疫治療中的應(yīng)用奠定了研究基礎(chǔ)。 方法： 通過RT-PCR的方法從人前列腺癌細(xì)胞系PC-3M中特異性擴(kuò)增hTERT基因,連接到真核表達(dá)載體pIRES-neo中,構(gòu)建真核表達(dá)質(zhì)粒pIRES-neo-hTERT。脂質(zhì)體法瞬時轉(zhuǎn)染B16細(xì)胞,通過G418加壓篩選單克隆細(xì)胞系,用western blot及免疫熒光和流式細(xì)胞術(shù)分別檢測B16細(xì)胞中hTERT基因的蛋白表達(dá)。 擴(kuò)增出hTERT基因片段eTERT (1609bp-2628bp),連接到真核表達(dá)載體pVAX1-IRES中,構(gòu)建hTERT廣譜抗腫瘤基因疫苗pVAX1-eTERTFcGB,脂質(zhì)體法瞬時轉(zhuǎn)染293T細(xì)胞,通過免疫熒光和流式細(xì)胞術(shù)檢測其表達(dá)情況。結(jié)果： 特異性擴(kuò)增得到大小與人hTERT基因相符的基因片段,序列測定證實(shí)與GenBank上登錄的序列一致；成功構(gòu)建真核表達(dá)質(zhì)粒pIRES-neo-hTERT。質(zhì)粒成功轉(zhuǎn)染B16細(xì)胞,經(jīng)加壓篩選,得到穩(wěn)定表達(dá)hTERT的B16細(xì)胞系。 成功擴(kuò)增出hTERT基因片段eTERT (1609bp-2628bp),并構(gòu)建hTERT廣譜抗腫瘤基因疫苗pVAX1-eTERTFcGB,流式細(xì)胞儀和免疫熒光的檢測結(jié)果顯示,重組基因疫苗質(zhì)粒pVAX1-eTERTFcGB在293T細(xì)胞中得到很好的表達(dá)。結(jié)論： 特異性擴(kuò)增出hTERT基因,成功構(gòu)建真核表達(dá)載體并篩選出穩(wěn)定表達(dá)hTERT的B16細(xì)胞系；成功構(gòu)建hTERT廣譜抗腫瘤基因疫苗pVAXl-eTERTFcGB,為進(jìn)一步研究hTERT基因疫苗的體內(nèi)外免疫功能奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392;R73-362

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本文編號：1915626