免疫增效型广谱抗肿瘤血管生成基因疫苗的基础研究

发布时间：2018-01-16 13:13

本文关键词：免疫增效型广谱抗肿瘤血管生成基因疫苗的基础研究　出处：《中国人民解放军军医进修学院》2010年硕士论文　论文类型：学位论文

【摘要】： 背景与目的：上世纪70年代由Judah Folkman提出的有关肿瘤血管生成的理论已被广泛接受,目前,抗肿瘤血管生成已经成为肿瘤学基础研究及临床治疗领域中最有前景的策略之一。大量研究证据表明,在众多的血管生成刺激因子中,内皮生长因子(VEGF)及其受体VEGFR2(鼠中称为flk-1,人中称为KDR)对于与肿瘤生长及转移密切相关的血管生成是最重要的,许多旨在通过打破VEGF/VEGFR2传导通路抑制肿瘤血管生成的主动免疫实验研究都取得了比较理想的结果,还有研究表明,与全长基因疫苗相比,编码VEGFR2胞外1-4区的基因疫苗同样能够有效降低小鼠血清中VEGF水平,从而抑制肿瘤的生长及转移。构建一种能够诱导有效免疫反应基因疫苗的关键是打破对靶抗原的自身免疫耐受,在这项研究中我们主要采取三项措施解决这一问题。首先,选取异种同源的小鼠VEGFR2胞外1-4 IgG样区域(简称mVEGFR2(1-4))作为疫苗的靶抗原；其次,将人白细胞介素12(hIL-12)融合基因插入到载体IRES序列的下游,构建一个下游可以同时表达hIL-12双亚基的双顺反子真核表达载体pVAX1-IRES-hIL12,hIL-12作为对体内细胞和体液免疫系统作用最强的免疫活性因子及一种抗血管生成因子,在基因疫苗中以免疫佐剂的形式发挥作用；最后,为了增强本疫苗的免疫粘附及抗原递呈功能,本研究利用真核表达载体pCI-Fc-GPI,实现了人Igk前导信号肽、人IgG-Fc段及GPI与靶抗原mVEGFR2(1-4)的共同表达,成功构建了免疫增效型广谱抗肿瘤血管生成基因疫苗pVAX1-sig-mVEGFR2(1-4)-Fc-GPI-IRES-hIL 12(简称pVAX1-mVEGFR2-hIL12)。方法：通过搭桥PCR获得人白细胞介素12 P35及P40双亚基的融合基因P35-F2A-P40,插入已构建好的DNA疫苗载体pVAX1-IRES下游,构建质粒载体pVAX1-IRES-hIL12;通过RT-PCR的方法从Balb/c小鼠胚胎组织中特异性扩增mVEGFR2(1-4)基因,连接到真核表达载体pVAX1-IRES-hIL12的上游,构建基因疫苗pVAX1-mVEGFR2-hIL12。脂质体法瞬时转染293T细胞,用ELISA、免疫荧光和流式细胞术分别检测293T细胞中hIL-12和mVEGFR2(1-4)基因的蛋白表达。结果：特异性扩增得到大小与mVEGFR2(1-4)基因相符的基因片段,序列测定证实与GenBank(GI:57923)上登录的序列一致；酶切鉴定和序列分析表明P35及P40双亚基融合基因P35-F2A-P40与设计完全一致,融合基因在体外细胞培养液检测中获得分泌表达；成功构建了广谱抗肿瘤血管生成基因疫苗pVAX1-mVEGFR2-hIL12。FACS及IMF检测证实重组基因疫苗得到有效表达。结论：成功构建了免疫增效型广谱抗肿瘤血管基因疫苗pVAX1-mVEGFR2-hIL12,为后续研究中进一步评价该疫苗抗肿瘤血管生成的效果奠定了必要基础,并为肿瘤免疫治疗领域提供了一种新的疫苗研究方向。
[Abstract]:Background and purpose: In -30s, the theory of tumor angiogenesis proposed by Judah Folkman has been widely accepted. Anti-angiogenesis has become one of the most promising strategies in the basic research and clinical treatment of oncology. Endothelial growth factor (VEGF) and its receptor VEGFR2 (called flk-1 in mice and KDRs in humans) are the most important for angiogenesis closely related to tumor growth and metastasis. Many active immunological studies aimed at inhibiting tumor angiogenesis by breaking the VEGF/VEGFR2 conduction pathway have achieved satisfactory results, and studies have shown that compared with full-length gene vaccines. The gene vaccine encoding the extracellular 1-4 region of VEGFR2 can also effectively reduce the level of VEGF in the serum of mice, thus inhibiting the growth and metastasis of tumor. The key to construct a gene vaccine that can induce an effective immune response is to break the autoimmune tolerance to target antigen. In this study, we mainly take three measures to solve this problem. The heterologous mouse VEGFR2 extracellular 1-4 IgG like region (mVEGFR2P1-4) was selected as the target antigen of the vaccine. Secondly, the fusion gene of human interleukin-12hIL-12 was inserted into the downstream of the vector IRES sequence. To construct a downstream eukaryotic expression vector pVAX1-IRES-hIL12 which can simultaneously express hIL-12 double subunits. HIL-12, as the most potent immunoreactive factor and an anti-angiogenic factor, plays an important role in gene vaccine as an immune adjuvant. Finally, in order to enhance the immune adherence and antigen presentation of the vaccine, the eukaryotic expression vector pCI-Fc-GPI was used to realize human Igk precursor signal peptide. The co-expression of human IgG-Fc fragment and GPI with target antigen mVEGFR2O1-4. The immune-synergistic broad-spectrum anti-angiogenesis gene vaccine pVAX1-sig-mVEGFR2(1-4)-Fc-GPI-IRES-hIL _ (12) was successfully constructed. For short, pVAX1-mVEGFR2-hIL12. Methods: The fusion gene P35-F2A-P40 of human interleukin 12 P35 and P40 double subunits was obtained by bypass PCR. The plasmid vector pVAX1-IRES-hIL12 was constructed by inserting the constructed DNA vaccine vector pVAX1-IRES downstream. The mVEGFR2O1-4) gene was specifically amplified from the embryonic tissues of Balb/c mice by RT-PCR. The gene vaccine pVAX1-mVEGFR2-hIL12was constructed by ligation to the upstream of eukaryotic expression vector pVAX1-IRES-hIL12. Liposome method was used to transfect 293T cells. The protein expression of hIL-12 and mVEGFR2O1-4 in 293T cells were detected by Elisa, immunofluorescence and flow cytometry, respectively. Results: The specific amplification showed that the size of the gene was consistent with that of the mVEGFR2O1-4) gene, which was confirmed by sequence determination to be the same as the sequence entered on GenBank Gi: 57923. Restriction endonuclease analysis and sequence analysis showed that P35 and P40 double subunit fusion genes P35-F2A-P40 were completely consistent with the design. The fusion gene was secreted and expressed in vitro cell culture medium. PVAX1-mVEGFR2-hIL12.FACS and IMF analysis showed that the recombinant gene vaccine was effectively expressed. Conclusion: The immune-synergistic broad-spectrum anti-angiogenic gene vaccine pVAX1-mVEGFR2-hIL12 was successfully constructed. It lays a necessary foundation for further evaluation of the anti-angiogenesis effect of the vaccine and provides a new vaccine research direction for tumor immunotherapy.
【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】：R392.1

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