精品文档---下载后可任意编辑hIL-12 腺病毒载体的构建及在人 hBMSCs 中的表达的开题报告摘要IL-12 是一种重要的免疫调节因子，能够作用于 T 细胞和 NK 细胞，发挥调节和刺激免疫功能的作用。本讨论旨在构建 hIL-12 腺病毒载体，并将其应用于人骨髓间充质干细胞（hBMSCs）中，观察其在细胞中的表达情况，为进一步的免疫治疗提供实验支持。首先，利用 PCR 技术克隆人 IL-12A 和 IL-12B 基因，将其与pCMV6-AC-GFP 载体进行重组，获得 hIL-12A/pCMV6-AC-GFP 和 hIL-12B/pCMV6-AC-GFP 质粒。然后，将两个质粒进行双酶切并连接，获得重组 hIL-12A/B/pCMV6-AC-GFP 质粒。接着，将其与 AdMax™易位载体进行重组，获得 hIL-12A/B/AdMax™质粒。最后，将重组质粒转染到 293A 细胞中，通过限制性内切酶切和 PCR 检测确认构建成功。利用Western blot 和 ELISA 检测封装感染后 hBMSCs 中的 hIL-12 表达情况，结果显示在 hBMSCs 中成功表达了 hIL-12，并且腺病毒载体能够高效介导 hBMSCs 中的基因转染。本讨论成功构建了 hIL-12 腺病毒载体，并成功表达在 hBMSCs 中。这为进一步讨论 hIL-12 在免疫治疗中的应用提供了实验依据。关键词：腺病毒；人 IL-12；骨髓间充质干细胞；基因表达AbstractIL-12 is an important immunoregulatory factor that can regulate and stimulate immune functions by acting on T cells and NK cells. The aim of this study was to construct a hIL-12 adenovirus vector and to apply it to human bone marrow mesenchymal stem cells (hBMSCs) to observe its expression in cells, providing experimental support for further immunotherapy.Firstly, human IL-12A and IL-12B genes were cloned by PCR, and were recombinant with pCMV6-AC-GFP vector to obtain hIL-12A/pCMV6-AC-GFP and hIL-12B/pCMV6-AC-GFP plasmids. Then, the two plasmids were double-digested and connected to obtain recombinant hIL-12A/B/pCMV6-AC-GFP plasmid. Subsequently, the recombinant hIL-12A/B/pCMV6-AC-GFP plasmid was recombined with AdMax™ transfection vectors to obtain hIL-12A/B/AdMax™ plasmid. Finally, the 精品文档---下载后可任意编辑recombinant plasmid was transfected into 293A cells, and confirmed to be successfully constructed by restriction enzyme digestion and PCR detection. The expression of hIL-12 in hBMSCs after packaging and infection was detected by Western blot and ELISA, and the results showed that hIL-12 was successfully expressed in hBMSCs, and the adenoviral vector could efficiently mediate gene transfection in hBMSCs.In summary, this study successfully constructed a hIL-12 adenovirus vector and expressed it in hBMSCs, providing experimental evidence for further research on the application of hIL-12 in immunotherapy.Keywords: adenovirus; human IL-12; bone marrow mesenchymal stem cells; gene expression
