TNF,IFN,MHCⅠ类分子基因“正反馈”式放大表达的重组腺病毒载体

Construction of adenoviral vector increasing tumor immunogenicity by enhancing gene expression of TNF,IFN,MHC Ⅰmolecule

目的：为使外源性 Ｔ Ｎ Ｆ， Ｉ Ｆ Ｎ 基因和内源性主要组织相容性复合物Ⅰ类（ Ｍ Ｈ ＣⅠ）分子基因表达呈现“正反馈”式放大效 应，构建在 Ｈ Ｌ Ａ Ｂ７ 启动子调控、 Ｉ Ｒ Ｅ Ｓ连接下协同表达 Ｔ Ｎ Ｆα和 Ｉ Ｆ Ｎβ基因的重组腺病毒载体。方法和结果：从ｐ Ｇ Ｌ３ Ｂ７ Ｔ Ｎ Ｆ及ｐ Ｉ Ｒ Ｉ Ｆ中切下 Ｂ７ｐｒｏ Ｔ Ｎ Ｆ和 Ｉ Ｒ Ｅ Ｓ Ｉ Ｆ Ｎβ基因片段，先后插入ｐ Ｂｌｕｅｓｃｒｉｐｔ Ｓｋ （＋ ）， 构建成ｐ Ｂ Ｌ Ｂ７ Ｔ Ｎ Ｉ Ｒ Ｉ Ｆ。回收 Ｔ Ｎ Ｆα Ｉ Ｒ Ｅ Ｓ Ｉ Ｆ Ｎβ基因片段，连入 Ａｄ Ｃ Ｍ Ｖ Ｌｉｎｋ１ 中，构建成 Ｃ Ｍ Ｖ 启动子驱动表达的 Ａｄ Ｃ Ｍ Ｖ Ｔ Ｎ Ｉ Ｒ Ｉ Ｆ（＋ ）。回收 Ｂ７ｐｒｏ Ｔ Ｎ Ｆ Ｉ Ｒ Ｅ Ｓ Ｉ Ｆ Ｎβ基因片段，连入缺失 Ｅ１ 和 Ｅ３ 区及启动子的 Ａｄ ＢｇｌⅡ中， 构建成 Ｈ Ｌ Ａ Ｂ７ 启动子驱动表达的 Ａｄ Ｂ７ Ｔ Ｎ Ｉ Ｒ Ｉ Ｆ（－ ）。结论：此两种载体为“正反馈”式放大肿瘤免疫原性提供一条新途径。

Objective: To increase tumor immunogenicity by enhancing gene expression of TNF,IFN,MHC Ⅰ molecule , adenoviral vectors containing HuTNFα and IFNβ cDNA driven by HLA B7 inducible promoter and CMV promoter were constructed, respectively. Methods and Results:The 2 fragments HLA B7 promoter TNF and IRES IFN were isolated from and pGL3B7TNF and pIRIF and insert to pBluescript Ⅱ Sk (+) to generate pBLB7TNIRIF.TNFα IRES IFNβ was isolated and ligated to AdCMVLink1 to generate AdCMVTNIRIF(+), in which gene transcription was driven by CMV promoter.B7pro TNFα IRES IFNβ was isolated and ligated to AdBglⅡto generate AdB7TNIRIF(-),in which gene expression was driven by HLA B7 promoter. Conclusion:The constructed vectors can be a new route for enlarging tumor immunogenicity.