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[目的]构建小鼠端粒酶反转录酶(mTERT)启动子调控的葡萄球菌肠毒素A(SEA)和CD80基因共表达重组腺病毒载体,并观察其介导的SEA和CD80在小鼠肝癌细胞Hepa1-6、结肠癌细胞CT26、黑色素瘤细胞B16和成纤维细胞NIH3T3中的表达情况。[方法]采用AdEasy腺病毒体系,亚克隆mTERT核心启动子区至穿梭质粒pShuttle2,并在其上游插入myc-Max反应元件MMRE,用来调控SEA及CD80基因的表达,构建SEA和CD80基因共表达重组腺病毒载体Ad-MMRE-mTERT-BIS,转化Ad293细胞制备病毒并纯化,然后将病毒以感染复数为100的浓度分别感染上述四种细胞。采用免疫荧光染色法及流式细胞术检测SEA和CD80在细胞膜表面的表达情况。[结果]以感染复数为100病毒量感染上述细胞后,CD80和SEA的不同肿瘤细胞上表达率不同,而病毒感染的NIH3T3细胞不表达SEA和CD80。[结论]成功地构建了mTERT启动子调控的SEA和CD80基因共表达重组腺病毒载体,能够调控SEA和CD80基因在不同肿瘤细胞中的靶向表达,但表达效率有所不同。为进一步采用同一腺病毒对不同肿瘤细胞的进行靶向基因治疗研究奠定了基础。
[Objective] To construct a recombinant adenovirus vector co-expressing staphylococcal enterotoxin A (SEA) and CD80 gene regulated by telomerase reverse transcriptase (mTERT) promoter in mice and to observe the expression of SEA and CD80 in mouse Liver cancer Hepa 1-6, Colon cancer CT26, Melanoma B16 and fibroblast NIH3T3 expression. [Methods] AdEasy adenovirus system was used to subclone mTERT core promoter region to shuttle plasmid pShuttle2 and insert myre-Max response element MMRE upstream of it to regulate the expression of SEA and CD80 genes and construct the co-expression of SEA and CD80 genes The recombinant adenovirus vector Ad-MMRE-mTERT-BIS was transformed into Ad293 cells to prepare and purify the virus, and then the viruses were respectively infected with the above four kinds of cells at a multiplicity of infection of 100. Immunofluorescence staining and flow cytometry were used to detect the expression of SEA and CD80 on the cell membrane surface. [Results] After infected with above 100 cells at a multiplicity of infection of 100, the expression rates of CD80 and SEA on different tumor cells were different, while the virus-infected NIH3T3 cells did not express SEA and CD80. [Conclusion] The recombinant adenovirus vector co - expressing SEA and CD80 regulated by mTERT promoter could be successfully constructed and could regulate the expression of SEA and CD80 in different tumor cells, but the expression efficiency was different. It laid the foundation for the further research on targeted gene therapy of different tumor cells using the same adenovirus.