论文部分内容阅读
目的:构建携带活化T细胞表达和分泌调节因子(regulated upon activation normal T-cell expressed and secreted,RANTES/CCL5)基因及氧依赖性降解结构域(oxygen-dependent degradation domain,ODD)融合基因的重组腺病毒,并观察其体外趋化活性。方法:PCR法将人RANTES基因与ODD融合,构建携带该融合基因的重组腺病毒SG511-CCL5-ODD;增殖实验观察重组腺病毒增殖特性,ELISA法观察常氧和缺氧条件下RANTES蛋白的表达;趋化试验观察重组腺病毒感染肝癌细胞后的趋化活性。结果:成功构建携带人RANTES-ODD融合基因的重组腺病毒SG511-CCL5-ODD;增殖实验表明重组腺病毒具有肿瘤选择性复制的特性;缺氧条件下重组病毒转染肝癌细胞后RANTES蛋白表达量均比常氧条件下高(P<0.05),显示ODD可有效调节RANTES蛋白表达;趋化试验表明重组腺病毒感染肝癌细胞具有趋化NK92细胞的作用。结论:重组腺病毒SG511-CCL5-ODD体外能有效感染肝癌细胞株HepG2和Hep3B,并在ODD调控下表达RANTES蛋白,有效发挥体外趋化NK92细胞的活性。
OBJECTIVE: To construct a recombinant adenovirus carrying the regulated upon activation normal T-cell expressed and secreted (RANTES / CCL5) gene and the oxygen-dependent degradation domain (ODD) fusion gene Virus and observe its chemotactic activity in vitro. METHODS: Human RANTES gene was fused with ODD by PCR to construct recombinant adenovirus SG511-CCL5-ODD carrying the fusion gene. Proliferation assay was used to observe the proliferation characteristics of recombinant adenovirus. The expression of RANTES protein under normoxia and hypoxia was observed by ELISA ; Chemotaxis assay to observe the chemotactic activity of recombinant adenovirus after infection of hepatoma cells. Results: The recombinant adenovirus SG511-CCL5-ODD carrying the human RANTES-ODD fusion gene was successfully constructed. The proliferation experiments showed that the recombinant adenovirus has the property of tumor selective replication. The expression of RANTES protein in hepatoma cells transfected with recombinant virus under hypoxia (P <0.05), indicating that ODD can effectively regulate the expression of RANTES protein. Chemotaxis assay showed that the recombinant adenovirus infected hepatoma cells have chemotactic effect on NK92 cells. CONCLUSION: The recombinant adenovirus SG511-CCL5-ODD can effectively infect HepG2 and Hep3B cells in vitro and express RANTES protein under the regulation of ODD, which can effectively activate the chemotactic NK92 cells in vitro.