氧依赖性降解结构域-RANTES融合基因腺病毒表达载体的构建及体外趋化活性观察

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。

Construction of adenovirus vector harboring RANTES regulated by oxygen-dependent degradation domain and its chemoattractant activity in vitro

Objective:To construct an adenovirus vector harboring the human RANTES gene regulated by oxygen-dependent degradation domain (ODD) and to observe its chemoattractant activity in vitro.Methods: The human RANTES gene was fused with ODD by PCR and the recombinant adenovirus was used to construct SG511-CCL5-ODD with the Gateway System.Viral replication experiments were performed to evaluate the selective replication ability of SG511-CCL5-ODD.The expression of RANTES protein was determined by ELISA under normal and hypoxia condition.Chemotactic test was used to analyze the chemoattractant ability of the expressed RANTES in liver cancer cells.Results: A recombinant adenovirus SG511-CCL5-ODD was constructed successfully.Cells infected with the recombinant virus expressed RANTES selectively.The expression of RANTES protein in the transfected liver cancer cells was higher under hypoxia condition than under normal condition (P<0.05),indicating ODD can effectively regulate RANTES protein expression.Chemotactic test showed that liver cancer cell infected with SG511-CCL5-ODD had the ability to recruit NK92 cells.Conclusion: Recombinant vector SG511-CCL5-ODD can effectively infect liver cancer cell line HepG2 and Hep3B,and can express RANTES protein under the regulation of ODD,demonstrating a chemoattractant activity in vitro.