ADAM17基因RNAi重组慢病毒的构建及鉴定

Construction and Indetification of Lentiviral Vector of RNA Interference of ADAM17 Gene

目的：研究解整合素金属蛋白酶-17（ADAM17）的特异性高效抑制剂,以控制炎症及其相关过程。方法：筛选确定ADAM17基因RNAi有效靶序列,合成靶序列的Oligo DNA,与慢病毒载体（GV115）连接,PCR筛选阳性克隆,测序鉴定。通过Western Blot检测细胞ADAM17的蛋白表达。用重组慢病毒转导脂多糖（LPS）刺激的U937细胞,观察重组慢病毒在蛋白质水平上对ADAM17的表达和活性的影响。结果：成功构建了ADAM17RNAi重组慢病毒,并能显著抑制ADAM17的蛋白表达,减少sTNF-α分泌。结论：ADAM17RNAi重组慢病毒降低了sTNF-α的分泌,对炎症有明显的抑制作用,为抗炎药物的设计和改造提供了新的依据和方法。

Objective：To construct a lentiviral vector of RNA interference（RNAi）of ADAM17-gene and study an effective inhibitor of ADAM17 to control inflammation and related processes.Methods：A lentivirus vector（GV115）expressing shRNA targeting the ADAM17 gene was constructed.ADAM17 expression was assessed by Western Blot.After stimulated with LPS,the U937 cells were transfected with the lentivirus.The sTNF-αwas assessed by ELISA and the mTNF-αwas assessed by Western Blot.Results：The ADAM17 shRNA lentivirus reduced ADAM17 expression,and prevented TNF-αmaturation in U937 cells.In summary,this study successfully constructed a shRNA lentivirus vector targeting the ADAM17 gene that had clear in vitro effects of TNF-αprocessing in response to an LPS challenge.Conclusion：These results may help inform the design and improvement of drugs designed to inhibit the function of ADAM17,and suggest a novel mean of controlling inflammation and related processes.