过表达大鼠TIPE2基因重组腺病毒载体的构建

Construction of a recombinant adenovirus overexpressing rat TIPE2 gene

背景:TIPE2抗炎蛋白,通过对T细胞受体(TCR)和T细胞TOLL样受体信号途径实行负向调节,从而对适应性免疫和固有免疫起到负性调控作用,有效地维持机体内环境的稳定。目的:使用人工合成腺病毒载体构建能过表达大鼠TIPE2基因的重组腺病毒。方法:利用RT-PCR的方法,从大鼠淋巴细胞中扩增出大鼠TIPE2基因,克隆到穿梭质粒pShuttle-clontech的表达框中,然后将包含TIPE2的完整表达框,进一步亚克隆到黑猩猩来源的腺病毒包装载体AdC68,转染HEK 293A细胞,包装出重组腺病毒。并以其感染HEK293A细胞,采用western blotting方法检测TIPE2基因的表达水平。结果与结论:PCR扩增、酶切鉴定和测序结果表明,所获取的cDNA为TIPE2的蛋白编码基因,western blotting结果表明,重组腺病毒能可高效表达TIPE2基因。说明成功完成AdC68-TIPE2重组腺病毒的构建,该腺病毒载体,能稳定表达大鼠TIPE2基因。

BACKGROUND：Tumor necrosis factor-α-induced protein-8 like-2 （TIPE2）, an anti-inflammatory protein, through the T cel receptor （TCR） and TOLL-like receptor signaling pathway, implements negative regulation of adaptive immunity and innate immunity, and thus effectively maintains the stable internal environment of the body.OBJECTIVE：To construct a recombinant adenovirus that can overexpress rat TIPE2 gene.METHODS：TIPE2 cDNA target gene was amplified from rat’s lymphocytes using RT-PCR, cloned into shuttle plasmid pShuttle-clontech, and then subcloned into artificial adenovirus vector AdC68. Hereafter, HEK 293 cel s were transfected to generate a recombinant adenovirus. HEK293A cel s were infected using this recombinant adenovirus, and then TIPE2 gene level was tested by western blot method.RESULTS AND CONCLUSION：Based on results of PCR, digestion identification and sequencing, the obtained cDNA was the coding sequence region of TIPE2. Western blot findings showed that the recombinant adenovirus could overexpress TIPE2 gene. These findings indicate that the recombinant adenovirus is constructed successful y and can express TIPE2 gene stably.