目的构建pEGFP-N3-TRIM22真核表达载体,观察其在293T细胞中的表达特点。方法用RT-PCR法扩增出TRIM22基因编码序列,克隆至真核表达载体pEGFP-N3,通过菌落PCR、双酶切及测序进行鉴定。将pEGFP-N3-TRIM22载体转染293T细胞,通过流式细胞仪...目的构建pEGFP-N3-TRIM22真核表达载体,观察其在293T细胞中的表达特点。方法用RT-PCR法扩增出TRIM22基因编码序列,克隆至真核表达载体pEGFP-N3,通过菌落PCR、双酶切及测序进行鉴定。将pEGFP-N3-TRIM22载体转染293T细胞,通过流式细胞仪、荧光显微镜观察TRIM22-EGFP的表达;通过激光共聚焦,经Lyso Tracker Deep Red及DAPI分别染色溶酶体及细胞核,观察TRIM22-EGFP在293T细胞的分布特点。结果经菌落PCR、双酶切及测序鉴定,成功构建pEGFP-N3-TRIM22真核表达载体。流式细胞仪检测发现,转染细胞中TRIM22-EGFP+细胞约70%左右。荧光显微镜检测发现,TRIM22-EGFP在293T细胞中能以弥散、小斑点或大颗粒团块3种形式存在。激光共聚焦检测发现,在不同的293T细胞中TRIM22-EGFP可分别优势表达于细胞核、细胞浆,或者在胞核及胞浆均有大量表达。结论 TRIM22-EGFP能以弥散、小斑点或大颗粒团块形式分布于细胞核或细胞浆中。展开更多
Tripartite motif containing 22 （TRIM22）, a member of the TRIM/RBCC family, has been reported to activate the nuclear factor-kappa B （NF-kB） pathway in unstimulated macrophage cell lines, but the detailed mechanism...Tripartite motif containing 22 （TRIM22）, a member of the TRIM/RBCC family, has been reported to activate the nuclear factor-kappa B （NF-kB） pathway in unstimulated macrophage cell lines, but the detailed mechanisms governing this activation remains unclear. We investigated this mechanism in HEK293T cells. We found that overexpression of TRIM22 could activate the NF-kB pathway and conversely, could inhibit the tumor necrosis factor receptor-associated factor 6 （TRAF6）-stimulated NF-kB pathway in HEK293T cells. Further experiments showed that TRIM22 could decrease the self-ubiquitination of TRAF6, and interact with and degrade transforming growth factor-13 activated kinase 1 binding protein 2 （TAB2）, and that these effects could be partially rescued by a TRIM22 RING domain deletion mutant. Collectively, our data indicate that overexpression of TRIM22 may negatively regulate the TRAF6-stimulated NF-rd3 pathway by interacting with and degrading TAB2.展开更多
文摘目的构建pEGFP-N3-TRIM22真核表达载体,观察其在293T细胞中的表达特点。方法用RT-PCR法扩增出TRIM22基因编码序列,克隆至真核表达载体pEGFP-N3,通过菌落PCR、双酶切及测序进行鉴定。将pEGFP-N3-TRIM22载体转染293T细胞,通过流式细胞仪、荧光显微镜观察TRIM22-EGFP的表达;通过激光共聚焦,经Lyso Tracker Deep Red及DAPI分别染色溶酶体及细胞核,观察TRIM22-EGFP在293T细胞的分布特点。结果经菌落PCR、双酶切及测序鉴定,成功构建pEGFP-N3-TRIM22真核表达载体。流式细胞仪检测发现,转染细胞中TRIM22-EGFP+细胞约70%左右。荧光显微镜检测发现,TRIM22-EGFP在293T细胞中能以弥散、小斑点或大颗粒团块3种形式存在。激光共聚焦检测发现,在不同的293T细胞中TRIM22-EGFP可分别优势表达于细胞核、细胞浆,或者在胞核及胞浆均有大量表达。结论 TRIM22-EGFP能以弥散、小斑点或大颗粒团块形式分布于细胞核或细胞浆中。
基金supported by a grant from the Key Projects in the National Science & Technology Pillar Program during the twelfth Five-Year Plan Period of China (2012ZX10001006-002)grants from the International Science & Technology Cooperation Program of China (2011DFA31030)Deutsche Forschungsgemeinschaft (SFB/Transregio TRR60)and Key Laboratory on Emerging Infectious Diseases and Biosafety in Wuhan
文摘Tripartite motif containing 22 （TRIM22）, a member of the TRIM/RBCC family, has been reported to activate the nuclear factor-kappa B （NF-kB） pathway in unstimulated macrophage cell lines, but the detailed mechanisms governing this activation remains unclear. We investigated this mechanism in HEK293T cells. We found that overexpression of TRIM22 could activate the NF-kB pathway and conversely, could inhibit the tumor necrosis factor receptor-associated factor 6 （TRAF6）-stimulated NF-kB pathway in HEK293T cells. Further experiments showed that TRIM22 could decrease the self-ubiquitination of TRAF6, and interact with and degrade transforming growth factor-13 activated kinase 1 binding protein 2 （TAB2）, and that these effects could be partially rescued by a TRIM22 RING domain deletion mutant. Collectively, our data indicate that overexpression of TRIM22 may negatively regulate the TRAF6-stimulated NF-rd3 pathway by interacting with and degrading TAB2.
