CARP1基因克隆、表达及其泛素连接酶活性的鉴定

Cloning and Expression of Human CARP1 Gene and Identification of its Ubiquitin Ligase Activity

目的:克隆并表达caspase-8/10相关RING结构域蛋白1(CARP1)基因,检测其泛素连接酶活性。方法:提取结肠癌HCT116细胞总RNA,RT-PCR扩增CARP1基因,将其克隆到真核表达载体pcDNA3-Flag中,序列正确的阳性克隆进行扩增,提取质粒转染至293T细胞中进行瞬时表达,通过体内泛素化检测其泛素化酶活性,通过萤光素酶报告基因的方法检测其对NF-κB转录活性的影响,利用细胞生长曲线检测CARP1基因对结肠癌细胞生长的影响。结果:免疫印迹实验表明CARP1基因在293T细胞中获得有效表达;免疫共沉淀实验表明,当CARP1存在时,受体相互作用蛋白1(RIP1)被泛素化;萤光素酶实验结果表明CARP1抑制肿瘤坏死因子α(TNFα)引起的NF-κB报道基因的激活;通过生长曲线发现CARP1能促进结肠癌细胞系HCT116生长,并能部分抵抗顺铂抑制细胞生长的作用。结论:构建的人CARP1基因真核表达载体在293T细胞中获得表达,表达产物具有RIP1泛素连接酶的活性,可抑制TNFα引起的NF-κB报告基因的激活,并且促进结肠癌细胞的生长,为进一步的基因功能研究奠定了基础。

Objective： To clone and express caspase-8/10-associated RING domain protein 1（CARP1） gene, and identify its ubiquitin ligase activity. Methods： CARP1 gene was amplified by RT-PCR from total RNA, which was extracted from HCTll6 cells by TRIzol. Then it was inserted into plasmid pcDNA3-Flag to construct CARPI eukaryotic expression plasmid. After the expression plasmid was transiently transfected into HEK293T cell lines by Vigofect, its expression was detected by immunoblotting, and ubiquitin ligase activity of CARP1 was detected by co-immunopreeipitation, and the function of suppression of NF-KB transcription was analyzed by luciferase report- er analysis. Results： The CARPI gene sequence was confirmed by sequencing and aligning. The immunoblotting re- sults showed that CARPI was expressed in 293T cell line. The co-immunoprecipitation results exhibited that recep- tor-interacting proteinl （RIP1） was ubiquitinylated at the presence of CARPI. The result of luciferase assay showed that the NF-κB activity was significantly downregulated by CARP1. Furthermore, HCT116 cells with CARP1 overexpression grew faster than control cells. Conclusion： The human CARPI gene was cloned and ex- pressed in 293T cell line. CARPI inhibits TNFa-mediated NF-κB activation, and promotes HCT116 cell growth in the presence or absence of cisplatin. These findings provide us a platform to explore CARPI function in tumor progression.