摘要
先天性免疫是机体防御病原体感染的第一道防线,而Akirin2是先天性免疫系统的一个重要功能基因。Akirin2蛋白严格定位于细胞核,参与NF-κB通路,但其作用机制尚未明确。小鼠Akirin2基因敲除实验表明,Akirin2是Toll样受体、肿瘤坏死因子和白介素1受体信号通路中一个重要的蛋白。体外细胞转染实验证实该蛋白的过量表达能明显促进IL-6和其它免疫因子的表达。为探讨Akirin2基因在猪体内的功能,本研究结合生物信息学方法和分子生物学实验,依照人Akirin2基因序列克隆到猪源Akirin2基因,并对该基因编码蛋白质的功能进行了系统的生物信息学分析,同时对其表达规律和特点进行了初探,获得了以下结果:
1.本研究依照人Akirin2基因序列克隆得到猪源Akirin2基因。生物信息学分析猪源Akirin2的序列特征与进化关系。结果显示:猪源Akirin2编码203个氨基酸,蛋白的N端和C端高度保守,猪源Akirin2与人源Akirin2相似度高达98%。
2.将Akirin2克隆到pET-32a原核表达载体上,转化到大肠杆菌表达菌BL21(DE3)中,在IPTG的诱导下使目的基因表达,通过SDS-PAGE分析,检测到43 ku大小的融合蛋白,与预期结果相一致。融合蛋白His-Akirin2,在0.7 mmol/L IPTG诱导6 h后,可溶性表达量较大。融合蛋白经过HiTrap Chelating HP层析小柱纯化。
3.将目的基因克隆到pEGFP-C1真核表达载体上,采用脂质体转染法转染进猪脐静脉血管内皮细胞(SUVECs)。荧光检测结果表明,在转染后24 h融合蛋白EGFP-Akirin2表达量最大。根据荧光发光位置判定猪源Akirin2蛋白定位于细胞核,与预期结果相一致。收集细胞后进行裂解,Western blot检测出融合蛋白EGFP-Akirin2。
Innate immune system is the first line of host defense that prevents host body from being infected by pathogen. Akirin2 is strictly localized to the nucleus, and it plays an essential role in the innate immune system. Although Akirin2 acts in NF-κB signaling pathway, its mechanism is unclear. Knockout of Akirin2 in mice shows that Akirin2 has an essential function downstream of the Toll-like receptor, tumor necrosis factor and IL-1βsignaling pathways. In vitro transfection demonstrated that over expression of Akirin2 can induce some inflammatory factors expressing, including IL-6. We in silico cloned porcine Akirin2 gene according to human Akirin2 sequence, and then conformed it by experiments. Our results are as following.
1. We cloned porcine Arikin2 gene, and analyzed the characteristics and evolution progression of porcine Akirin2 by using bioinformatics software. Our results showed that porcine Akirin2 encoded 203 amino acids, and that the N and C-terminal of Arikin2 protein was highly conserved. There is similarity of 98% between porcine and human Akirin2.
2. We subcloned Akirin2 into prokaryotic expression vetor pET-32a, and induced and expressed it in E. coli BL21(DE3) using IPTG. SDS-PAGE analysis showed that we got fusion protein of 43ku. We selected the optional conditions for expressing His-Akirin2 fusion protein with final concentration of IPTG at 0.7 mmol/L for 6h. The fusion protein was purified by HiTrap Chelating HP.
3. We inserted porcine Akirin2 into eukaryotic expression vetor pEGFP-C1 which contains a green fluorescence reporter gene and transfected this plasmid into SUVECs (swine umbilical vein endothelial cells) by lipofectamine-2000. Fluorescence microscope showed that fusion protein EGFP-Akirin2 was at the best expression level at 24 hours post transfection. According to the fluorescence position of the cells, we concluded that porcine Akirin2 was localized to the nucleus. Western blot analysis confirmed the expression of EGFP-Akirin2 in the cells in vitro.
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