猪IFITM3基因PiggyBac载体构建及其在PK15细胞中的表达

发布时间：2018-05-25 16:19

本文选题：猪 + IFITM3基因　；参考：《河南农业大学》2015年硕士论文

【摘要】：干扰素是一种具有抗病毒、抑制细胞分裂、调节免疫和促进细胞凋亡等多种生物学活性的细胞因子,干扰素通过与其受体结合,诱导细胞内干扰素刺激基因(Interferon Stimulated Genes,ISGs)产生,表达相应的蛋白质从而完成多种生物学功能。干扰素诱导的跨膜蛋白3(Interferon-induced transmembrane protein 3,IFITM3)基因是一种重要的干扰素刺激基因。IFITM3具有调节细胞生长分化、细胞增殖和抵御病毒入侵等多种生物学功能。目前对于IFITM3基因的研究主要集中在人和鼠方面,着重于IFITM3基因在肿瘤组织和癌细胞中表达丰度的变化。有关猪IFITM3基因的研究集中在其抗病毒作用方面,具体机制尚不清楚。PiggyBac(PB)转座子能快速高效地将目的基因插入真核细胞基因组中,为在细胞中评价目的基因的功能提供了便利。本文旨在构建猪IFITM3基因的PB转座子重组载体,并初步在PK15细胞中对其表达进行评价,为进一步研究该基因在细胞水平的抗病毒机制奠定前期实验基础。利用RT-PCR方法从猪脾脏组织中对IFITM3基因ORF区进行克隆,分析IFITM3基因在猪各组织中的表达,以p ET-21b(+)为骨架构建原核重组表达载体,利用SDS-PAGE和Western Blot分析IFITM3基因在大肠杆菌中的融合表达,用PB转座子构建真核重组表达载体PB-IFITM3,将IFITM3基因转染整合到PK15细胞基因组中,QRT-PCR方法检测IFITM3等基因在PK15细胞中的mRNA表达水平,评价IFITM3基因过表达对LPS介导的炎性信号分子表达的影响。结果显示:克隆的猪IFITM3基因长度为438 bp,编码145个氨基酸;IFITM3基因在猪脾脏和肺脏中表达丰度较高;原核表达载体所表达的蛋白大小为22.3ku,其中一部分为可溶性蛋白,另一部分以包涵体形式存在;构建了PB-IFITM3真核重组表达载体,实现了IFITM3基因在PK15细胞中的过表达;QRT-PCR结果显示:IFITM3基因过表达后用LPS处理,能进一步上调IFITM3基因表达,且依赖于作用时间;TLR4、NFκB、IFN-α、IFN-β和TNF-α基因表达显著下调(P0.05),与LPS作用呈现时间依赖性;TBK1、p38 MAPK的基因表达水平略有下调,但在4h和8h时不明显,12h下调明显。提示IFITM3基因过表达能在一定程度上抑制LPS激活的TLR4信号通路。
[Abstract]:Interferon is a kind of cytokine with many biological activities, such as anti-virus, inhibiting cell division, regulating immunity and promoting cell apoptosis. Interferon induces the production of Interferon Stimulated genes by binding to its receptor. Expression of the corresponding protein to complete a variety of biological functions. IFITM3 is an important interferon stimulating gene. IFITM3 has many biological functions, such as regulating cell growth and differentiation, cell proliferation and resisting virus invasion. At present, the study of IFITM3 gene is mainly focused on human and mouse, focusing on the changes of the expression abundance of IFITM3 gene in tumor tissues and cancer cells. The research on porcine IFITM3 gene is focused on its antiviral effect. The specific mechanism is not clear. The transposon can rapidly and efficiently insert the target gene into the eukaryotic cell genome, which provides convenience for evaluating the function of the target gene in the cell. The aim of this study was to construct the recombinant vector of porcine IFITM3 gene, and to evaluate its expression in PK15 cells, so as to lay a foundation for further study on the antiviral mechanism of porcine IFITM3 gene at cell level. The ORF region of IFITM3 gene was cloned from porcine spleen tissue by RT-PCR method, and the expression of IFITM3 gene in pig tissues was analyzed. The prokaryotic expression vector was constructed with pET-21b () as the skeleton. The fusion expression of IFITM3 gene in Escherichia coli was analyzed by SDS-PAGE and Western Blot. The eukaryotic expression vector PB-IFITM3 was constructed by PB transposon. The IFITM3 gene was transfected into PK15 cell genome to detect the mRNA expression level of IFITM3 and other genes in PK15 cells. To evaluate the effect of overexpression of IFITM3 gene on the expression of inflammatory signaling molecules mediated by LPS. The results showed that the length of the cloned porcine IFITM3 gene was 438bp, encoding 145 amino acids, IFITM3 gene was highly expressed in the spleen and lung of pigs, and the protein expressed by the prokaryotic expression vector was 22.3ku. some of which were soluble proteins. In the other part, the eukaryotic expression vector of PB-IFITM3 was constructed, and the overexpression of IFITM3 gene in PK15 cells was realized by QRT-PCR. The results showed that the expression of IFITM3 gene could be upregulated further after the overexpression of IFITM3 gene was treated with LPS. The expression of IFN- 尾 and TNF- 伪 down-regulated the expression of TLR4N- 伪 -IFN- 尾 and TNF- 伪 in a time-dependent manner, and the expression level of TBK1p38 MAPK was down-regulated in a time-dependent manner with LPS, but it was not significantly down-regulated at 4h and 8h. These results suggest that overexpression of IFITM3 gene can inhibit the TLR4 signaling pathway activated by LPS to some extent.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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