人及小鼠干扰素诱导蛋白IFIT1的基因克

发布时间：2019-02-21 21:01

【摘要】：干扰素是一类由单核细胞和淋巴细胞产生的细胞因子,具有广谱抗病毒、抗菌、抗肿瘤以及调节机体免疫功能等多种生物活性,是构成机体先天免疫系统的重要成分。IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1)是第一个被发现的受干扰素调控基因表达的ISGs蛋白,能够通过多种方式参与并调控机体的生理活动。本研究选择人的IFIT1编码基因HIFIT1 (GenBank登录号：BC007091.1)及小鼠的IFIT1编码基因mifit1 (GenBank登录号：CT010226.1)进行克隆表达和制备抗体。HIFIT1基因的开放阅读框(ORF)为1437 bp,编码一条含478个氨基酸、分子量为55.36 kDa的多肽链。而mifit1基因ORF为1392 bp,编码一条含463个氨基酸、分子量为53.74 kDa的多肽链。提取人的293T细胞及鼠的3T3细胞总RNA后通过逆转录PCR分别扩增得到HIFIT1和mifit1完整的ORF,将这两个ORFs分别插入到表达载体pGEX-4T-1上形成重组质粒,转化入Escherichia coli DH5a细胞中,成功表达了重组的HIFIT1蛋白(rHIFIT1)和重组的MIFIT1蛋白(rMIFIT1),表观分子量分别约为81 kDa和79 kDa,与它们作为GST融合蛋白的理论预测值相符。用这两种重组蛋白分别腹腔注射免疫BALB/c小鼠和新西兰兔,利用间接酶联吸附试验(ELISA)测定免疫前后鼠血清及兔血清抗体水平的消长,结果表明3次免疫后所有免疫动物血清中特异性IgG抗体滴度均达100以上。通过蛋白免疫印迹(Western Blot)分析,结果表明rHIFIT1免疫鼠血清能够与293T细胞的天然HIFIT1蛋白相结合,rMIFIT1免疫兔血清能够识别3T3细胞的天然MIFIT1蛋白,从而显示rHIFIT1蛋白及rMIFIT1蛋白具有很好的免疫原性和反应原性,能够替代其相应的天然蛋白作抗原制备抗体。利用间接免疫荧光抗体实验(IFAT)技术分别检测天然HIFIT1蛋白在人293T细胞上的定位和MIFIT1蛋白在鼠3T3细胞上的定位显示天然HIFIT1蛋白和MIFIT1蛋白均主要分布在相应细胞的细胞质内。将经rHIFIT1蛋白免疫的BALB/c小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,通过多次筛选得到9株抗rHIFIT1的阳性杂交瘤细胞,并制备获得含有抗rHIFIT1抗体的小鼠腹水。本实验成功构建了HIFIT1和MIFIT1的原核表达系统,并制备了HIFIT1的多克隆抗体、单克隆抗体及MIFIT1多克隆抗体,促进了IFIT1蛋白分子生物学及免疫学方面的研究,为相关疾病的免疫诊断及免疫治疗奠定了基础。
[Abstract]:Interferon is a class of cytokines produced by monocytes and lymphocytes. It has many biological activities, such as broad-spectrum anti-virus, anti-bacterial, anti-tumor and regulating the immune function of the body. IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1) is the first known ISGs protein regulated by interferon, which can participate in and regulate the physiological activities of the body in many ways. In this study, human IFIT1 encoding gene HIFIT1 (GenBank accession number: BC007091.1) and mouse IFIT1 encoding gene mifit1 (GenBank accession number: CT010226.1) were selected for cloning, expression and preparation of antibodies. The open reading frame (ORF) of HIFIT1 gene was 1437 bp,. Encode a polypeptide chain containing 478 amino acids with a molecular weight of 55.36 kDa. Mifit1 gene ORF 1392 bp, encodes a polypeptide containing 463 amino acids with a molecular weight of 53.74 kDa. The total RNA of human 293T cells and mouse 3T3 cells were extracted, and then amplified by reverse transcription PCR. HIFIT1 and mifit1 complete ORF, were inserted into the expression vector pGEX-4T-1 to form recombinant plasmids, which were transformed into Escherichia coli DH5a cells. The recombinant HIFIT1 protein (rHIFIT1) and the recombinant MIFIT1 protein (rMIFIT1) were successfully expressed, with apparent molecular weights of about 81 kDa and 79 kDa, respectively, which were consistent with their theoretical predicted values as GST fusion proteins. BALB/c mice and New Zealand rabbits were immunized with these two recombinant proteins intraperitoneally respectively. The levels of antibody in serum and rabbit serum were measured by indirect enzyme-linked adsorption assay (ELISA) before and after immunization. The results showed that the titers of specific IgG antibody in serum of all immunized animals were above 100 after three times of immunization. Western blot (Western Blot) analysis showed that rHIFIT1 immunized rat serum could bind to the natural HIFIT1 protein of 293T cells, and rMIFIT1 immunized rabbit serum could recognize the natural MIFIT1 protein of 3T3 cells. The results showed that rHIFIT1 protein and rMIFIT1 protein had good immunogenicity and reactivity, and they could be used as antigens to prepare antibodies instead of their natural proteins. The localization of natural HIFIT1 protein on human 293T cells and the localization of MIFIT1 protein on mouse 3T3 cells by indirect immunofluorescence antibody assay (IFAT) showed that the natural HIFIT1 protein and MIFIT1 protein were mainly distributed in the cytoplasm of the corresponding cells. The spleen cells of BALB/c mice immunized with rHIFIT1 protein were fused with SP2/0 cells of mouse myeloma cells. Nine strains of anti-rHIFIT1 hybridoma cells were obtained by multiple screening, and ascites containing anti-rHIFIT1 antibody were prepared. The prokaryotic expression systems of HIFIT1 and MIFIT1 were successfully constructed, and the polyclonal antibody, monoclonal antibody and MIFIT1 polyclonal antibody of HIFIT1 were prepared, which promoted the study of molecular biology and immunology of IFIT1 protein. It lays a foundation for immune diagnosis and immunotherapy of related diseases.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392

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