猪ISG43和ISG60蛋白的原核表达及多克隆抗体制备

发布时间：2018-02-09 07:48

本文关键词： ISG43 ISG60 蛋白表达多克隆抗体　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：ISG60属于IFIT家族成员,是一类干扰素诱导基因,在进化上高度保守。在干扰素、病毒、脂多糖等条件的刺激下,ISG60基因高效表达,主要作用于翻译的起始、病毒的复制、I型干扰素的反馈调节、细胞的迁移和增殖等。ISG43于1999年首次被Liu等发现并报道其去泛素化功能,又称为UBP43,是泛素特异性蛋白酶(UBP)家族成员。2002年,Malakhov等首次发现UBP43的去ISG化ISGylation功能,部分缺失UBP43基因的细胞会发生干扰素过敏导致细胞凋亡,由此可见ISG43是维持ISG化动态平衡的关键。本研究克隆了 ISG60和ISG43基因,将这些基因连接到pMD18-T载体,测序正确后再用Xho I和BamH I双酶切法插入PET-32a(+)克隆位点,成功构建了 PET-ISG60和PET-ISG43重组原核表达质粒。将重组质粒转化Rosetta(DE3)衍生菌并用不同浓度的IPTG进行诱导,经SDS-PAGE 和 Western-Blot 分析显示,ISG43 在 0.5 mM IPTG 诱导下表达最好,ISG60在0.05mMIPTG诱导下表达最好,ISG43和ISG60两者的重组蛋白都能以包涵体和可溶性蛋白两种形式进行表达。通过可溶性蛋白形式纯化蛋白获得有活性的免疫原,以纯化的ISG43和ISG60重组活性蛋白作为抗原与弗式佐剂一起免疫小鼠。经过四次免疫后获得了反应性和特异性较高的多克隆抗体。用Western Blot检测,产生的抗体能与原核表达蛋白发生反应,又能与PK-15细胞中经PolyI:C诱导产生的相应蛋白显示良好反应性。抗体在稀释比例为1:2000、1:4000、1:8000情况下都能与原核表达蛋白发生特异性反应。同样,抗体在稀释比例为1:600和1:1200时也能与PK-15细胞中表达的相应蛋白发生反应。
[Abstract]:ISG60, a member of the IFIT family, is a family of interferon-inducible genes, which is highly conserved in evolution. It is highly expressed under the stimulation of interferon, virus, lipopolysaccharide and plays an important role in the initiation of translation. The feedback regulation of interferon type I, cell migration and cell proliferation of virus replication. ISG43 was first discovered by Liu et al in 1999 and its deubiquitin function was reported. Also known as UBP43, UBP43 is a member of the UBP family of ubiquitin specific protease. In 2002, Malakhov et al first discovered the function of ISG free ISGylation in UBP43. Some cells with UBP43 gene deletion may develop interferon allergy and lead to apoptosis. In this study, ISG60 and ISG43 genes were cloned, ligated to pMD18-T vector, sequenced correctly, then inserted into PET-32a () cloning site by Xho I and BamH I double enzyme digestion. The recombinant prokaryotic expression plasmids of PET-ISG60 and PET-ISG43 were successfully constructed. The recombinant plasmids were transformed into Rosetta-DE3) derived bacteria and induced by different concentrations of IPTG. SDS-PAGE and Western-Blot analysis showed that ISG43 expressed best when induced by 0.5 mMIPTG. The recombinant protein of ISG43 and ISG60 could be expressed as inclusion body and soluble protein under the induction of 0.05 mMIPTG. The recombinant protein could be expressed in two forms: inclusion body and soluble protein. Form purified protein to obtain active immunogen, The purified ISG43 and ISG60 recombinant active proteins were used as antigens to immunize mice with Freund adjuvant. After four times of immunization, reactive and specific polyclonal antibodies were obtained. Western Blot was used to detect these antibodies. The antibody can react with prokaryotic expression protein and the corresponding protein induced by PolyI:C in PK-15 cells. The antibody can react specifically with prokaryotic expression protein when the dilution ratio is 1: 2000 / 1: 4 000 or 1: 8 000. At dilution ratios of 1: 600 and 1: 1200, antibodies also reacted with the corresponding proteins expressed in PK-15 cells.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4;Q78

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