鸡ISG12基因的克隆表达及抗病毒活性分析

发布时间：2018-06-15 16:27

本文选题：干扰素 + 干扰素刺激基因12　；参考：《山东农业大学》2015年硕士论文

【摘要】：动物的抗病毒作用主要通过机体的免疫系统产生的特异性免疫和非特异性免疫来实现。干扰素(Interferon,IFN)系统作为非特异性免疫系统的重要组成部分,是动物抗病毒感染的第一道防线,在先天性免疫和获得性免疫中起关键作用。IFN本身对病毒无灭活作用,它主要通JAK-STAT信号途径诱导IFN刺激基因(Instimulatedgene,ISG)的表达建立动物细胞的抗病毒状态,抑制病毒的复制和增殖。作为一种ISG,ISG12能受IFN-α诱导上调表达,它是一种新发现的先天免疫调节器,它能够调节抗炎细胞核受体,并在细胞中起着特定的作用,一些研究发现,ISG12基因可能参与了JAK/STAT信号途径的信号传递。ISG12基因在病毒感染过程中已经被监测到,但是针对其抗病毒活性的研究却很少。为了研究ISG12的抗病毒活性,根据已发表的ISG12蛋白的mRNA基因序列,设计一对特异性引物,采用反转录-聚合酶链式反应(RT-PCR)的方法从鸡胚成纤维细胞(CEF)中扩增一条分子量为352bp的特异性基因片段,经测序分析,该基因序列与Genbank中登录的禽属ISG12基因序列相似性为100%,证明已经成功扩增到正确的ISG12基因。将该基因亚克隆到原核表达载体pET-32a构建出重组表达质粒p32a-C12,工程菌p32a-C12/BL21经IPTG诱导表达,SDS-PAGE分析获得了分子量约30 ku的目的蛋白,重组蛋白经Ni2+琼脂糖凝胶柱层析纯化得到的单一的目的蛋白。将纯化的ISG12蛋白梯度稀释(50μg/mL、25μg/mL、12.5μg/mL)后处理CEF细胞12h,结果50μg/m L和25μg/mL的ISG12蛋白都会引起细胞凋亡,而12.5μg/m L的ISG12蛋白在眼观上对细胞没有影响。因此将ISG12蛋白稀释到高(12.5μg/mL)低(2.5μg/mL)两个浓度处理CEF细胞和鸡外周血淋巴细胞(Peripheral blood lymphocyte,PBL)12h,利用MTS法检测ISG12蛋白对细胞活性的影响。结果发现高浓度(12.5μg/mL)的ISG12蛋白能够显著降低PBL细胞的活性,而低浓度(2.5μg/mL)的ISG12蛋白能够显著提高两种细胞的活性。因此,我们得出结论,ISG12蛋白对细胞活力的影响具有剂量依赖性。以禽流感病毒(Avian influenza virus,AIV)1215株作为病毒模型进行抗病毒活性试验。试验一:将病毒感染ISG12蛋白预处理12h后的CEF细胞和PBL细胞;试验二:将ISG12蛋白与病毒混合后一起感作CEF细胞和PBL细胞。以管家基因为参照,利用荧光定量PCR检测病毒的载量。结果显示AIV 1215株在CEF细胞上的复制水平几乎没有差异,而AIV 1215株在PBL细胞上的复制水平显著降低。综上所述,我们得出结论,合适浓度的重组ISG12蛋白在体外能够抑制AIV 1215株在PBL细胞上的增殖但是在CEF细胞上却没有明显作用,这或许与ISG12蛋白抗病毒的机理有关,具体原因还有待进一步研究。
[Abstract]:The antiviral effect of animals is mainly realized by the specific immunity and non-specific immunity produced by the immune system. As an important part of the nonspecific immune system, IFNs are the first line of defense against virus infection in animals. IFNs play a key role in innate and acquired immunity. It mainly induces the expression of IFN- stimulated gene (ISG) through JAK-STAT signaling pathway to establish the antiviral state of animal cells and to inhibit the replication and proliferation of viruses. ISG- ISG12, a newly discovered innate immune regulator, can regulate anti-inflammatory nuclear receptors and play a specific role in cells, which can be up-regulated by IFN- 伪. Some studies have found that the ISG12 gene may be involved in the signal transduction of JAK / stat signaling pathway. ISG12 gene has been detected in the process of virus infection, but little research has been done on its antiviral activity. In order to study the antiviral activity of ISG12, a pair of specific primers were designed according to the published gene sequence of ISG12 protein. A specific gene fragment with molecular weight of 352bp was amplified from chicken embryo fibroblasts by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. The similarity between the sequence of ISG12 gene and that of Genbank is 100, which proves that the correct ISG12 gene has been amplified successfully. The gene was subcloned into prokaryotic expression vector pET-32a to construct the recombinant expression plasmid p32a-C12. The target protein with molecular weight of about 30 ku was obtained by IPTG induced expression of p32a-C12 / BL21. The recombinant protein was purified by Ni 2 agarose gel column chromatography. CEF cells were treated with 50 渭 g / mL of ISG12 protein and 25 渭 g / mL of ISG12 protein for 12 h. The results showed that 50 渭 g / mL of ISG12 protein and 25 渭 g / mL of ISG12 protein could induce apoptosis, but 12.5 渭 g / mL ISG12 protein had no effect on the cells. Therefore, the ISG12 protein was diluted to 12.5 渭 g 路mL ~ (-1) and 2.5 渭 g 路mL ~ (-1) for 12 h, and the effect of ISG12 protein on cell activity was detected by MTS method. The results showed that the ISG12 protein at high concentration (12.5 渭 g / mL) significantly decreased the activity of PBL cells, while the ISG12 protein at low concentration of 2.5 渭 g / mL significantly increased the activity of the two kinds of cells. Therefore, we conclude that the effect of ISG 12 protein on cell viability is dose-dependent. The antiviral activity of avian influenza virus strain Avian influenza virus1215 was tested. In experiment 1, CEF cells and PBL cells were pretreated with ISG12 protein for 12 h, and CEF cells and PBL cells were sensitized by mixing ISG12 protein with virus. The viral load was detected by fluorescent quantitative PCR using butler base as reference. The results showed that there was almost no difference in the replication level of AIV 1215 strain on CEF cells, while the replication level of AIV 1215 strain on PBL cells was significantly decreased. In conclusion, we conclude that the recombinant ISG12 protein at the appropriate concentration can inhibit the proliferation of AIV 1215 strain in vitro, but not in CEF cells, which may be related to the antiviral mechanism of ISG12 protein. The specific reasons need to be further studied.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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