鸡IL-18和IFN-γ原核表达及抗IBDV活性分析

发布时间：2018-07-15 14:06

【摘要】：禽病毒性疾病如禽传染性法氏囊病(Infectious Bursal Disease,IBD)一直困扰着养禽业的健康、快速发展。化学药物对病毒的抑制效果不明显,而且还容易产生耐药性和药物残留,新型抗病毒化学药物的开发严重滞后;尽管多数禽类病毒性疾病可用疫苗进行免疫预防,但效果不是都很理想,且在疫苗的使用过程中,一些免疫力低下的个体容易诱发严重的疾病,出现免疫应答不平衡等现象。因此,有必要开展新型细胞因子制剂的研究。它可以从增强免疫力,抗病毒活性等多方面达到预期的效果。已证明,鸡白细胞介素18(Chicken interleukin-18,ChIL-18)和鸡干扰素γ(Chicken interferon,ChIFN-γ)对多种病毒感染具有抑制作用。因此,有必要研究重组蛋白ChIL-18和ChIFN-γ以及二者混合后的抗病毒效果及其作用机制。本研究首先对ChIL-18和ChIFN-γ的全蛋白基因进行分析,设计引物后分别扩增ChIL-18和ChIFN-γ基因并将该基因插入pET28a原核表达载体,构建重组质粒。将该质粒转化到大肠杆菌BL21中,经IPTG诱导表达ChIL-18和ChIFN-γ蛋白,经纯化后进行生物活性检测。为了鉴定ChIL-18和ChIFN-γ蛋白的抗病毒活性,我们将200 ng/ml ChIL-18和ChIFN-γ分别或共同接种到鸡胚胎成纤维细胞中。24小时后,用2.5 MOI的IBDV感染鸡胚成纤维细胞。结果表明,联合组的抗病毒效果优于ChIL-18或ChIFN-γ组。为了研究蛋白在鸡体内的抗病毒效果,我们将200μg ChIL-18和ChIFN-γ分别或共同注射14日龄雏鸡,同时用PBS作阴性对照,24小时后再次注射,1天后对雏鸡进行7×104PFU的IBDV攻毒,检测鸡法氏囊组织的病毒复制水平以及病理变化。结果表明,ChIL-18和ChIFN-γ可以抵抗IBDV,特别是共同作用组的IBDV增殖受到明显的抑制,表明混合组具有更好的抗病毒效果。蛋白注射雏鸡后7天内,应用试剂盒检测蛋白在体外刺激脾脏淋巴细胞增殖及其分泌的细胞因子和体内血清中细胞因子的变化情况。结果表明,混合组蛋白可促进鸡脾脏淋巴细胞增殖并分泌高水平IL-6,IL-12及IL-17,与PBS组比差异极显著,而IL-4和IL-10的水平变化不大,血清中的细胞因子变化情况与淋巴细胞中相似。表明混合组诱导的细胞免疫作用优于ChIL-18和ChIFN-γ组。利用鸡外周血巨噬细胞分析ChIL-18和ChIFN-γ对细胞内几种Toll样受体(Toll-like receptors,TLR)及下游信号通路、主要组织相容性复合物(majorhistocompatibility complex,MHC)、NO的影响,结果混合组蛋白可以诱导巨噬细胞中TLR2、TLR 3、TLR 4和MHC-II的表达量升高,MAPK和NFκB信号通路被激活,NO的分泌水平显著升高,这些结果表明混合组蛋白具有更好的抗病毒能力。综上结果表明,ChIL-18和ChIFN-γ以及二者协同可诱导机体对病毒感染产生有效的防御作用,为新型细胞因子制剂的研究提供参考。
[Abstract]:Avian viral diseases such as infectious bursal disease (IBD) have been puzzling the healthy and rapid development of poultry industry. The inhibitory effect of chemical drugs on virus is not obvious, and it is easy to produce drug resistance and drug residues. The development of new antiviral chemical drugs is seriously delayed; although most avian viral diseases can be immunized with vaccines, However, the effect is not very good, and in the use of vaccine, some individuals with low immunity are prone to induce serious diseases, such as imbalance of immune response. Therefore, it is necessary to develop new cytokine preparations. It can enhance immunity, antiviral activity and other aspects to achieve the desired results. It has been proved that Chicken Interleukin-18 (ChIL-18) and Chicken Interferon- 纬 (ChIFN- 纬) can inhibit the infection of many viruses. Therefore, it is necessary to study the antiviral effect and mechanism of recombinant protein ChIL-18 and ChIFN- 纬. ChIL-18 and ChIFN- 纬 genes were amplified by primer design and inserted into prokaryotic expression vector pET28a to construct recombinant plasmid. The plasmid was transformed into Escherichia coli BL21 and induced by IPTG to express ChIL-18 and ChIFN- 纬 proteins. In order to identify the antiviral activity of ChIL-18 and ChIFN- 纬 proteins, we inoculated 200 ng/ml ChIL-18 and ChIFN- 纬 into chicken embryonic fibroblasts for .24 hours, then infected chicken embryonic fibroblasts with 2.5 moi IBDV. The results showed that the antiviral effect of the combined group was better than that of the ChIL-18 or ChIFN- 纬 group. In order to study the antiviral effect of ChIL-18 and ChIFN- 纬, 200 渭 g ChIL-18 and ChIFN- 纬 were injected into 14-day-old chicks, respectively, and PBS was used as negative control for 24 hours. The viral replication and pathological changes in the bursa of chicken were detected. The results showed that ChIL-18 and ChIFN- 纬 could resist the proliferation of IBDV, especially in the co-acting group, which indicated that the mixed group had better antiviral effect. The proliferation and secretion of cytokines and cytokines in serum of spleen lymphocytes stimulated by protein in vitro were detected by kit within 7 days after injection of chicken protein. The results showed that the mixed histone could promote the proliferation and secretion of IL-6, IL-12 and IL-17 in spleen lymphocytes, which was significantly different from that in PBS group, but the levels of IL-4 and IL-10 did not change. The changes of cytokines in serum were similar to those in lymphocytes. The results showed that the cellular immunity induced by the mixed group was better than that of the ChIL-18 and ChIFN- 纬 groups. The effects of ChIL-18 and ChIFN- 纬 on several Toll-like receptors (TLRs) and their downstream signaling pathways, majorhistocompatibility complexes (MHCs) and nitric oxide (no) in chicken peripheral blood macrophages were analyzed. Results the mixed histone could induce the expression of TLR2TLR3TLR4 and MHC-II in macrophages. The MAPK and NF 魏 B signaling pathway were activated to increase the level of no secretion. These results indicated that the mixed histone had better antiviral ability. The results show that ChIL-18, ChIFN- 纬 and their synergism can induce effective defense against virus infection, and provide a reference for the study of novel cytokines.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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