丹顶鹤α干扰素的克
发布时间：2018-10-07 17:38
【摘要】：α[干扰素(Interferon-α, IFN-α)是机体白细胞产生的一种细胞因子,具有抗病毒繁殖、抗肿瘤生长和免疫调节等多种生物学功能,是机体防御系统的重要组成部分。近些年来,动物干扰素的研究主要集中于经济动物,并取得了一定进展,目前已有商品化的鸡、犬、猪重组干扰素产品在市场上应用。迄今为止,国内外尚未有关于丹顶鹤(Grus japonensis)干扰素方面的相关报道。丹顶鹤是大型涉禽,在湿地环境中属于食物链的上层,是湿地生物多样性的关键物种,被称为“湿地之神”。我国是世界上丹顶鹤栖息的主要国家,国家林业局已经把丹顶鹤作为唯一的国鸟候选鸟上报国务院,是国家一级保护动物。近年来,由于环境变迁和人为干扰,禽流感、新城疫、马立克氏疾病时常暴发,野生丹顶鹤数量逐年下降,全球现存数量仅为2600只左右,而东亚的大陆种仅1500只左右。本研究利用简并引物和基因组步移PCR技术首次克隆出丹顶鹤α干扰素(crIFN-a)基因,该基因全长741 bp,编码246个氨基酸,与哺乳动物IFN-a的同源性小于45%,与禽IFN-a基因的同源性在60～80%之间,其中与鹦鹉的亲缘关系最近,同源性高达80.7%。根据表达载体的酶切位点设计crIFN-a成熟肽基因表达引物,目的基因亚克隆到原核表达载体pET32a(+)中,在大肠杆菌BL21中进行表达。经SDS-PAGE电泳分析,crIFN-α的融合蛋白主要以包涵体的形式存在,大小约43KDa。该包涵体经纯化、透析复性处理后加到鸡胚成纤维细胞上,而后进行水泡性口炎病毒(VSV)攻击,结果发现重组crIFN-α可以抑制水泡性口炎病毒的复制,生物活性最高时达到1.87x105U/mg；被兔抗crIFN-α多克隆抗体中和后的重组crIFN-α将不再具备抗病毒功能。间接免疫荧光结果表明,crIFN-α基因也可在鸡胚成纤维细胞的细胞质中得到表达。此外,我们在克隆crIFN-α的过程中曾设计了2对简并引物(AIF-TY1a和AIF-TY2b, POAU和AIF-TY2b),其应用结果表明,AIF-TY1a和AIF-TY2b在扩增禽IFN-α时更为特异,为克隆禽α干扰素未知序列提供了重要参考。通过本研究,我们首次获得了crIFN-α基因,在大肠杆菌和鸡胚成纤维细胞中表达相应蛋白,并对原核表达的重组蛋白进行了抗病毒活性分析,为基因工程干扰素的开发提供了理论依据,为丹顶鹤干扰素的工业化生产奠定了基础。
[Abstract]:Interferon- 伪 (IFN- 伪) is a cytokine produced by human leukocytes. It has many biological functions, such as anti-virus reproduction, anti-tumor growth and immunomodulation, and is an important part of the body's defense system. In recent years, the research of animal interferon is mainly focused on economic animals, and has made some progress. At present, the commercial recombinant interferon products of chicken, dog and pig have been used in the market. Up to now, there are no reports about (Grus japonensis) interferon in red crowned cranes at home and abroad. Red crowned crane is a large wading bird, which belongs to the upper layer of food chain in wetland environment and is the key species of wetland biodiversity. It is called "God of Wetlands". China is the main country where red-crowned cranes inhabit in the world. The State Forestry Administration has reported the red-crowned crane as the only candidate bird to the State Council, which is a national first-class protected animal. In recent years, avian influenza, Newcastle disease and Marek's disease often break out due to environmental changes and human disturbance. The number of wild red-crowned cranes is decreasing year by year, and the number of existing cranes in the world is only about 2600, while the continental species in East Asia are only about 1500. In this study, interferon alpha (crIFN-a) gene was first cloned by degenerate primers and genomic step PCR technique. The full-length 741 bp, gene encoded 246 amino acids. The homology with mammalian IFN-a was less than 45%, and the homology with avian IFN-a gene was between 60% and 80%. CrIFN-a mature peptide gene expression primers were designed according to the restriction site of the expression vector. The target gene was subcloned into prokaryotic expression vector pET32a () and expressed in Escherichia coli BL21. The fusion protein of crIFN- 伪 was mainly in the form of inclusion body by SDS-PAGE electrophoresis, and the size of the fusion protein was about 43KDa. The inclusion body was purified, treated with hemodialysis and added to chicken embryo fibroblasts, and then attacked with blistering stomatitis virus (VSV). The results showed that recombinant crIFN- 伪 could inhibit the replication of vesicular stomatitis virus, and the biological activity reached 1.87 x 105 U / mg. The recombinant crIFN- 伪 neutralized by rabbit anti crIFN- 伪 polyclonal antibody will no longer have anti viral function. The results of indirect immunofluorescence showed that crIFN- 伪 gene could also be expressed in the cytoplasm of chicken embryo fibroblasts. In addition, two pairs of degenerate primers (AIF-TY1a, AIF-TY2b, POAU and AIF-TY2b) were designed in the process of cloning crIFN- 伪. The results showed that AIF-TY1a and AIF-TY2b were more specific in the amplification of avian IFN- 伪, which provided an important reference for cloning the unknown sequence of avian interferon 伪. In this study, we obtained the crIFN- 伪 gene for the first time, expressed the corresponding protein in E. coli and chicken embryo fibroblasts, and analyzed the antiviral activity of the recombinant protein expressed in E. coli and chicken embryo fibroblasts. It provides a theoretical basis for the development of genetic engineering interferon and lays a foundation for the industrial production of red crowned crane interferon.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.93
本文编号：2255071
[Abstract]:Interferon- 伪 (IFN- 伪) is a cytokine produced by human leukocytes. It has many biological functions, such as anti-virus reproduction, anti-tumor growth and immunomodulation, and is an important part of the body's defense system. In recent years, the research of animal interferon is mainly focused on economic animals, and has made some progress. At present, the commercial recombinant interferon products of chicken, dog and pig have been used in the market. Up to now, there are no reports about (Grus japonensis) interferon in red crowned cranes at home and abroad. Red crowned crane is a large wading bird, which belongs to the upper layer of food chain in wetland environment and is the key species of wetland biodiversity. It is called "God of Wetlands". China is the main country where red-crowned cranes inhabit in the world. The State Forestry Administration has reported the red-crowned crane as the only candidate bird to the State Council, which is a national first-class protected animal. In recent years, avian influenza, Newcastle disease and Marek's disease often break out due to environmental changes and human disturbance. The number of wild red-crowned cranes is decreasing year by year, and the number of existing cranes in the world is only about 2600, while the continental species in East Asia are only about 1500. In this study, interferon alpha (crIFN-a) gene was first cloned by degenerate primers and genomic step PCR technique. The full-length 741 bp, gene encoded 246 amino acids. The homology with mammalian IFN-a was less than 45%, and the homology with avian IFN-a gene was between 60% and 80%. CrIFN-a mature peptide gene expression primers were designed according to the restriction site of the expression vector. The target gene was subcloned into prokaryotic expression vector pET32a () and expressed in Escherichia coli BL21. The fusion protein of crIFN- 伪 was mainly in the form of inclusion body by SDS-PAGE electrophoresis, and the size of the fusion protein was about 43KDa. The inclusion body was purified, treated with hemodialysis and added to chicken embryo fibroblasts, and then attacked with blistering stomatitis virus (VSV). The results showed that recombinant crIFN- 伪 could inhibit the replication of vesicular stomatitis virus, and the biological activity reached 1.87 x 105 U / mg. The recombinant crIFN- 伪 neutralized by rabbit anti crIFN- 伪 polyclonal antibody will no longer have anti viral function. The results of indirect immunofluorescence showed that crIFN- 伪 gene could also be expressed in the cytoplasm of chicken embryo fibroblasts. In addition, two pairs of degenerate primers (AIF-TY1a, AIF-TY2b, POAU and AIF-TY2b) were designed in the process of cloning crIFN- 伪. The results showed that AIF-TY1a and AIF-TY2b were more specific in the amplification of avian IFN- 伪, which provided an important reference for cloning the unknown sequence of avian interferon 伪. In this study, we obtained the crIFN- 伪 gene for the first time, expressed the corresponding protein in E. coli and chicken embryo fibroblasts, and analyzed the antiviral activity of the recombinant protein expressed in E. coli and chicken embryo fibroblasts. It provides a theoretical basis for the development of genetic engineering interferon and lays a foundation for the industrial production of red crowned crane interferon.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.93
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