抗牛γ-干扰素单克隆抗体制备和鉴定

发布时间：2018-01-10 05:26

本文关键词：抗牛γ-干扰素单克隆抗体制备和鉴定　出处：《东北农业大学》2009年硕士论文　论文类型：学位论文

【摘要】： γ-干扰素(IFN-γ)主要是在特定的抗原、有丝分裂原(ConA、PHA)的刺激下由活化T淋巴细胞或NK细胞产生的一类具有广谱抗病毒、抑制细胞增殖和免疫调节等多种生物学活性的糖蛋白。自Cerretti于1986年首次克隆出乳牛IFN-γ基因后,重组牛IFN-γ(rBoIFN-γ)先后在原核表达系统、真核表达系统、巴斯德毕赤氏酵母系统、牛I型疱疹病毒载体(BHV-1)中成功获得了表达,并对其免疫学和生物学活性进行了研究。国内外关于牛IFN-γ的研究和应用方面的工作已经开展很多,但不论是对免疫细胞功能及机体细胞免疫应答方面的研究,还是对疾病状态下IFN-γ产生水平的判定,都需要一种简便、快速的检测手段。应用牛IFN-γ单克隆抗体作为研究、检测IFN-γ的手段,在免疫机制的研究、免疫功能检测等领域具有广泛的应用。本研究通过常规PCR方法从pMD18-T-BoIFN-γ质粒中扩增出BoIFN-γ成熟蛋白基因,并将其插入原核表达载体pET30a和pGEX-6p-1中,经DNA测序确认后,分别转化入Rosetta(DE3)pLysS中,经IPTG诱导分别表达出约18ku和42ku左右的融合蛋白。以纯化的rHis-BoIFN-γ蛋白作为免疫原,免疫BALB/c小鼠,用纯化的rGST-BoIFN-γ蛋白做为检测抗原,应用淋巴细胞杂交瘤技术,通过间接ELISA方法进行阳性杂交瘤细胞的筛选,共筛选出4株稳定分泌抗rBoIFN-γ的单克隆抗体的细胞株,分别命名为3C9、3D10、2C12、5C4,免疫球蛋白类均为IgM,轻链为κ链;杂交瘤细胞株连续传代15次以上及冻存后复苏,细胞生长状态良好,效价稳定;腹水效价分别为1:51200、1:51200、1:12800、1:6400。Western blot结果表明,4株单抗均可特异性的识别rGST-BoIFN-γ蛋白,而不与GST标签蛋白反应。间接ELISA试验证明,4株单抗只与rBoIFN-γ反应,而不与GST蛋白和rGoIFN-α、rGoIFN-γ、rBoIFN-α等细胞因子发生交叉反应。间接免疫荧光结果表明,4株单抗均与BHK21细胞表达的rBoIFN-γ发生强荧光信号,进一步证明了单抗的特异性。阻断ELISA检测结果表明3D10株单抗能够与天然干扰素发生反应,证实了该单抗具有实际的应用价值。本研究所制备的4株抗BoIFN-γ的单克隆抗体,为进一步研究BoIFN-γ单抗在牛免疫学以及牛疫病诊断中的应用奠定了基础。
[Abstract]:Interferon 纬 (IFN- 纬) is a kind of broad-spectrum anti-virus produced by activated T lymphocytes or NK cells stimulated by specific antigen, mitogen Cona PHAs. Glycoproteins that inhibit cell proliferation and immune regulation. The IFN- 纬 gene of dairy cattle was first cloned by Cerretti in 1986. The recombinant bovine IFN- 纬 rBoIFN- 纬) was successively used in prokaryotic expression system, eukaryotic expression system and Pichia pastoris system. Bovine herpesvirus type I (BHV-1) was successfully expressed. The immunological and biological activities of bovine IFN- 纬 have been studied. A lot of work has been done on the research and application of bovine IFN- 纬 at home and abroad. However, whether the study on immune cell function and cellular immune response, or to determine the level of IFN- 纬 production under the condition of disease, need a simple and convenient. Using bovine IFN- 纬 monoclonal antibody as research method, detecting IFN- 纬 is widely used in the research of immune mechanism and immune function test. In this study, BoIFN- 纬 mature protein gene was amplified from plasmid pMD18-T-BoIFN- 纬 by conventional PCR method. It was inserted into prokaryotic expression vectors pET30a and pGEX-6p-1, and then transformed into Rosetta(DE3)pLysS after confirmed by DNA sequencing. The fusion proteins of about 18 ku and 42 ku were induced by IPTG. The purified rHis-BoIFN- 纬 protein was used as immunogen to immunize BALB/c mice. The purified rGST-BoIFN- 纬 protein was used as the detection antigen and the lymphocyte hybridoma technique was used to screen the positive hybridoma cells by indirect ELISA method. Four cell lines secreting monoclonal antibodies against rBoIFN- 纬 were selected and named as 3C9, 3D10, 2C125C4, and immunoglobulins were all IgM. The light chain is 魏 chain; The hybridoma cell line was subcultured for more than 15 times and resuscitated after cryopreservation. The cell growth state was good and the titer was stable. The titer of ascites was 1: 51200g 1: 51200g 1: 12800 1: 6400.Western blot results showed. The four McAbs could specifically recognize rGST-BoIFN- 纬 protein but not react with GST tag protein. Indirect ELISA test showed that the four McAbs only reacted with rBoIFN- 纬. The results of indirect immunofluorescence showed that GST protein and rGoIFN- 伪 rGoIFN- 纬 rBoIFN- 伪 were not cross-reacted with other cytokines. The four McAbs showed strong fluorescence signal with rBoIFN- 纬 expressed in BHK21 cells. Further confirmed the specificity of the monoclonal antibody. The result of blocking ELISA showed that 3D10 McAb could react with natural interferon. Four monoclonal antibodies against BoIFN- 纬 were prepared. It lays a foundation for further research on the application of BoIFN- 纬 monoclonal antibody in bovine immunology and the diagnosis of bovine epidemic disease.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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