本文關(guān)鍵詞：抗牛γ-干擾素單克隆抗體制備和鑒定　出處：《東北農(nóng)業(yè)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 牛γ-干擾素 單克隆抗體 干擾素檢測

【摘要】： γ-干擾素(IFN-γ)主要是在特定的抗原、有絲分裂原(ConA、PHA)的刺激下由活化T淋巴細(xì)胞或NK細(xì)胞產(chǎn)生的一類具有廣譜抗病毒、抑制細(xì)胞增殖和免疫調(diào)節(jié)等多種生物學(xué)活性的糖蛋白。自Cerretti于1986年首次克隆出乳牛IFN-γ基因后,重組牛IFN-γ(rBoIFN-γ)先后在原核表達(dá)系統(tǒng)、真核表達(dá)系統(tǒng)、巴斯德畢赤氏酵母系統(tǒng)、牛I型皰疹病毒載體(BHV-1)中成功獲得了表達(dá),并對其免疫學(xué)和生物學(xué)活性進(jìn)行了研究。國內(nèi)外關(guān)于牛IFN-γ的研究和應(yīng)用方面的工作已經(jīng)開展很多,但不論是對免疫細(xì)胞功能及機(jī)體細(xì)胞免疫應(yīng)答方面的研究,還是對疾病狀態(tài)下IFN-γ產(chǎn)生水平的判定,都需要一種簡便、快速的檢測手段。應(yīng)用牛IFN-γ單克隆抗體作為研究、檢測IFN-γ的手段,在免疫機(jī)制的研究、免疫功能檢測等領(lǐng)域具有廣泛的應(yīng)用。 本研究通過常規(guī)PCR方法從pMD18-T-BoIFN-γ質(zhì)粒中擴(kuò)增出BoIFN-γ成熟蛋白基因,并將其插入原核表達(dá)載體pET30a和pGEX-6p-1中,經(jīng)DNA測序確認(rèn)后,分別轉(zhuǎn)化入Rosetta(DE3)pLysS中,經(jīng)IPTG誘導(dǎo)分別表達(dá)出約18ku和42ku左右的融合蛋白。以純化的rHis-BoIFN-γ蛋白作為免疫原,免疫BALB/c小鼠,用純化的rGST-BoIFN-γ蛋白做為檢測抗原,應(yīng)用淋巴細(xì)胞雜交瘤技術(shù),通過間接ELISA方法進(jìn)行陽性雜交瘤細(xì)胞的篩選,共篩選出4株穩(wěn)定分泌抗rBoIFN-γ的單克隆抗體的細(xì)胞株,分別命名為3C9、3D10、2C12、5C4,免疫球蛋白類均為IgM,輕鏈為κ鏈;雜交瘤細(xì)胞株連續(xù)傳代15次以上及凍存后復(fù)蘇,細(xì)胞生長狀態(tài)良好,效價(jià)穩(wěn)定;腹水效價(jià)分別為1:51200、1:51200、1:12800、1:6400。Western blot結(jié)果表明,4株單抗均可特異性的識別rGST-BoIFN-γ蛋白,而不與GST標(biāo)簽蛋白反應(yīng)。間接ELISA試驗(yàn)證明,4株單抗只與rBoIFN-γ反應(yīng),而不與GST蛋白和rGoIFN-α、rGoIFN-γ、rBoIFN-α等細(xì)胞因子發(fā)生交叉反應(yīng)。間接免疫熒光結(jié)果表明,4株單抗均與BHK21細(xì)胞表達(dá)的rBoIFN-γ發(fā)生強(qiáng)熒光信號,進(jìn)一步證明了單抗的特異性。阻斷ELISA檢測結(jié)果表明3D10株單抗能夠與天然干擾素發(fā)生反應(yīng),證實(shí)了該單抗具有實(shí)際的應(yīng)用價(jià)值。本研究所制備的4株抗BoIFN-γ的單克隆抗體,為進(jìn)一步研究BoIFN-γ單抗在牛免疫學(xué)以及牛疫病診斷中的應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Interferon 緯 (IFN- 緯) 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- 緯 gene of dairy cattle was first cloned by Cerretti in 1986. The recombinant bovine IFN- 緯 rBoIFN- 緯) 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- 緯 have been studied. A lot of work has been done on the research and application of bovine IFN- 緯 at home and abroad. However, whether the study on immune cell function and cellular immune response, or to determine the level of IFN- 緯 production under the condition of disease, need a simple and convenient. Using bovine IFN- 緯 monoclonal antibody as research method, detecting IFN- 緯 is widely used in the research of immune mechanism and immune function test. In this study, BoIFN- 緯 mature protein gene was amplified from plasmid pMD18-T-BoIFN- 緯 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- 緯 protein was used as immunogen to immunize BALB/c mice. The purified rGST-BoIFN- 緯 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- 緯 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- 緯 protein but not react with GST tag protein. Indirect ELISA test showed that the four McAbs only reacted with rBoIFN- 緯. The results of indirect immunofluorescence showed that GST protein and rGoIFN- 偽 rGoIFN- 緯 rBoIFN- 偽 were not cross-reacted with other cytokines. The four McAbs showed strong fluorescence signal with rBoIFN- 緯 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- 緯 were prepared. It lays a foundation for further research on the application of BoIFN- 緯 monoclonal antibody in bovine immunology and the diagnosis of bovine epidemic disease.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 曹黎黎;穩(wěn)定分泌抗牛γ-干擾素雜交瘤細(xì)胞株的建立與鑒定[D];東北農(nóng)業(yè)大學(xué);2011年

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本文編號：1404046