發(fā)布時(shí)間：2018-04-26 02:28

  本文選題：豬紅細(xì)胞CR1-like + 單克隆抗體�。� 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：目的：為進(jìn)一步研究動物紅細(xì)胞CR1天然分布狀態(tài)及其功能調(diào)控機(jī)制,本試驗(yàn)以豬為研究對象,制備豬CR1-like單克隆抗體,應(yīng)用該抗體對CR1-like分子在豬紅細(xì)胞膜表面的分布進(jìn)行深入研究,以期為課題組進(jìn)一步研究動物紅細(xì)胞免疫功能的分子機(jī)制提供理論數(shù)據(jù)和技術(shù)手段。方法：運(yùn)用Gene ious軟件分析豬CR1-like基因cDNA序列的生物信息學(xué)特征,合成免疫抗原肽段；采用單克隆抗體制備技術(shù)建立分泌豬CR1-like單克隆抗體的小鼠雜交瘤細(xì)胞,以親和層析、中壓蛋白純化的技術(shù)純化所分泌的單克隆抗體；通過SDS-PAGE方法對所產(chǎn)單抗的蛋白特性及純度進(jìn)行鑒定,并應(yīng)用間接免疫熒光技術(shù)研究了該單抗的免疫活性。利用該抗體,應(yīng)用激光共聚焦顯微鏡觀察天然豬紅細(xì)胞CR1-like膜分布狀態(tài)；人工制備免疫復(fù)合物C3b-beads為報(bào)告因子,以流式細(xì)胞儀檢測膜流動性變化對豬紅細(xì)胞CR1-like發(fā)揮免疫粘附功能的影響。結(jié)果：獲得了分泌豬CR1-like單克隆抗體的小鼠雜交瘤細(xì)胞株,并成功生產(chǎn)、純化出小鼠抗豬CR1-like單克隆抗體；應(yīng)用所產(chǎn)單抗間接免疫熒光染色豬紅細(xì)胞,于熒光顯微鏡下觀察,豬紅細(xì)胞表面可見綠色熒光；激光共聚焦顯微鏡觀察豬CR1-like分布狀態(tài),經(jīng)膜預(yù)固定處理的豬紅細(xì)胞表面其綠色熒光分布成片狀；相比之下,未經(jīng)膜固定處理的豬紅細(xì)胞表面其熒光點(diǎn)呈顆粒狀分布,較膜固定組分布明顯成簇化。流式細(xì)胞儀檢測發(fā)現(xiàn),膜固定組平均熒光強(qiáng)度值為302.35,未固定組為638.44,陰性對照組為4.7,統(tǒng)計(jì)得：膜固定組、未固定組均明顯高于陰性對照組,差異極顯著(P0.01),同時(shí)未固定組明顯高于膜固定組,差異極顯著(P0.01)。結(jié)論：本研究成功獲得可穩(wěn)定傳代生產(chǎn)豬CR1-like單克隆抗體的雜交瘤細(xì)胞株(型號：CRT-2 CL-5):生產(chǎn)、純化出具有生物活性的豬CR1-like單克隆抗體；天然狀態(tài)下豬紅細(xì)胞表面的CR1-like分布呈分散狀態(tài),發(fā)揮免疫功能時(shí),CR1-like表現(xiàn)出多價(jià)高效的結(jié)合特性,其分布狀態(tài)表現(xiàn)為顆粒樣成簇分布；紅細(xì)胞膜流動性是影響豬紅細(xì)胞CR1-like發(fā)揮免疫粘附功能的重要生理基礎(chǔ)。
[Abstract]:Objective: to further study the natural distribution of CR1 in animal erythrocytes and its functional regulation mechanism, porcine monoclonal antibodies against CR1-like were prepared in this study. The distribution of CR1-like molecules on porcine erythrocyte membrane was studied by using this antibody in order to provide theoretical data and technical means for further study on the molecular mechanism of animal erythrocyte immune function. Methods: Gene ious software was used to analyze the bioinformatics characteristics of porcine CR1-like gene cDNA sequence, to synthesize the immune antigen peptide, and to establish murine hybridoma cells secreting porcine CR1-like monoclonal antibody by affinity chromatography. The monoclonal antibody was purified by the technique of medium pressure protein purification, the protein characteristics and purity of the monoclonal antibody were identified by SDS-PAGE method, and the immune activity of the monoclonal antibody was studied by indirect immunofluorescence technique. The distribution of CR1-like membrane in natural porcine red blood cells was observed by laser confocal microscopy, and the immune complex C3b-beads was prepared as a reporter factor. The effect of membrane fluidity on immune adherence of porcine erythrocyte CR1-like was detected by flow cytometry. Results: murine hybridoma cell lines secreting monoclonal antibodies to porcine CR1-like were obtained, and mouse monoclonal antibodies against porcine CR1-like were successfully produced and purified. Green fluorescence was observed on the surface of porcine red blood cells. The distribution of porcine CR1-like was observed by laser confocal microscopy. The fluorescence points on the surface of porcine red blood cells treated without membrane fixation showed granular distribution, which was more obvious than that of membrane fixation group. Flow cytometry showed that the average fluorescence intensity of membrane fixation group was 302.35, that of unfixed group was 638.44, and that of negative control group was 4.7. The statistical results showed that the average fluorescence intensity of membrane fixation group was significantly higher than that of negative control group. The difference was very significant (P 0.01), and the difference was significantly higher in the group without fixation than in the group of membrane fixation, and the difference was very significant (P 0.01). Conclusion: the hybridoma cell line (type 1: CRT-2 CL-5), which can be used to produce monoclonal antibody against porcine CR1-like, was successfully obtained in this study. The monoclonal antibody against porcine CR1-like with biological activity was purified from the hybridoma cell line (type 1: CRT-2 CL-5). The distribution of CR1-like on the surface of porcine red blood cells was dispersed in natural state, and the distribution of CR1-like showed multivalent and efficient binding characteristics, and the distribution of CR1-like was granulocylike cluster distribution. Erythrocyte membrane fluidity is an important physiological basis to affect the immune adherence function of porcine erythrocyte CR1-like.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.4

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本文編號：1804085