本文關(guān)鍵詞： 單克隆抗體 純化 層析 乙肝核心抗原　出處：《浙江工業(yè)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：乙型肝炎是嚴(yán)重危害人類健康的病毒性傳染病,也是各種病毒性肝炎中對人類危害最大的疾病。在我國,乙肝表面抗原(HBsAg)陽性者約1.2億人,慢性乙型肝炎患者達(dá)3000多萬人,給社會和家庭造成嚴(yán)重負(fù)擔(dān),已成為當(dāng)今社會非常突出的重要問題。乙肝核心抗原(HBcAg)是乙肝病毒的核心部分,是判斷乙肝病毒復(fù)制和病程活動的重要指標(biāo),但其檢測所需的單克隆抗體主要依賴于國外進(jìn)口,價格昂貴。本實(shí)驗(yàn)制備了含抗HBcAg單克隆抗體的小鼠腹水,考察了各因素對抗體制備的影響,并采用不同方法進(jìn)行純化,通過比較與優(yōu)化,建立了良好的純化HBcAg單抗方法,為其臨床應(yīng)用于乙肝患者的治療和快速診斷奠定了基礎(chǔ),并為其它單克隆抗體的純化提供了一定的參考價值。主要實(shí)驗(yàn)結(jié)果如下: 1.弗氏不完全佐劑預(yù)處理的小鼠比石蠟預(yù)處理的小鼠腹水產(chǎn)量高:10～12周齡小鼠腹水產(chǎn)量高于6～8周齡小鼠;雌性小鼠腹水產(chǎn)量顯著高于雄性小鼠腹水產(chǎn)量。腹水經(jīng)辛酸-硫酸銨沉淀法純化后抗體純度大于硫酸銨沉淀法。 2.樣品以磷酸緩沖液(pH7.0,20 mmol/L PB+1.2 mol/L(NH_4)_2SO_4對倍稀釋后上樣于HiTrap Octyl FF預(yù)裝疏水層析柱,采用pH7.0,20 mmol/L PB+1.2 mol/L(NH_4)_2SO_4～pH7.0,20 mmol/L PB+0 mol/L(NH_4)_2SO_4,12CV梯度洗脫,然后依次用蒸餾水、0.5 mol/LNaOH、70%乙醇清洗介質(zhì),可獲得純度大于75%的mAb,抗體回收率達(dá)80%。 3.樣品以磷酸緩沖液(pH7.0,20 mmol/L PB+3 mol/L NaCl)對倍稀釋后上樣于HiTrap rProtein A FF預(yù)裝親和層析柱,pH3.0,0.1mol/LGIy-HCl一步洗脫,回收抗體的純度大于95%,抗體回收率達(dá)75%。 4.樣品以磷酸緩沖液(pH8.0,20 mmol/L PB+0.5 mol/L NaCl)對倍稀釋后上樣于HisTrap HP預(yù)裝金屬螯合親和層析柱(鎳柱),20mmol/L NaAC-HAC+0.5 mol/L NaCl(pH6.0、5.5、5.0、4.5、4.0)分步洗脫,可獲得純度大于85%的mAb,抗體回收率達(dá)80%。 上述疏水作用層析法、蛋白A親和層析法,金屬螯合親和層析法純化后抗體的生物學(xué)活性均沒有下降。
[Abstract]:Hepatitis B virus infectious disease is a serious hazard to human health, the most harmful disease to human and viral hepatitis. In China, hepatitis B surface antigen (HBsAg) positive about 120 million people, chronic hepatitis B patients up to about 30000000 people, to the family and society caused serious burden, has become an important problem Society is very prominent. The hepatitis B core antigen (HBcAg) is the core part of hepatitis B virus, is an important indicator of hepatitis B virus replication and disease activity, but its monoclonal antibody testing should mainly rely on foreign imports, the price is expensive. Ascites containing monoclonal antibodies against HBcAg were prepared in this experiment, the effects of various factors on the preparation of antibody, and purified by different methods, through comparison and optimization, establish a good method of purification of HBcAg monoclonal antibody, for the treatment of its clinical application in patients with hepatitis B and fast The diagnosis lays the foundation and provides some reference for the purification of other monoclonal antibodies. The main results are as follows:
1. incomplete Freund's adjuvant pretreated mice than paraffin pretreated Mice Ascites high yield: 10~12 week old mice ascites was higher than that of 6~8 week old mice; female mice ascites was significantly higher than that of male mice ascites. Ascites production by octanoic acid ammonium sulfate precipitation method of purified antibody purity was higher than ammonium sulfate precipitation.
2. samples with phosphate buffer (pH7.0,20 mmol/L PB+1.2 mol/L (NH_4) _2SO_4 of diluted sample in HiTrap Octyl FF with hydrophobic chromatography, using pH7.0,20 mmol/L PB+1.2 mol/L (NH_4) _2SO_4 ~ pH7.0,20 mmol/L PB+0 mol/L (NH_4) _2SO_4,12CV gradient elution, then followed by distilled water, 0.5 mol/LNaOH, 70% ethanol cleaning medium purity greater than 75% mAb, the rate of recovery was 80%.
The 3. sample was diluted with phosphoric acid buffer (pH7.0,20 mmol/L PB+3 mol/L NaCl), then it was added to HiTrap rProtein A FF preloaded affinity chromatography column and pH3.0,0.1mol/LGIy-HCl was eluted. The purity of the recovered antibody was more than 95%, and the recovery rate of the antibody reached to 75%..
4. samples with phosphate buffer (pH8.0,20 mmol/L PB+0.5 mol/L NaCl) on the diluted sample in HisTrap HP with metal chelate affinity chromatography (Nie Zhu), 20mmol/L NaAC-HAC+0.5 mol/L NaCl (pH6.0,5.5,5.0,4.5,4.0) step by step elution, the purity of more than 85% mAb, the rate of recovery was 80%.
The hydrophobic interaction chromatography, protein A affinity chromatography, and metal chelating affinity chromatography did not decrease the biological activity of the purified antibody.

【學(xué)位授予單位】：浙江工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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