抗乙肝核心抗原单克隆抗体分离纯化的研究

发布时间：2018-03-01 00:08

本文关键词： 单克隆抗体纯化层析乙肝核心抗原　出处：《浙江工业大学》2009年硕士论文　论文类型：学位论文

【摘要】：乙型肝炎是严重危害人类健康的病毒性传染病,也是各种病毒性肝炎中对人类危害最大的疾病。在我国,乙肝表面抗原(HBsAg)阳性者约1.2亿人,慢性乙型肝炎患者达3000多万人,给社会和家庭造成严重负担,已成为当今社会非常突出的重要问题。乙肝核心抗原(HBcAg)是乙肝病毒的核心部分,是判断乙肝病毒复制和病程活动的重要指标,但其检测所需的单克隆抗体主要依赖于国外进口,价格昂贵。本实验制备了含抗HBcAg单克隆抗体的小鼠腹水,考察了各因素对抗体制备的影响,并采用不同方法进行纯化,通过比较与优化,建立了良好的纯化HBcAg单抗方法,为其临床应用于乙肝患者的治疗和快速诊断奠定了基础,并为其它单克隆抗体的纯化提供了一定的参考价值。主要实验结果如下: 1.弗氏不完全佐剂预处理的小鼠比石蜡预处理的小鼠腹水产量高:10～12周龄小鼠腹水产量高于6～8周龄小鼠;雌性小鼠腹水产量显著高于雄性小鼠腹水产量。腹水经辛酸-硫酸铵沉淀法纯化后抗体纯度大于硫酸铵沉淀法。 2.样品以磷酸缓冲液(pH7.0,20 mmol/L PB+1.2 mol/L(NH_4)_2SO_4对倍稀释后上样于HiTrap Octyl FF预装疏水层析柱,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梯度洗脱,然后依次用蒸馏水、0.5 mol/LNaOH、70%乙醇清洗介质,可获得纯度大于75%的mAb,抗体回收率达80%。 3.样品以磷酸缓冲液(pH7.0,20 mmol/L PB+3 mol/L NaCl)对倍稀释后上样于HiTrap rProtein A FF预装亲和层析柱,pH3.0,0.1mol/LGIy-HCl一步洗脱,回收抗体的纯度大于95%,抗体回收率达75%。 4.样品以磷酸缓冲液(pH8.0,20 mmol/L PB+0.5 mol/L NaCl)对倍稀释后上样于HisTrap HP预装金属螯合亲和层析柱(镍柱),20mmol/L NaAC-HAC+0.5 mol/L NaCl(pH6.0、5.5、5.0、4.5、4.0)分步洗脱,可获得纯度大于85%的mAb,抗体回收率达80%。上述疏水作用层析法、蛋白A亲和层析法,金属螯合亲和层析法纯化后抗体的生物学活性均没有下降。
[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.

【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】