全人源抗狂犬病病毒单克隆抗体的制备与鉴定

发布时间：2018-03-09 10:43

本文选题：人源IgM转基因小鼠　切入点：狂犬病病毒　出处：《南京医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：狂犬病是由狂犬病病毒(rabies virus, RV)感染而引发的人兽共患中枢神经系统传染病,是迄今为止人类唯一病死率高达100%的急性乙类传染病。狂犬病呈全球性分布,我国是受狂犬病危害最为严重的国家之一,位居全球第二位。流行病学调查显示,近年来我国被犬咬伤和死于狂犬病的患者人数逐年增加,已成为严重的公共卫生和社会问题。 WHO建议狂犬病三级暴露后的治疗包括接种狂犬疫苗和注射抗狂犬病免疫球蛋白。目前大多使用的两种抗狂犬病免疫球蛋白(rabies immunoglobulin, RIG)包括人抗狂犬病免疫球蛋白(human rabies immunoglobulin, HRIG)和马抗狂犬病免疫球蛋白(equine rabies immunoglobulin, ERIG),但血源性免疫球蛋白成本高且产量极低,难以满足预防用药需求,并且血源产品质量难以控制,存在潜在病毒再感染的风险以及容易发生过敏反应。全人源或人源化治疗性单克隆抗体药物为狂犬病接触后的预防和治疗提供了新的解决方法,以全人源治疗性单克隆抗体药物取代目前大量使用的血源性免疫球蛋白,已成为国内外临床医生和生物制药专家的共识。本课题组在国内首次采用从英国医学研究理事会(MRC)和剑桥大学引进的人源IgM转基因小鼠,结合杂交瘤技术及酶联免疫吸附(ELISA)高通量交叉筛选技术(HTS),制备全人源抗狂犬病病毒单克隆抗体,并对其生物学活性进行鉴定,为进一步研发人源抗狂犬病病毒免疫预防性中和抗体及其免疫治疗应用奠定基础。研究方法 1.以灭活狂犬病病毒CTN株作为抗原,采用皮下多点免疫及腹腔免疫法免疫人源IgM转基因小鼠。 2.采用杂交瘤技术结合酶联免疫吸附(ELISA)高通量交叉筛选技术(HTS)制备、筛选全人源抗狂犬病病毒杂交瘤细胞株。 3.采用硫酸铵沉淀法结合Protein L亲和层析柱纯化全人源抗狂犬病病毒单克隆抗体,SDS-PAGE鉴定单抗纯度,以及单抗重链、轻链分子量大小。通过双抗体夹心ELISA鉴定单抗的人源性和抗体类型。 4.采用间接ELISA、斑点杂交实验(Dot Blot)检测单抗的特异性,BiaCoreX-100测定单抗结合抗原的亲和力。 5.狂犬病病毒糖蛋白的表达及纯化,并通过Western Blot分析单抗与糖蛋白的结合特性。研究结果 1.建立了5株稳定分泌全人源抗狂犬病病毒单抗的杂交瘤细胞株,分别命名为6B2、8G2、9E3、9F2、16B1。 2.纯化5株全人源抗狂犬病病毒单抗,SDS-PAGA结果显示纯化后的单抗纯度较高,单抗重链和轻链分子量分别为75kDa、25kDa。双抗体夹心ELISA结果显示5株单抗均为人源性免疫球蛋白IgM类型的抗体。 3.间接ELISA、斑点杂交实验结果表明5株单抗均能特异性识别灭活狂犬病病毒CTN株。其中3株单抗8G2、9E3、9F2与狂犬病病毒CTN株抗原结合的亲和力分别为2.62×10-10M、2.62×10-10M、4.06×10-11M。 4.表达并纯化狂犬病病毒糖蛋白,Western Blot结果显示其中3株单抗8G2、9E3、9F2可与狂犬病病毒糖蛋白特异性结合。结论建立了5株稳定分泌全人源抗狂犬病病毒IgM单抗的杂交瘤细胞株,5株单抗均能特异性识别灭活狂犬病病毒CTN株,其中3株显示出较高的亲和力,并且能与狂犬病病毒糖蛋白特异性结合。
[Abstract]:Rabies is by rabies virus (rabies virus, RV) zoonotic infectious disease of central nervous system caused by infection, is by far the only human fatality rate of acute B infectious diseases as high as 100%. Rabies is global distribution, our country is affected by rabies harm is one of the most serious countries, ranked second in the world of epidemiology. Survey shows that in recent years China has been bitten by dogs and the number of patients died of rabies has increased year by year, has become a serious public health and social problems.
WHO suggested three level after exposure to rabies treatment including rabies vaccine and anti rabies immune globulin injection. Two kinds of anti rabies immune globulin currently mostly used (rabies immunoglobulin, RIG) including human anti rabies immune globulin (human rabies, immunoglobulin, HRIG) and horse anti rabies immune globulin (equine rabies immunoglobulin. ERIG), but the blood borne immune globulin of high cost and low yield, difficult to meet the needs of preventive medication, and blood product quality is difficult to control, there is a potential risk of infection and the virus more prone to allergic reactions.
Human or humanized monoclonal antibody drug therapy provides a new solution for the prevention and treatment of rabies after contact, instead of blood borne immune globulin is widely used in the whole human therapeutic monoclonal antibody drugs, has become the domestic and foreign clinicians and biopharmaceutical expert consensus.
The research group in the country for the first time from the UK Medical Research Council (MRC) and human IgM transgenic mice introduced by University of Cambridge, with hybridoma technique and enzyme-linked immunosorbent assay (ELISA) for high-throughput screening of cross technology (HTS), the preparation of human anti rabies virus monoclonal antibody, and to identify its the biological activity for further development of human anti rabies virus neutralizing antibodies and immune preventive immunotherapy to lay the foundation.
research method
1. using the inactivated rabies virus CTN strain as an antigen, the human IgM transgenic mice were immunized by subcutaneous multipoint immunization and intraperitoneal immunization.
2. hybridoma technique combined with enzyme linked immunosorbent assay (ELISA) high throughput cross screening (HTS) was used to screen all human hybridoma cell lines against rabies virus.
3., ammonium sulfate precipitation combined with Protein L affinity column was used to purify the whole human rabies virus monoclonal antibody. SDS-PAGE was used to identify the purity of McAb and the size of mAb heavy chain and light chain. The double antibody sandwich ELISA was used to identify the type of human antibody and monoclonal antibody.
4. the specificity of the monoclonal antibody was detected by indirect ELISA, dot blot hybridization (Dot Blot), and BiaCoreX-100 was used to determine the affinity of McAb binding antigen.
5. the expression and purification of glycoproteins of rabies virus and the binding properties of McAbs to glycoproteins were analyzed by Western Blot.
Research results
1. a hybridoma cell line, which secretes all human monoclonal antibodies against rabies virus, was established, named 6B2,8G2,9E3,9F2,16B1., respectively.
2. purified 5 human rabies virus monoclonal antibodies. SDS-PAGA showed that the purity of purified McAbs was high. The molecular weights of heavy chain and light chain of McAb were 75kDa and 25kDa. double antibody sandwich ELISA respectively. The results showed that all 5 mAbs were human immunoglobulin IgM type antibodies.
3. indirect ELISA and dot blot analysis showed that 5 McAbs could specifically identify inactivated rabies virus CTN strain, and the affinity of 3 McAb 8G2,9E3,9F2 to rabies virus CTN strain was 2.62 * 10-10M, 2.62 x 10-10M, 4.06 x 10-11M..
4. expression and purification of rabies virus glycoprotein, Western Blot results showed that 3 of the monoclonal antibody 8G2,9E3,9F2 could be specific binding to rabies virus glycoprotein.
conclusion
5 hybridoma cell lines secreting all human rabies virus IgM monoclonal antibody were established. 5 McAbs could specifically identify inactivated rabies virus CTN strain, 3 of them showed high affinity and could be specifically combined with rabies virus glycoprotein.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】