本文選題：人源IgM轉(zhuǎn)基因小鼠　切入點：狂犬病病毒　出處：《南京醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：狂犬病是由狂犬病病毒(rabies virus, RV)感染而引發(fā)的人獸共患中樞神經(jīng)系統(tǒng)傳染病,是迄今為止人類唯一病死率高達100%的急性乙類傳染病。狂犬病呈全球性分布,我國是受狂犬病危害最為嚴重的國家之一,位居全球第二位。流行病學調(diào)查顯示,近年來我國被犬咬傷和死于狂犬病的患者人數(shù)逐年增加,已成為嚴重的公共衛(wèi)生和社會問題。 WHO建議狂犬病三級暴露后的治療包括接種狂犬疫苗和注射抗狂犬病免疫球蛋白。目前大多使用的兩種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)和馬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產(chǎn)量極低,難以滿足預(yù)防用藥需求,并且血源產(chǎn)品質(zhì)量難以控制,存在潛在病毒再感染的風險以及容易發(fā)生過敏反應(yīng)。 全人源或人源化治療性單克隆抗體藥物為狂犬病接觸后的預(yù)防和治療提供了新的解決方法,以全人源治療性單克隆抗體藥物取代目前大量使用的血源性免疫球蛋白,已成為國內(nèi)外臨床醫(yī)生和生物制藥專家的共識。 本課題組在國內(nèi)首次采用從英國醫(yī)學研究理事會(MRC)和劍橋大學引進的人源IgM轉(zhuǎn)基因小鼠,結(jié)合雜交瘤技術(shù)及酶聯(lián)免疫吸附(ELISA)高通量交叉篩選技術(shù)(HTS),制備全人源抗狂犬病病毒單克隆抗體,并對其生物學活性進行鑒定,為進一步研發(fā)人源抗狂犬病病毒免疫預(yù)防性中和抗體及其免疫治療應(yīng)用奠定基礎(chǔ)。 研究方法 1.以滅活狂犬病病毒CTN株作為抗原,采用皮下多點免疫及腹腔免疫法免疫人源IgM轉(zhuǎn)基因小鼠。 2.采用雜交瘤技術(shù)結(jié)合酶聯(lián)免疫吸附(ELISA)高通量交叉篩選技術(shù)(HTS)制備、篩選全人源抗狂犬病病毒雜交瘤細胞株。 3.采用硫酸銨沉淀法結(jié)合Protein L親和層析柱純化全人源抗狂犬病病毒單克隆抗體,SDS-PAGE鑒定單抗純度,以及單抗重鏈、輕鏈分子量大小。通過雙抗體夾心ELISA鑒定單抗的人源性和抗體類型。 4.采用間接ELISA、斑點雜交實驗(Dot Blot)檢測單抗的特異性,BiaCoreX-100測定單抗結(jié)合抗原的親和力。 5.狂犬病病毒糖蛋白的表達及純化,并通過Western Blot分析單抗與糖蛋白的結(jié)合特性。 研究結(jié)果 1.建立了5株穩(wěn)定分泌全人源抗狂犬病病毒單抗的雜交瘤細胞株,分別命名為6B2、8G2、9E3、9F2、16B1。 2.純化5株全人源抗狂犬病病毒單抗,SDS-PAGA結(jié)果顯示純化后的單抗純度較高,單抗重鏈和輕鏈分子量分別為75kDa、25kDa。雙抗體夾心ELISA結(jié)果顯示5株單抗均為人源性免疫球蛋白IgM類型的抗體。 3.間接ELISA、斑點雜交實驗結(jié)果表明5株單抗均能特異性識別滅活狂犬病病毒CTN株。其中3株單抗8G2、9E3、9F2與狂犬病病毒CTN株抗原結(jié)合的親和力分別為2.62×10-10M、2.62×10-10M、4.06×10-11M。 4.表達并純化狂犬病病毒糖蛋白,Western Blot結(jié)果顯示其中3株單抗8G2、9E3、9F2可與狂犬病病毒糖蛋白特異性結(jié)合。 結(jié)論 建立了5株穩(wěn)定分泌全人源抗狂犬病病毒IgM單抗的雜交瘤細胞株,5株單抗均能特異性識別滅活狂犬病病毒CTN株,其中3株顯示出較高的親和力,并且能與狂犬病病毒糖蛋白特異性結(jié)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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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相關(guān)期刊論文 前2條

1 李琛;林紅;劉新建;王忠燦;周鎮(zhèn)先;陳樂如;管曉虹;朱進;;人源抗狂犬病毒免疫型抗體庫的構(gòu)建及特異性抗體篩選與鑒定[J];南京醫(yī)科大學學報(自然科學版);2010年05期

2 ;Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus[J];Journal of Biomedical Research;2010年05期

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本文編號：1588183