本文關(guān)鍵詞：蜱傳腦炎病毒中和抗體研究　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蜱傳腦炎病毒 全人源單克隆抗體 流式分選 單細(xì)胞PCR

【摘要】：蜱傳腦炎(tick-borne encephalitis,TBE)又名森林腦炎,是經(jīng)硬蜱媒介傳播的自然疫源性傳染病,主要侵犯中樞神經(jīng)系統(tǒng),病原體是蜱傳腦炎病毒(tick-borne encephalitis virus,TBEV)。據(jù)世界衛(wèi)生組織統(tǒng)計(jì),全球每年有超過(guò)10000例蜱傳腦炎感染患者;在我國(guó),蜱傳腦炎已被列為5大職業(yè)性傳染病之一。近年來(lái)隨著國(guó)家經(jīng)濟(jì)高速發(fā)展、旅游業(yè)爆發(fā)式增長(zhǎng),遠(yuǎn)足、探險(xiǎn)等戶外活動(dòng)興起,進(jìn)入森林疫區(qū)人群數(shù)量呈現(xiàn)逐年上升趨勢(shì),蜱傳腦炎的發(fā)病率也在逐年升高,威脅著廣大人民群眾的生命健康。此外,蜱傳腦炎病毒還是重要的生物戰(zhàn)劑之一,美國(guó)CDC將其列為病毒C類生物武器。蜱傳腦炎致殘率高,臨床上尚無(wú)特異性療法,主要采用支持治療和一般對(duì)癥治療為主。疫苗接種是預(yù)防蜱傳腦炎的主要手段之一。當(dāng)前,大部分蜱傳腦炎病毒抗體多是研究用的鼠源性單抗,尚未有全人源TBEV單抗報(bào)道。在本研究中,我們擬通過(guò)流式分選-單細(xì)胞PCR技術(shù),從蜱傳腦炎疫苗免疫志愿者體內(nèi)篩選獲得全人源中和單克隆抗體,為蜱傳腦炎治療和預(yù)防提供新的途徑。實(shí)驗(yàn)選取2名健康男性志愿者(25-40歲),在告知并簽署知情同意書(shū)后按照蜱傳腦炎滅活疫苗(長(zhǎng)春生物制品研究所有限公司研制)說(shuō)明書(shū)要求,分別在第0天、14天對(duì)志愿者進(jìn)行2次免疫,又在第42天加強(qiáng)免疫1次。采集志愿者第0天、21天、28天、49天和63天血液樣本,ELISA檢測(cè)血清抗體滴度,結(jié)果表明兩名志愿者均產(chǎn)生了針對(duì)TBEV的特異抗體。用Ficoll-hypaque法分離志愿者第49天血液樣本20 m L,獲得的外周血單核細(xì)胞(peripheral blood mononuclear cells,PBMCs)經(jīng)熒光抗體標(biāo)記后,用流式細(xì)胞分選儀分選出CD19+/CD3-/CD20-/CD27high/CD38high特異的單個(gè)抗體分泌細(xì)胞。我們先后共計(jì)分選獲得1632個(gè)單細(xì)胞克隆,占上樣細(xì)胞總數(shù)的0.02%;經(jīng)反轉(zhuǎn)錄和巢式PCR擴(kuò)增抗體可變區(qū)基因,擴(kuò)增出重、輕鏈配對(duì)克隆349個(gè),配對(duì)克隆的擴(kuò)增效率約21.38%;對(duì)巢式PCR產(chǎn)物進(jìn)行測(cè)序和IMGT/V-QUEST分析,最終獲得有用克隆175個(gè)。對(duì)175個(gè)單克隆巢式PCR產(chǎn)物進(jìn)一步擴(kuò)增抗體基因可變(V)區(qū),通過(guò)重疊延伸PCR技術(shù)與抗體啟動(dòng)子-前導(dǎo)序列和恒定區(qū)-多聚A尾序列連接,構(gòu)建出抗體基因重、輕鏈線性表達(dá)框。將抗體線性表達(dá)框基因轉(zhuǎn)染到Expi 293F細(xì)胞進(jìn)行表達(dá)后,ELISA方法篩選到14株具有結(jié)合活性的單克隆抗體。我們進(jìn)一步驗(yàn)證了這14株結(jié)合單抗在BHK-21細(xì)胞中對(duì)TBEV活病毒的中和效果,發(fā)現(xiàn)兩株具有一定中和保護(hù)能力的單抗1C4和3E12。在抗體濃度達(dá)到200μg/m L以上時(shí),1C4和3E12兩株單抗對(duì)細(xì)胞能夠起到完全保護(hù)效果,其中1C4的保護(hù)效果要強(qiáng)于3E12,1C4的IC50值為75μg/m L,而3E12的IC50值為108μg/m L。為對(duì)篩選到的抗體作進(jìn)一步研究,我們?cè)谠讼到y(tǒng)表達(dá)純化了TBEV抗原E和NS1蛋白。TBEV在病毒分類學(xué)上屬黃病毒科(Flavivirade)黃病毒屬(Flavivirus),為單股正鏈RNA包膜病毒。其抗原成分包括結(jié)構(gòu)蛋白E和非結(jié)構(gòu)蛋白NS1。我們將TBEV抗原E和NS1基因克隆到p ET-32a表達(dá)載體上,轉(zhuǎn)化BL21感受態(tài)細(xì)胞進(jìn)行誘導(dǎo)表達(dá)和純化,兩個(gè)蛋白均以包涵體形式表達(dá)。NS1蛋白純化過(guò)程以尿素為變性劑,將包涵體溶解后過(guò)鎳柱純化,然后通過(guò)逐步減少尿素濃度進(jìn)行透析復(fù)性,最后獲得純化的NS1蛋白;E蛋白用相同方法進(jìn)行純化時(shí),在復(fù)性過(guò)程中總是會(huì)變成沉淀析出,于是我們嘗試采用比較溫和的變性劑——十二烷基肌氨酸鈉溶解包涵體,然后在含氧化還原體系(GSH和GSSG)的TE緩沖液中進(jìn)行透析復(fù)性,最后獲得E抗原蛋白。我們將純化好的NS1蛋白和E蛋白與志愿者陽(yáng)性血清分別進(jìn)行Western Blot和ELISA檢測(cè),發(fā)現(xiàn)陽(yáng)性血清只與E蛋白反應(yīng)而不與NS1蛋白起作用,說(shuō)明本研究中使用的滅活疫苗有效成分包含抗原E蛋白,而不包含NS1蛋白。將E抗原蛋白包被后與中和單抗1C4、3E12進(jìn)行ELISA驗(yàn)證,結(jié)果表明具有中和活性的1C4和3E12兩株單抗與原核表達(dá)的E抗原蛋白具有較弱的結(jié)合活性,這可能是因?yàn)閮芍陠慰贡旧碛H和力較低�？紤]到原核表達(dá)的抗原蛋白缺乏糖基化修飾;此外,蜱傳腦炎病毒膜蛋白pr M在病毒復(fù)制成熟過(guò)程中也起重要作用,所以我們又分別將抗原pr M-E和NS1基因克隆到真核表達(dá)載體p DC316,并在293T細(xì)胞中進(jìn)行了表達(dá)。用志愿者陽(yáng)性血清對(duì)293T細(xì)胞裂解物進(jìn)行Western Blot鑒定,可觀察到pr M-E蛋白在55 k Da處出現(xiàn)特異性條帶,說(shuō)明pr M-E基因得到表達(dá)并能與陽(yáng)性血清反應(yīng);而NS1蛋白無(wú)目的條帶,考慮是因?yàn)镹S1蛋白不是滅活疫苗的有效抗原成分,志愿者血清中缺乏抗NS1抗體所致。在Western Blot實(shí)驗(yàn)中,我們發(fā)現(xiàn)pr M-E抗原在真核表達(dá)的細(xì)胞裂解物沉淀,在與篩選到的中和單抗單獨(dú)反應(yīng)時(shí)均難以出現(xiàn)明顯目的條帶;而將兩株單抗混合在一起時(shí),則可以看到明顯特異條帶。這說(shuō)明我們篩到的兩株中和單抗可能分別結(jié)合于抗原E蛋白的不同表位,盡管單獨(dú)使用時(shí)抗原抗體相互作用比較弱,但混合在一起仍可看出明顯特異條帶。綜上所述,本研究基于流式分選-單細(xì)胞PCR技術(shù)成功從蜱傳腦炎滅活疫苗免疫志愿者體內(nèi)篩選獲得2株具有中和活性的單克隆抗體,該抗體可進(jìn)一步研究用作潛在的治療蜱傳腦炎感染的被動(dòng)免疫制劑;通過(guò)原核和真核表達(dá)系統(tǒng)表達(dá)了TBEV抗原蛋白E和NS1,其中只有E抗原能與志愿者陽(yáng)性血清有結(jié)合反應(yīng),說(shuō)明滅活疫苗的有效成分包含抗原E蛋白;此外,篩選到的中和單抗與抗原E蛋白具有結(jié)合活性,說(shuō)明兩株中和單抗的靶抗原是E抗原。
[Abstract]:Tick borne encephalitis (tick-borne encephalitis, TBE) also known as forest encephalitis, by Ixodes media natural infectious disease mainly affects the central nervous system, is a pathogen of tick borne encephalitis virus (tick-borne encephalitis, virus, TBEV). According to the WHO statistics, there are more than 10000 cases of tick borne encephalitis infection worldwide every year; in my in China, tick borne encephalitis has been listed as one of the 5 major occupation of infectious diseases. In recent years, with the rapid development of the national economy, the tourism industry explosive growth, hiking and other outdoor activities, the rise of exploration into the forest, epidemic population quantity increased year by year, the incidence rate of tick borne encephalitis also increased year by year, threaten the the life and health of the people. In addition, tick borne encephalitis virus is one of the important biological warfare agents, the United States CDC listed it as a class C virus biological weapons. Tick borne encephalitis with high rate of disability, there is no specific clinical The main therapy, supportive treatment and symptomatic treatment. Vaccination is one of the main means of prevention of tick borne encephalitis. At present, most of tick borne encephalitis virus antibody is a mouse monoclonal antibody for the research, has not yet been fully human monoclonal antibody TBEV reported. In this study, we proposed by FACS - Single cell PCR, encephalitis vaccine volunteers from ticks screened human neutralizing monoclonal antibody, provide a new way for the treatment and prevention of tick borne encephalitis. Methods: 2 healthy male volunteers (aged 25-40), to inform and signed informed consent in accordance with the tick borne encephalitis vaccine research (Changchun Biological Products Co. Ltd.) specification requirements, respectively in zeroth days, 14 days of volunteers were immunized 2 times and 1 times in the forty-second days of immunization. Collect zeroth days, 21 days, 28 days, 49 days and 63 days blood samples, ELISA. Measurement of serum antibody titers, results showed that two volunteers had antibodies to specific TBEV. The separation of volunteers forty-ninth days blood samples of 20 m L with Ficoll-hypaque method, peripheral blood mononuclear cells obtained (peripheral blood mononuclear cells, PBMCs) by fluorescent antibody labeled with a single antibody, select CD19+/CD3-/CD20-/CD27high/CD38high specific flow cytometry separation of secretory cells. We have a total of 1632 single cell clones obtained from the sample cell, accounted for 0.02% of the total; amplified antibody variable region gene reverse transcription and nested PCR, amplified heavy and light chain paired 349 clones, 21.38% clones matched about amplification efficiency; were sequenced and analyzed by IMGT/V-QUEST for nested PCR the final product, obtained 175 clones. Useful for 175 monoclonal antibody gene products were further amplified by nested PCR variable (V) region, by overlap extension PCR and antibody Promoter - leader sequence and constant region - A poly-t linker sequence, construct the antibody light chain gene, linear expression box. The antibody linear expression box gene was transfected into Expi 293F cells. After ELISA screening method to 14 strains of monoclonal antibody binding activity. We further validate the 14 strains with monoclonal antibody of TBEV live virus in BHK-21 cells and the effect, found two strains of neutralizing monoclonal antibody 1C4 and 3E12. capacity of more than 200 mu g/m L in antibody concentration, 1C4 and 3E12 two monoclonal antibodies to cell can play a full protection effect, the stronger the protecting effect of 1C4 on 3E12,1C4 IC50 the value is 75 g/m L, 3E12 and IC50 value of 108 g/m L. for the screening of the antibody for further study, we in the prokaryotic expression and purification of TBEV antigen and E NS1 protein.TBEV in the virus taxonomy belong to the Flaviviridae family (Flavivirade) Flavivirus (Flavivirus), is a single stranded RNA virus. Its antigen components including structural protein E and non structural protein NS1. we will TBEV antigen E and NS1 gene was cloned into P ET-32a expression vector, expression and purification of cells were induced into BL21 state of feeling, two proteins were expressed in the form of inclusion bodies.NS1 protein purification process using urea as the denaturant, dissolving the inclusion body after nickel column purification, and then gradually reduce the concentration of urea was dialyzed, finally purified NS1 protein; E protein was purified by the same method, the renaturation process always become precipitation, so we try to use the denaturant compared mild: Twelve alkyl sodium sarcosinate insoluble body, and then in the oxidation reduction system containing (GSH and GSSG) in TE buffer for dialysis renaturation, finally obtained E protein. We purified NS1 protein And E protein and the positive serum of volunteers respectively by Western Blot and ELISA testing, found that only positive serum reacted with E proteins but not with NS1 protein, that is used in this study and effective components of vaccine contains E protein antigen, which does not contain NS1 protein. E protein antigen coated with neutralizing monoclonal antibody 1C4,3E12 the ELISA verification, the results showed that the neutralizing activity of 1C4 and 3E12 two monoclonal antibodies with weak E antigen binding activity and protein expression, this may be because the two mAbs itself low affinity. Considering the prokaryotic expression of the antigen protein lack of glycosylation; in addition, tick borne encephalitis virus membrane PR M protein also plays an important role in virus replication during maturation, so we are pr M-E and NS1 antigen gene was cloned into the eukaryotic expression vector p DC316, and expressed in 293T cells. With positive blood volunteers Clear Western Blot identification of 293T cell lysate, observed PR M-E protein specific bands at 55 K Da, PR M-E gene expression and can react with positive serum; and NS1 protein band, is because the NS1 protein is not inactivated vaccine effective antigen components, volunteers the serum caused by the lack of anti NS1 antibody in Western Blot. In the experiment, we found that the precipitation of PR M-E antigen in cell lysates eukaryotic expression, the neutralizing monoclonal antibodies and screening to the individual reactions are difficult to appear obvious band; and the two mAbs mixed together, you can see clearly the specific with different tables. This shows that we screened two strains of neutralizing monoclonal antibodies may be bound to the antigen E protein, although when used alone the antigen antibody interaction is relatively weak, but mixed together can still be seen obvious specific bands from the above mentioned. This study, based on flow cytometry - single cell PCR from tick borne encephalitis Inactivated Vaccine Immunized volunteers in vivo screening 2 strains of monoclonal antibodies with neutralizing activity, further study for passive immunization potential treatment of tick borne encephalitis infection by the antibody; prokaryotic and eukaryotic expression system of TBEV antigen protein E and NS1, which can have only E antigen binding reaction with positive serum of volunteers, this shows that the effective ingredient of inactivated vaccine containing antigen E protein; in addition, the screening of the neutralizing monoclonal antibodies and antigen binding activity of E protein, said target antigen indicated that the two strains of neutralizing antibodies is E antigen.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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