蜱传脑炎病毒中和抗体研究

发布时间：2018-01-03 21:50

本文关键词：蜱传脑炎病毒中和抗体研究　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：蜱传脑炎(tick-borne encephalitis,TBE)又名森林脑炎,是经硬蜱媒介传播的自然疫源性传染病,主要侵犯中枢神经系统,病原体是蜱传脑炎病毒(tick-borne encephalitis virus,TBEV)。据世界卫生组织统计,全球每年有超过10000例蜱传脑炎感染患者;在我国,蜱传脑炎已被列为5大职业性传染病之一。近年来随着国家经济高速发展、旅游业爆发式增长,远足、探险等户外活动兴起,进入森林疫区人群数量呈现逐年上升趋势,蜱传脑炎的发病率也在逐年升高,威胁着广大人民群众的生命健康。此外,蜱传脑炎病毒还是重要的生物战剂之一,美国CDC将其列为病毒C类生物武器。蜱传脑炎致残率高,临床上尚无特异性疗法,主要采用支持治疗和一般对症治疗为主。疫苗接种是预防蜱传脑炎的主要手段之一。当前,大部分蜱传脑炎病毒抗体多是研究用的鼠源性单抗,尚未有全人源TBEV单抗报道。在本研究中,我们拟通过流式分选-单细胞PCR技术,从蜱传脑炎疫苗免疫志愿者体内筛选获得全人源中和单克隆抗体,为蜱传脑炎治疗和预防提供新的途径。实验选取2名健康男性志愿者(25-40岁),在告知并签署知情同意书后按照蜱传脑炎灭活疫苗(长春生物制品研究所有限公司研制)说明书要求,分别在第0天、14天对志愿者进行2次免疫,又在第42天加强免疫1次。采集志愿者第0天、21天、28天、49天和63天血液样本,ELISA检测血清抗体滴度,结果表明两名志愿者均产生了针对TBEV的特异抗体。用Ficoll-hypaque法分离志愿者第49天血液样本20 m L,获得的外周血单核细胞(peripheral blood mononuclear cells,PBMCs)经荧光抗体标记后,用流式细胞分选仪分选出CD19+/CD3-/CD20-/CD27high/CD38high特异的单个抗体分泌细胞。我们先后共计分选获得1632个单细胞克隆,占上样细胞总数的0.02%;经反转录和巢式PCR扩增抗体可变区基因,扩增出重、轻链配对克隆349个,配对克隆的扩增效率约21.38%;对巢式PCR产物进行测序和IMGT/V-QUEST分析,最终获得有用克隆175个。对175个单克隆巢式PCR产物进一步扩增抗体基因可变(V)区,通过重叠延伸PCR技术与抗体启动子-前导序列和恒定区-多聚A尾序列连接,构建出抗体基因重、轻链线性表达框。将抗体线性表达框基因转染到Expi 293F细胞进行表达后,ELISA方法筛选到14株具有结合活性的单克隆抗体。我们进一步验证了这14株结合单抗在BHK-21细胞中对TBEV活病毒的中和效果,发现两株具有一定中和保护能力的单抗1C4和3E12。在抗体浓度达到200μg/m L以上时,1C4和3E12两株单抗对细胞能够起到完全保护效果,其中1C4的保护效果要强于3E12,1C4的IC50值为75μg/m L,而3E12的IC50值为108μg/m L。为对筛选到的抗体作进一步研究,我们在原核系统表达纯化了TBEV抗原E和NS1蛋白。TBEV在病毒分类学上属黄病毒科(Flavivirade)黄病毒属(Flavivirus),为单股正链RNA包膜病毒。其抗原成分包括结构蛋白E和非结构蛋白NS1。我们将TBEV抗原E和NS1基因克隆到p ET-32a表达载体上,转化BL21感受态细胞进行诱导表达和纯化,两个蛋白均以包涵体形式表达。NS1蛋白纯化过程以尿素为变性剂,将包涵体溶解后过镍柱纯化,然后通过逐步减少尿素浓度进行透析复性,最后获得纯化的NS1蛋白;E蛋白用相同方法进行纯化时,在复性过程中总是会变成沉淀析出,于是我们尝试采用比较温和的变性剂——十二烷基肌氨酸钠溶解包涵体,然后在含氧化还原体系(GSH和GSSG)的TE缓冲液中进行透析复性,最后获得E抗原蛋白。我们将纯化好的NS1蛋白和E蛋白与志愿者阳性血清分别进行Western Blot和ELISA检测,发现阳性血清只与E蛋白反应而不与NS1蛋白起作用,说明本研究中使用的灭活疫苗有效成分包含抗原E蛋白,而不包含NS1蛋白。将E抗原蛋白包被后与中和单抗1C4、3E12进行ELISA验证,结果表明具有中和活性的1C4和3E12两株单抗与原核表达的E抗原蛋白具有较弱的结合活性,这可能是因为两株单抗本身亲和力较低。考虑到原核表达的抗原蛋白缺乏糖基化修饰;此外,蜱传脑炎病毒膜蛋白pr M在病毒复制成熟过程中也起重要作用,所以我们又分别将抗原pr M-E和NS1基因克隆到真核表达载体p DC316,并在293T细胞中进行了表达。用志愿者阳性血清对293T细胞裂解物进行Western Blot鉴定,可观察到pr M-E蛋白在55 k Da处出现特异性条带,说明pr M-E基因得到表达并能与阳性血清反应;而NS1蛋白无目的条带,考虑是因为NS1蛋白不是灭活疫苗的有效抗原成分,志愿者血清中缺乏抗NS1抗体所致。在Western Blot实验中,我们发现pr M-E抗原在真核表达的细胞裂解物沉淀,在与筛选到的中和单抗单独反应时均难以出现明显目的条带;而将两株单抗混合在一起时,则可以看到明显特异条带。这说明我们筛到的两株中和单抗可能分别结合于抗原E蛋白的不同表位,尽管单独使用时抗原抗体相互作用比较弱,但混合在一起仍可看出明显特异条带。综上所述,本研究基于流式分选-单细胞PCR技术成功从蜱传脑炎灭活疫苗免疫志愿者体内筛选获得2株具有中和活性的单克隆抗体,该抗体可进一步研究用作潜在的治疗蜱传脑炎感染的被动免疫制剂;通过原核和真核表达系统表达了TBEV抗原蛋白E和NS1,其中只有E抗原能与志愿者阳性血清有结合反应,说明灭活疫苗的有效成分包含抗原E蛋白;此外,筛选到的中和单抗与抗原E蛋白具有结合活性,说明两株中和单抗的靶抗原是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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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