基于单B细胞的汉坦病毒全人源中和单抗的研制

发布时间：2018-03-07 04:05

本文选题：肾综合征出血热　切入点：汉坦病毒　出处：《中国人民解放军军事医学科学院》2016年硕士论文　论文类型：学位论文

【摘要】：肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)是由汉坦病毒引起的急性传染性疾病,在世界范围内广泛流行,疫源地分布于5大洲70多个国家,已成为世界公共卫生问题。我国是世界上HFRS疫情最严重的国家,年报告发病人数约为4万-6万,占世界报告病例总数的90%以上。HFRS严重危害人民的生命健康安全,是我国重点防治的急性传染病之一。近些年来合并较多并发症的HFRS病例在临床上越来越多地出现,这些病例极易进展至多器官功能衰竭、颅内出血和弥散性血管内凝血,这些亦是该病导致死亡的主要原因,因此虽然近几年肾综合征出血热的年发病率有所下降,但针对该病的防控形势却越来越严峻。目前市场上还没有肾综合征出血热的特效治疗药物,临床上主要以对症治疗为主。研究表明抗汉坦病毒中和抗体可以快速有效地中和体内的汉坦病毒,达到治疗的目的,所以通过抗汉坦病毒中和抗体治疗肾综合征出血热,已经成为国内外临床医学和生物制药专家的共识。本实验尝试利用基于单细胞PCR技术的全人源单克隆抗体制备技术,进行抗汉坦病毒全人源中和抗体的研制。首先采集了4名肾综合征出血热患者的恢复期外周血共80m L,通过密度梯度沉降法,从这些外周血中分离出淋巴细胞。然后根据B细胞表面分子标记特征对分离的淋巴细胞进行荧光标记,通过流式细胞分选仪分选CD3-/CD20-/CD19+/C D27+/CD38+的单个浆细胞,共分选获得2200个B淋巴细胞。接下来分两步从单个B细胞中扩增抗体基因。由于单B细胞中的抗体基因序列是未知的,而抗体基因又具有丰富的亚型,因此第一步以覆盖绝大部分抗体可变区基因家族的18条引物进行RT-PCR,在一个反应体系中同时扩增单个B细胞中抗体的轻链可变区基因和重链可变区基因;第二步再以RT-PCR的产物做为模板,分三个反应体系进行巢式PCR,分别扩增H链、κ链以及λ链可变区基因,重链扩增VDJ重链基因区段,轻链扩增VJ轻链基因区段,混合引物包括针对每一个抗体可变区基因家族的特异引物。最终从2200个单个B淋巴细胞中获得了427个H链可变区基因片段、880个κ链可变区基因片段458个λ链可变区基因片段,阳性比例分别为20%、40%和21%,轻链中κ链和λ链的占比分别为66%和34%,基本符合人的表达κ链和λ链的B细胞的比例,其中轻重链基因天然配对且唯一的抗体基因是130对。获取抗体基因后需要进行抗体表达和筛选,但是传统构建表达载体转染哺乳动物细胞表达抗体的方式费时费力,为了加速初期筛选,我们尝试利用线性表达框表达130对抗体基因。构建线性表达框时将CMV启动子、抗体信号肽序列、抗体轻/重链的可变区基因序列、抗体轻/重链恒定区序列和poly A信号序列等转录翻译必需元件通过一步重叠延伸PCR构建成一个完整的线性片段,将轻/重链线性片段共转染HEK-293悬浮细胞,130个抗体中120个获得表达,与传统的克隆方法相比,通过线性表达框表达抗体,大大降低了工作量,在3-4天内便可获得可用于后续筛选的抗体。线性表达框表达抗体的水平多数可以达到1.2μg/m L以上,可以满足抗体特异性筛选的需要。随后以灭活的汉坦病毒作为抗原,通过ELISA对成功表达的抗体进行了特异性检测,结果显示,120个单抗中有51个可以特异性结合灭活的汉坦病毒,占42.5%。随后我们从这51个单抗中挑选30个特异性较高的单抗,将其轻重链构建到表达载体中,并瞬时转染到HEK-293悬浮细胞中进行表达纯化,30个都获得了表达,纯化后抗体浓度在0.5-1.2mg/m L之间。抗体中和活性检测我们采用的是荧光抗体病毒中和试验,通过FITC标记的抗汉坦病毒单抗检测病毒感染的细胞定量病毒,进而判断单抗的中和作用。通过检测我们筛选到2个具有中和活性的抗体(20C8、11H11),两者中和效果都存在剂量依赖关系,其中11H11中和作用明显强于20C8,0.16μg的11H11可以中和70%200 TCID50病毒,而中和同样的病毒需要0.64μg的20C8。以灭活的汉坦病毒作为抗原,采用ELISA方法初步测定抗体与抗原的结合力,20C8的结合EC50约为8.3×10-8 mol/L,11H11的结合EC50约为1.8×10-6 mol/L。汉坦病毒结构蛋白G1和G2蛋白是两个主要的中和抗原,预期以这两个蛋白作为抗原筛选特异性抗体,分别用HEK-293悬浮细胞和毕赤酵母表达这两个蛋白的膜外区,结果在毕赤酵母中G2蛋白有少量表达,而G1没有检测到表达产物;G1和G2在HEK-293悬浮细胞中都可以实现表达,但是G1和G2主要存在于细胞膜上,没有分泌到细胞外。汉坦病毒中和抗原G1/G2的表达一直是世界性难题,后续需要在现有工作基础上开展进一步研究,为确定20C8和11H11的抗原表位,并深入评价这两株抗体的亲和力和特异性奠定基础。利用本课题所建立的单B细胞全人源抗体制备技术,可以从传染病患者或者疫苗免疫者外周血出发制备预防或治疗性抗体,是研制突发传染病病原体中和抗体的有效手段;本研究筛选到的两个具有中和活性的抗汉坦病毒单抗,经过进一步的评价和优化,可能成为潜在的治疗HFRS的被动免疫制剂。
[Abstract]:Hemorrhagic fever with renal syndrome (hemorrhagic fever with renal syndrome, HFRS) is an acute infectious disease caused by Hantavirus, widely popular in the world, the foci located in 5 continents in more than 70 countries, the world has become a public health problem. China is the most serious epidemic situation of HFRS in the world, the incidence of about the number of years 40 thousand -6 million, accounting for more than 90% of the total number of reported cases in the world.HFRS serious harm to people's health and safety, is one of the acute infectious disease prevention in China. In recent years, with more complications of HFRS cases appears more and more in clinical practice, these cases can easily progress of multiple organ failure, intracranial hemorrhage and disseminated intravascular coagulation, the disease is the leading cause of death, although it has declined in recent years the incidence of hemorrhagic fever with renal syndrome rate, but for the disease prevention and control situation But more and more serious. There is no specific treatment of hemorrhagic fever with renal syndrome syndrome on the market, the main clinical symptomatic treatment. The research showed that neutralizing anti hantavirus antibody effectively neutralize hantaviruses, achieve the purpose of treatment, so by neutralizing anti hantavirus antibody in the treatment of hemorrhagic fever with renal syndrome has become. The consensus of clinical medicine and bio pharmaceutical experts at home and abroad. The experiment of using technology fully human monoclonal antibody preparation of single cell based on PCR technology, research of anti hantavirus human neutralizing antibody. The first collection of 4 patients with hemorrhagic fever with renal syndrome recovery of peripheral blood 80m L by density gradient sedimentation, isolated lymphocytes from the peripheral blood. And then were labeled according to B cell surface markers characteristic of lymphocyte separation by flow cytometry. A single cytoplasm sorter sorting CD3-/CD20-/CD19+/C D27+/CD38+, were obtained from the 2200 B lymphocytes. The following two step amplification antibody gene from a single B cell. The antibody gene sequence of single B cells is unknown, but the antibody gene has subtype and rich, so the first step is to cover the vast gene RT-PCR most of the family of the antibody variable region 18 primers, and the amplified gene of light chain variable region gene single antibody in B cells and the heavy chain variable region in a reaction system; the second step to the product of the RT-PCR as a template, three reaction system were amplified by nested PCR, H chain gene kappa chain and the lambda chain variable region heavy chain amplification of VDJ heavy chain gene, light chain amplification VJ light chain gene, gene specific primers for mixed primers including each antibody variable region of the family. From the end of 2200 single B lymph The gene fragment of 427 H chain variable region gene fragment of 880 cells, a kappa chain variable region 458 lambda chain variable region gene fragment, the positive rates were 20%, 40% and 21%, light chain kappa chain and lambda chain accounted for 66% and 34%, consistent with B cell kappa chain the expression of the lambda chain and the ratio of the antibody light and heavy chain genes and only natural pairing of 130. To obtain antibody genes needed after antibody expression and screening, but the traditional construction of expression vector was transfected into mammalian cells expressing antibody method is time-consuming and laborious, in order to accelerate the early screening, we try to use the linear expression frame expression 130 the antibody gene. The linear expression frame when CMV promoter and signal peptide sequence of antibody, antibody light / heavy chain variable region gene sequence, antibody light / heavy chain constant region sequence and poly A sequence signal transcription and translation essential element by one step heavy Overlap extension PCR constructs a complete linear fragment, the light / heavy chain linear fragments were transfected into HEK-293 cell suspension, 120 130 antibody was expressed, compared with the traditional method of cloning, expression of antibody box by linear expression, greatly reduces the workload in 3-4 days can be obtained for subsequent antibody screening. Linear expression frame antibody level in most can reach more than 1.2 g/m L, can meet the needs of specific antibody screening. Then using inactivated Hantavirus as antigen by antibody ELISA on the successful expression of the specific detection, results showed that the 120 mAbs have 51 Specific combined with the inactivated hantavirus, accounting for 42.5%. from the 51 then we chose 30 monoclonal antibodies with high specificity monoclonal antibody, the light chain constructed into the expression vector, then transiently transfected into HEK-293 cells suspension Purification, 30 have been expressed, purified antibody concentration in 0.5-1.2mg/m L. Detection of antibody neutralizing activity we used the fluorescent antibody virus neutralization test, virus by cell quantitative anti hantavirus monoclonal antibody FITC labeled virus infection, and then judge neutralization monoclonal antibody. Through the detection we screened 2 neutralizing the activity of antibody (20C8,11H11), and both effects are dose-dependent, and the effect was stronger than the 20C8,0.16 11H 11 g 11H11 70%200 TCID50 can neutralize the virus, and the virus neutralization also requires 0.64 g 20C8. with inactivated hantavirus antigen, antibody and antigen binding assay using ELISA method, combined with EC50 20C8 is about 8.3 * 10-8 mol/L, with EC50 11H11 is about 1.8 * 10-6 mol/L. structural protein of Hantaan virus G1 and G2 proteins are two major neutralizing antibody The original, is expected to two of the protein as a screening of specific antibody antigen, respectively HEK-293 cell suspension and Pichia expression of extracellular domain of these two proteins results in Pichia pastoris G2 protein expression, while G1 did not detect the expression product; G1 and G2 can realize the expression in HEK-293 cell suspension however, G1 and G2 are mainly in the cell membrane, not secreted into the cell. The expression of Hantaan virus neutralizing antigen G1/G2 has been a worldwide problem, the need to carry out further research work on the existing basis, to determine the 20C8 and 11H11 antigen epitope, and in-depth evaluation of the two strains of antibody affinity and the specificity of the foundation. The preparation techniques of using the single cell B human antibody preparation, preparation of preventive or therapeutic antibodies from patients with infectious disease or vaccine of peripheral blood, is the development of infectious disease disease Two effective neutralizing antibodies against hantavirus are screened out. The further evaluation and optimization of these antibodies may be potential passive agents for the treatment of HFRS.

【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】：2016
【分类号】：R512.8

【相似文献】