人源抗狂犬病毒基因工程抗体研究

发布时间：2019-05-16 19:12

【摘要】：狂犬病是由狂犬病毒(Rabies Virus, RV)引起的人兽共患疾病,发病后死亡率几乎达百分之百。狂犬病毒属弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus),基因组编码五种结构蛋白,其中由G基因编码的糖蛋白,是病毒与宿主细胞结合的配体,能诱导机体产生中和抗体,与病毒的毒力、致病性密切相关。根据世界卫生组织(World Health Organization, WHO)推荐,对狂犬病III级暴露后的预防主要是采用狂犬疫苗注射结合抗狂犬病毒免疫球蛋白(rabies immune globulin, RIG)的方法。目前使用的两类RIG为人抗狂犬病毒免疫球蛋白(human rabies immune globulin, HRIG)和马抗狂犬病毒免疫球蛋白(Equine rabies immune globulin, ERIG)。但ERIG副反应比较严重,而且对某些疫苗的抗体反应有抑制,HRIG价格昂贵,供应量有限并且有潜在的病原威胁。抗狂犬病毒单克隆抗体(monoclonal antibodies, mAbs)具有中和效果高,安全性好,成本低,可大量生产等优点,能够取代RIG用于狂犬病的暴露后预防。本研究以噬菌体表面展示技术为平台,筛选人源抗狂犬病毒中和抗体,实验有以下三部分：一,人源抗狂犬病毒Fab噬菌体抗体库的构建与筛选我们首先采集具有高滴度狂犬病毒抗体的疫苗注射者外周血,分离淋巴细胞,然后提取总RNA并合成cDNA,接着用特异性的PCR引物扩增特异性轻链和重链Fd段基因,在对PCR产物鉴定和纯化后,用轻链基因与噬菌粒pComb3构建了轻链库,随后将重链基因克隆入轻链库中构建Fab噬菌体抗体库。最后使用辅助噬菌体对Fab噬菌体抗体库进行包装,检测库容量,结果表明我们所构建的Fab噬菌体抗体库的容量为4.5×108,轻链插入率和重链插入率均为100%,完全满足我们筛库的需要。在成功构建Fab噬菌体抗体库的基础上,用纯化的狂犬病毒颗粒aG和CTN株对Fab噬菌体抗体库进行富集筛选,利用ELISA、IFA对所获人源单克隆抗体Fab段基因的功能特性进行鉴定,并通过序列测定确定所获抗体的基因序列,然后与Genebank报道的抗体序列进行比较,根据抗体基因序列推断出其氨基酸序列,与vbase database比较,确定其CDR区。结果我们获得11株Fab抗体,序列测定结果证明其序列均为新的抗体序列,间接免疫荧光试验证明均针对狂犬病毒糖蛋白。二,人源抗狂犬病毒全抗体表达及功能鉴定在原核表达Fab抗体的基础上,我们利用昆虫杆状病毒载体技术平台实现了全抗体的真核表达。我们将11株Fab抗体中5株Fab抗体(RVFab1、RVFab3、 RVFab5、RVFab8、RVFab9)的轻链和重链Fd段基因插入杆状病毒载体pAC-L-Fc,然后将构建好的重组杆状病毒质粒转染昆虫细胞,通过IFA检测转染效果,接着纯化和扩增重组病毒,对表达产物进行纯化。利用SDS-PAGE、 ELISA、IFA等方法纯化的抗狂犬病毒IgG全抗进行功能鉴定,结果表明5株人源单克隆抗体均特异性针对狂犬病毒糖蛋白。利用非竞争ELISA检测5株抗体的亲和力,均高达10-9M。利用快速免疫荧光灶抑制中和试验对单抗的中和活性进行检测,结果表明5株单抗均具有较好的中和活性。三,人源抗狂犬病毒中和抗体表位研究通过竞争ELISA对5株抗体与抗原结合表位的相互关系进行鉴定,结果表明,5株抗体所识别的抗原表位彼此间存在相互重叠的关系。进一步利用竞争ELISA对5株抗体与一株针对狂犬病毒糖蛋白I号中和表位的抗体RVIgG57进行鉴定,结果表明,5株抗体与RVIgG57所识别的抗原表位均不存在相互重叠的关系。通过生物信息学分析狂犬病毒糖蛋白关键抗原位点,并结合相关文献报道,设计针对狂犬病毒糖蛋白三个主要中和位点的点突变,通过IFA分析人源抗体与中和位点突变体的结合活性。结果表明：我们获得的5株抗体均针对狂犬病毒糖蛋白II号中和表位,而RVIgG57针对狂犬病毒糖蛋白I号中和表位,与文献描述一致。综上所述,本研究运用噬菌体抗体库技术,成功构建了人源抗狂犬病毒Fab噬菌体抗体库,筛选获得了11株特异性针对狂犬病毒糖蛋白的人源Fab抗体。将其中5株构建为IgG全抗体,显示均具有高亲和力和高中和活性,并且针对狂犬病毒糖蛋白II号中和抗原表位。这一结果的获得为单克隆抗体取代RIG做为狂犬病暴露后预防的被动免疫制剂奠定了基础。
[Abstract]:Rabies are caused by the rabies virus (RV), and the mortality rate is almost 100% after the onset of the disease. The rabies virus belongs to the genus Rhabdoviridae, and the genome encodes five structural proteins, wherein the glycoprotein encoded by the G gene is a ligand which is bound by the virus and the host cell, can induce the production of neutralizing antibodies in the body, and is closely related to the virulence and the pathogenicity of the virus. According to the recommendations of the World Health Organization (WHO), the prevention of rabies III exposure is mainly the use of rabies vaccine injection in combination with the method of anti-rabies virus immune globulin (RIG). Two types of RIG are currently used as human anti-rabies virus immune globulin (HRG) and horse anti-rabies virus immunoglobulin (ERIG). However, the side reactions of the ERIG are serious, and the antibody response to some vaccines is inhibited, and the HRG is expensive, the supply is limited, and the potential pathogenic threat is present. The anti-rabies virus monoclonal antibody (mAbs) has the advantages of high neutralization effect, good safety, low cost, large production and the like, and can replace the RIG for preventing the rabies. in that study, the phage surface display technology is used as a platform to screen the human anti-rabies virus neutralizing antibody, and the experiment has the following three parts:1. the construction and the screening of a human anti-rabies virus Fab phage antibody library first collect the peripheral blood of the vaccine injection with the high-titer rabies virus antibody, the lymphocyte is separated, the total RNA is extracted and the cDNA is synthesized, the specific light chain and the heavy chain Fd segment gene are amplified by specific PCR primers, and the light chain library is constructed by the light chain gene and the phagemid pComb3 after the PCR product is identified and purified; The heavy chain gene was then cloned into the light chain library to construct the Fab phage antibody library. Finally, using the auxiliary phage to package the Fab phage antibody library, the library capacity was detected. The results showed that the capacity of the Fab phage antibody library was 4.5-108, the light chain insertion rate and the heavy chain insertion rate were 100%, which completely met the needs of our screen library. on the basis of successfully constructing the Fab phage antibody library, the Fab phage antibody library is enriched and screened by purified rabies virus particles aG and CTN strains, and the functional characteristics of the obtained Fab fragment genes of the human monoclonal antibody are identified by ELISA and IFA, The gene sequence of the obtained antibody is determined by the sequence measurement, and then compared with the antibody sequence reported by Genbank, the amino acid sequence of the antibody is deduced according to the sequence of the antibody gene, and the CDR region thereof is determined based on the comparison with the vbase database. As a result,11 Fab antibodies were obtained, and the results of the sequence determination showed that the sequence was a new antibody sequence, and the indirect immunofluorescence test proved to be specific to the rabies virus glycoprotein. 2. The full-antibody expression and function of the human anti-rabies virus are based on the prokaryotic expression of the Fab antibody, and the eukaryotic expression of the whole antibody is realized by using the insect baculovirus vector technology platform. The light chain and the heavy chain Fd section gene of the five Fab antibodies (RVFab1, RVFab3, RVFab5, RVFab8, RVFab9) in the 11 Fab antibodies were inserted into the baculovirus vector pAC-L-Fc, then the constructed recombinant baculovirus plasmid was transfected into the insect cell, the transfection effect was detected by IFA, and then the recombinant virus was purified and amplified, The expression product was purified. The anti-rabies virus IgG was purified by SDS-PAGE, ELISA, IFA and other methods. The results showed that 5 strains of human monoclonal antibodies were specific to the glycoprotein of rabies virus. The affinity of five antibodies was detected by non-competitive ELISA, which was as high as 10-9M. The neutralization test was used to detect the neutralizing activity of the monoclonal antibody, and the results showed that the 5 McAbs had better neutralizing activity. 3. The anti-rabies virus and the antibody epitope of the human anti-rabies virus were identified by the competitive ELISA. The results showed that the antigen epitopes recognized by the five anti-rabies virus were in overlapping relationship with each other. The anti-rabies virus glycoprotein I and the antibody RVIgG57 were identified by competitive ELISA. The results showed that there were no overlapping relationship between the five antibody and the antigen epitope recognized by the RVIgG57. The key antigenic site of the rabies virus glycoprotein was analyzed by bioinformatics, and the point mutation of the three main neutralizing sites of the rabies virus glycoprotein was designed and the binding activity of the human antibody with the neutralizing site was analyzed by IFA. The results showed that 5 of the antibodies we obtained were for the neutralizing epitope of the rabies virus glycoprotein II, while the RVIgG57 is the neutralizing epitope of the rabies virus glycoprotein I and is consistent with the literature description. In conclusion, the phage antibody library was successfully constructed by using the phage antibody library technique, and 11 human Fab antibodies specific to the rabies virus glycoprotein were selected. Five of these were constructed as IgG full-antibodies, showing high affinity and high school and activity, and for rabies virus glycoprotein II and epitope. The result is that the obtained monoclonal antibody replaces the RIG as the basis for the passive immune preparation after the rabies exposure.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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