基于双特异性抗体的热带传染病快速侦检方法的研究

发布时间：2018-04-11 16:53

本文选题：热带传染病 + 快速诊断　；参考：《第二军医大学》2017年硕士论文

【摘要】：热带传染病是威胁我军指战员身体健康的重要疾病。部队官兵热带传染病自我防护意识比较薄弱且进入新环境因缺乏免疫力等而极易感染热带传染病。由于部队执行多样化的军事任务,迫切需要建立热带传染病侦检、诊治和防控相关技术,应对热带传染病的威胁。疟疾属于虫媒传染病,由疟原虫寄生人体红细胞引起,被WHO列入的8大热带病之一,广泛分布于热带亚热带地区,且近年来疟原虫对抗疟药普遍具有抗药性,潜在危害极大。恙虫病是由恙虫病东方体感染人体而引起的虫媒传染病,近年来在我国的流行区域有不断扩大延伸的趋势。我国南海岛屿地处热带,常年多雨,温暖潮湿,是各种病原体滋生的温床,有调查显示该地区大部分区域是恙虫病的疫源地。由于驻守海岛的部队多为外来人群,对该类传染病的免疫力低,发病率较高,严重影响了部队各项任务的完成,是我国南方战区造成非战斗减员的重要传染病。部队军事作业及战时的特点都要求建立快速、便捷、无需或较少借助仪器的诊断方法。为了建立这样一种满足现场使用要求的热带传染病快速侦检方法,我们引入了基因工程双特异性抗体BsAb,它是将两种针对不同抗原的单克隆抗体A和B的重、轻链可变区以非共价键、共价键或亮氨酸拉链、螺旋-转角-螺旋等蛋白质结构域连接形成的重组抗体分子。利用基因工程双特异性抗体可同时结合两种不同的抗原的特性,如构建抗人红细胞/抗病原体抗原的双特异性抗体,该BsAb可同时分别结合人红细胞和血液中病原体,形成可见的红细胞的集聚,从而对病原体感染做出诊断检测。本课题在制备了抗恶性疟原虫裂殖子表面抗原、抗恙虫病东方体外膜抗原以及抗人红细胞膜表面非凝集抗原的单克隆抗体杂交瘤细胞的基础上,通过RT-PCR分别获得cDNA,以其为模板,利用设计的多对引物PCR扩增得到各单克隆抗体的重链和轻链可变区目的基因片段VH、VL。设计拼接引物,利用重叠延伸PCR法,将抗疟原虫裂殖子表面抗原的单克隆抗体的重链和轻链可变区目的基因片段通过连接肽序列拼接得到抗-疟原虫裂殖子表面抗原的单链抗体scFv目的基因片段;将抗人红细胞膜表面非凝集抗原的单克隆抗体的重链和轻链可变区目的基因片段与抗恙虫病东方体外膜抗原的单克隆抗体的重链和轻链可变区目的基因片段通过两个短连接肽序L10(SAKTTPKLGG)和一个长连接肽序列LL(RADAAAA(G4S)4)拼接得到抗-人红细胞膜表面非凝集抗原/抗-恙虫病东方体外膜抗原的双特异性抗体BsAb目的基因片段。同时分别在其N端引入毕赤酵母真核表达系统中信号肽前导序列,在其C端引入6×His标签序列。将这些基因片段分别克隆至毕赤酵母分泌表达载体pPIC9K,电转化毕赤酵母菌GS115并诱导表达,先通过SDS-PAGE电泳分析确定疑似目的蛋白条带后,再应用Western blot检测目的蛋白的表达。获得的确定有目的蛋白表达的上清后续可经镍柱纯化得到电泳纯重组抗体。本研究中,我们通过重叠延伸PCR获得抗-疟原虫裂殖子表面抗原的单链抗体scFv和抗-人红细胞膜表面非凝集抗原/抗-恙虫病东方体外膜抗原的双特异性抗体BsAb目的基因片段。将抗-疟原虫裂殖子表面抗原的单链抗体scFv克隆至毕赤酵母分泌表达载体pPIC9K上进行诱导表达,SDS-PAGE和Western blot检测分析重组蛋白的表达,得到的重组蛋白大小与理论分子量相符。间接免疫荧光实验结果表明该单链抗体可与疟原虫裂殖子表面蛋白识别并结合,从而检测到疟原虫感染,具有与亲本抗体相似的识别结合能力。将抗-人红细胞膜表面非凝集抗原/抗-恙虫病东方体外膜抗原的双特异性抗体重组基因片段克隆至毕赤酵母真核分泌表达载体pPIC9K进行诱导表达,SDS-PAGE和Western blot检测重组蛋白的表达,得到的重组蛋白大小与理论分子量相符。红细胞集聚实验结果表明该双特异性抗体具有与红细胞膜表面非凝集抗原识别并结合的活性,为后续将该基因工程双特异性抗体用于恙虫病等感染性疾病病原体的快速诊断以及种属鉴定等提供技术支撑。
[Abstract]:Tropical infectious diseases are the main disease threatening the health of the army. The troops of self - protection awareness is relatively weak and tropical infectious diseases into the new environment because of the lack of immunity and vulnerable to tropical infectious diseases. Because the troops carry out diversified military tasks, the urgent need to establish a tropical infectious disease detection, diagnosis and prevention and control technology to deal with the threat of infectious diseases. Tropical malaria belongs to insect borne diseases caused by Plasmodium parasites in human red blood cells, one of the 8 major tropical diseases were included in the WHO, is widely distributed in tropical and subtropical regions, and in recent years, Plasmodium antimalarial drugs generally have drug resistance, the potential harm is great. Tsutsugamushi disease is infectious disease by tsutsugamushi Oriental body disease caused by the infection of human, in recent years in epidemic areas in our country has the expanding trend in the South China Sea. The island is located in the tropics, perennial rainy, warm and moist, is A hotbed of various pathogens, a survey shows that most of this area is the focus of tsutsugamushi disease. As for foreign troops stationed in the island population, the infectious disease is low, high incidence rate, serious impact on the military tasks, is an infectious disease in South China caused by the non combat attrition theater an important characteristic of the military operation. And wartime are required to establish a fast, convenient, without or less using instrument diagnostic method. In order to establish such a satisfying tropical infectious disease rapid detection method using the site requirements, I have introduced the engineered bispecific antibody BsAb, it is two different antigens monoclonal antibody A and B heavy and light chain variable region by non covalent bonds, covalent bond or leucine zipper, recombinant antibody molecule protein domain helix turn helix connection. The use of gene Bispecific antibody can be combined with the engineering characteristics of two different antigens, such as the construction of bispecific antibody against human red blood cell / pathogen antigen, the BsAb can also bind to the pathogen of human red blood cells respectively and blood, the formation of agglomeration visible red blood cells, so as to make a diagnosis of pathogen infection detection in this article. The preparation of Plasmodium falciparum merozoite surface antigen, anti Orientia outer membrane antigen and anti human RBC membrane surface agglutination antigen monoclonal antibody hybridoma cells, cDNA were obtained by RT-PCR with the template, using several pairs of primers designed by PCR was amplified by heavy chain monoclonal antibody and the light chain variable region gene fragment VH, VL. splicing design primers, using overlap extension PCR method, the heavy chain of monoclonal antibodies against Plasmodium falciparum merozoite surface antigen and a light chain variable region objective base Because the fragment by connecting peptide sequence scFv scFv gene fragment of anti Plasmodium merozoite surface antigen; the purpose of the heavy chain and light chain variable region gene fragment to the heavy chain of monoclonal antibody against human erythrocyte membrane surface agglutination antigen and a light chain variable region gene fragment with anti Orientia membrane resistance the original monoclonal antibody by two short peptide sequence L10 (SAKTTPKLGG) and a peptide sequence of LL long connection (RADAAAA (G4S) 4) spliced anti human erythrocyte membrane surface agglutination antigen / anti Orientia membrane antigen bispecific antibody BsAb fragment respectively. At the same time the N is introduced into Pichia pastoris eukaryotic expression system of signal peptide in its preamble sequence, C is introduced into 6 * His tag sequence. These gene fragments were cloned into Pichia pastoris expression vector pPIC9K secretion, electroporation of Pichia yeast Strain GS115 and induced by SDS-PAGE electrophoresis analysis, to determine the suspected target protein bands, the expression and application of the target protein was detected by Western blot. The determination of the target protein expression was subsequent purification by Ni column electrophoresis pure recombinant antibody. In this study, we obtained by overlap extension PCR scFv scFv anti merozoite surface antigen and anti human RBC membrane surface agglutination antigen / anti Orientia membrane antigen bispecific antibody BsAb fragment. The anti Plasmodium merozoite surface antigen single chain antibody scFv was cloned into pPIC9K vector for expression of Pichia pastoris, and SDS-PAGE Western blot expression analysis of recombinant protein, the recombinant protein has a molecular mass and size theory. The experimental results show that the indirect immunofluorescence antibody and malaria Insect merozoite surface protein recognize and bind to the detection of Plasmodium infection, thus, with the recognition of similar and parental antibody binding ability. The anti human erythrocyte membrane surface agglutination antigen / anti Orientia membrane antigen bispecific antibody gene fragment was cloned into Pichia pastoris eukaryotic expression vector pPIC9K was induced the expression, expression of recombinant protein SDS-PAGE and Western blot, the recombinant protein with the theoretical molecular weight of red blood cells. The experimental results show that the concentration of bispecific antibody with surface and erythrocyte agglutination antigen recognition and non binding activity, for the follow-up of the gene engineering bispecific antibody is used to provide technical support for rapid diagnosis of tsutsugamushi infectious disease pathogens and species identification.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R824

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