抗白念珠菌人鼠嵌合抗体真核表达载体的构建与表达
发布时间：2019-05-18 17:33
【摘要】： 近年来,随着HIV感染者的增加、异种移植术的开展、肿瘤发病率的上升,免疫受损人群不断扩大,真菌感染性疾病越来越被关注[1]。其中白念珠菌感染是发病率最高、影响最为广泛的一类真菌疾病[2]。而目前的抗真菌药物大多毒副作用较大,而且真菌的耐药性迅速增加。因此临床迫切需要安全和特效的抗真菌药物[3]。 人们一直在探索抗真菌生物学治疗手段,其中基因工程抗体是一种重要的策略[4]。近年来基因工程抗体技术的迅猛发展,为许多疾病的临床诊断和治疗提供了新的思路。嵌合抗体是目前研究较多、也较为成熟的基因工程抗体。它在降低鼠源性单抗的免疫原性的同时,最大限度地保留了亲代鼠源单抗的抗原结合特异性和亲和性;在人体内半衰期较鼠源单抗长,生物学功能也较强;还可根据需要更换IgC区基因的类别、型或亚型,从而改变抗体Fc段的功能。自从1984年Morrison[5]首次成功研制人鼠嵌合抗体以来,现已有多种嵌合抗体用于临床,取得了良好的效果[6]。众多临床试验表明,嵌合抗体应用于人体是安全的,且较改型的人源化抗体能更好地保留亲本鼠单抗的亲和力和特异性[6, 7]。国外多个实验室的研究提示抗白念珠菌抗体具有良好的抗真菌作用,并预示了良好的临床应用前景[8, 9]。本研究组在前期工作中获得了单克隆天然抗体3B4,并发现3B4对白念珠菌有高特异亲和性和明确的抗白念珠菌作用[10]。 本研究采用基因工程技术对鼠源性单抗3B4进行了人源化改造,将鼠源抗体可变区与人抗体恒定区相连接,构建了抗白念珠菌人鼠嵌合抗体真核表达载体,进行了真核表达,并初步鉴定了嵌合抗体的生物学活性。一、抗白念珠菌人鼠嵌合抗体真核表达载体的构建 从含有单克隆天然抗白念珠菌抗体3B4可变区基因的载体pMDT-V2H和pUC-VL中PCR克隆鼠源单抗3B4的可变区VH和VL基因,依次插入含有人免疫球蛋白κ轻链恒定区基因和γ1重链恒定区基因的真核表达载体pMH-CA中,将抗白念珠菌鼠源单抗的可变区与人IgG1恒定区连接。经酶切和DNA序列测定正确后,成功构建人鼠嵌合基因工程抗体的真核表达载体PHK。 二、抗白念珠菌人鼠嵌合抗体的真核表达和生物活性鉴定采用电穿孔方法转染小鼠浆细胞瘤细胞系J558L细胞进行嵌合抗体的真核表达,RT-PCR、ELISA方法对抗体表达进行鉴定。RT-PCR和DNA序列测定显示人鼠嵌合抗体重链和轻链在mRNA水平正确地转录和剪切,ELISA证实了抗白念珠菌人鼠嵌合抗体的表达以及对角蛋白和白念珠菌芽管的结合活性。 本研究成功构建了抗白念珠菌人鼠嵌合抗体的真核表达载体,成功转染真核细胞,并表达出具有结合活性的基因工程抗体。为下一步嵌合抗体的功能和临床应用研究奠定了基础。
[Abstract]:In recent years, with the increase of HIV infection, the development of xenotransplantation, the increase of tumor incidence, the expansion of immune impairment population, fungal infectious diseases have attracted more and more attention [1]. Among them, candida albicans infection is the highest incidence and the most widely affected fungal disease [2]. At present, most of the antifungal drugs have great toxic and side effects, and the drug resistance of fungi is increasing rapidly. Therefore, there is an urgent need for safe and special antifungal drugs [3]. People have been exploring antifungal biological therapy, in which genetic engineering antibody is an important strategy [4]. In recent years, the rapid development of genetic engineering antibody technology has provided new ideas for the clinical diagnosis and treatment of many diseases. Chimerism antibody is a kind of genetic engineering antibody which has been studied more and more mature at present. It not only decreased the immunogenicity of mouse monoclonal antibody, but also retained the antigen binding specificity and affinity of parental mouse monoclonal antibody to the maximum extent, and the half-life of mouse monoclonal antibody was longer than that of mouse monoclonal antibody in human body, and its biological function was stronger than that of mouse monoclonal antibody. The type, type or subgroup of IgC region gene can also be changed according to the need, thus changing the function of antibody Fc segment. Since 1984 Morrison [5] successfully developed human mouse chimerism antibody, a variety of chimeric antibodies have been used in clinical practice, and good results have been obtained [6]. Many clinical trials have shown that chimerism antibody is safe to be used in human body, and the modified humanized antibody can better retain the affinity and specificity of parental mouse monoclonal antibody [6,7]. Studies in many laboratories abroad suggest that anti-candida albicans antibody has a good antifungal effect and predict a good clinical application prospect [8,9]. In the previous work, the monoclonal natural antibody 3B4 was obtained, and it was found that 3B4 had high specific affinity and definite anti-candida effect on candida albicans [10]. In this study, mouse monoclonal antibody 3B4 was humanized by genetic engineering. The variable region of mouse antibody was connected with the constant region of human antibody, and the eukaryotic expression vector of human mouse chimeric antibody against candida albicans was constructed. The biological activity of chimeric antibody was preliminarily identified. 1. Construction of eukaryotic expression vector of human mouse chimeric antibody against candida albicans VH and VL genes of mouse monoclonal antibody 3B4 were cloned from vectors pMDT-V2H and pUC-VL containing monoclonal natural anti-candida antibody 3B4 variable region gene. The variable region of mouse monoclonal antibody against candida albicans was inserted into the eukaryotic expression vector pMH-CA containing human immunoglobulin kappa light chain constant region gene and gamma 1 heavy chain constant region gene in turn, and the variable region of anti-candida albicans mouse monoclonal antibody was connected to the constant region of human IgG1. After restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector PHK. of human mouse chimerism genetic engineering antibody was successfully constructed. 2. Eukaryotic expression and biological activity identification of chimeric antibody against candida albicans human mouse chimerism antibody was transformed into mouse plasma cell tumor cell line J558L by electroporation for eukaryotic expression of chimerism antibody, and RT-PCR, was used to express chimeric antibody in mouse plasmacytoma cell line J558L by electroporation. The expression of antibody was identified by ELISA. RT-PCR and DNA sequencing showed that the heavy chain and light chain of human mouse chimerism antibody were transcribed and cut correctly at mRNA level. ELISA confirmed the expression of chimeric antibody against candida albicans and the binding activity of keratin to the bud tube of candida albicans. In this study, the eukaryotic expression vector of human mouse chimeric antibody against candida albicans was successfully constructed, which was successfully transfected into eukaryotic cells and expressed genetically engineering antibody with binding activity. It lays a foundation for the further study of the function and clinical application of chimerism antibody.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2480197
[Abstract]:In recent years, with the increase of HIV infection, the development of xenotransplantation, the increase of tumor incidence, the expansion of immune impairment population, fungal infectious diseases have attracted more and more attention [1]. Among them, candida albicans infection is the highest incidence and the most widely affected fungal disease [2]. At present, most of the antifungal drugs have great toxic and side effects, and the drug resistance of fungi is increasing rapidly. Therefore, there is an urgent need for safe and special antifungal drugs [3]. People have been exploring antifungal biological therapy, in which genetic engineering antibody is an important strategy [4]. In recent years, the rapid development of genetic engineering antibody technology has provided new ideas for the clinical diagnosis and treatment of many diseases. Chimerism antibody is a kind of genetic engineering antibody which has been studied more and more mature at present. It not only decreased the immunogenicity of mouse monoclonal antibody, but also retained the antigen binding specificity and affinity of parental mouse monoclonal antibody to the maximum extent, and the half-life of mouse monoclonal antibody was longer than that of mouse monoclonal antibody in human body, and its biological function was stronger than that of mouse monoclonal antibody. The type, type or subgroup of IgC region gene can also be changed according to the need, thus changing the function of antibody Fc segment. Since 1984 Morrison [5] successfully developed human mouse chimerism antibody, a variety of chimeric antibodies have been used in clinical practice, and good results have been obtained [6]. Many clinical trials have shown that chimerism antibody is safe to be used in human body, and the modified humanized antibody can better retain the affinity and specificity of parental mouse monoclonal antibody [6,7]. Studies in many laboratories abroad suggest that anti-candida albicans antibody has a good antifungal effect and predict a good clinical application prospect [8,9]. In the previous work, the monoclonal natural antibody 3B4 was obtained, and it was found that 3B4 had high specific affinity and definite anti-candida effect on candida albicans [10]. In this study, mouse monoclonal antibody 3B4 was humanized by genetic engineering. The variable region of mouse antibody was connected with the constant region of human antibody, and the eukaryotic expression vector of human mouse chimeric antibody against candida albicans was constructed. The biological activity of chimeric antibody was preliminarily identified. 1. Construction of eukaryotic expression vector of human mouse chimeric antibody against candida albicans VH and VL genes of mouse monoclonal antibody 3B4 were cloned from vectors pMDT-V2H and pUC-VL containing monoclonal natural anti-candida antibody 3B4 variable region gene. The variable region of mouse monoclonal antibody against candida albicans was inserted into the eukaryotic expression vector pMH-CA containing human immunoglobulin kappa light chain constant region gene and gamma 1 heavy chain constant region gene in turn, and the variable region of anti-candida albicans mouse monoclonal antibody was connected to the constant region of human IgG1. After restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector PHK. of human mouse chimerism genetic engineering antibody was successfully constructed. 2. Eukaryotic expression and biological activity identification of chimeric antibody against candida albicans human mouse chimerism antibody was transformed into mouse plasma cell tumor cell line J558L by electroporation for eukaryotic expression of chimerism antibody, and RT-PCR, was used to express chimeric antibody in mouse plasmacytoma cell line J558L by electroporation. The expression of antibody was identified by ELISA. RT-PCR and DNA sequencing showed that the heavy chain and light chain of human mouse chimerism antibody were transcribed and cut correctly at mRNA level. ELISA confirmed the expression of chimeric antibody against candida albicans and the binding activity of keratin to the bud tube of candida albicans. In this study, the eukaryotic expression vector of human mouse chimeric antibody against candida albicans was successfully constructed, which was successfully transfected into eukaryotic cells and expressed genetically engineering antibody with binding activity. It lays a foundation for the further study of the function and clinical application of chimerism antibody.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 白银,俞莉章,吕英谦,艾军魁,周丽君,张春丽,王琰;提高抗人膀胱癌人-鼠嵌合抗体的表达和抗体的功能鉴定[J];中华医学杂志;2003年04期
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