抗白念珠菌人鼠嵌合抗體真核表達(dá)載體的構(gòu)建與表達(dá)
發(fā)布時(shí)間：2019-05-18 17:33
【摘要】： 近年來(lái),隨著HIV感染者的增加、異種移植術(shù)的開(kāi)展、腫瘤發(fā)病率的上升,免疫受損人群不斷擴(kuò)大,真菌感染性疾病越來(lái)越被關(guān)注[1]。其中白念珠菌感染是發(fā)病率最高、影響最為廣泛的一類真菌疾病[2]。而目前的抗真菌藥物大多毒副作用較大,而且真菌的耐藥性迅速增加。因此臨床迫切需要安全和特效的抗真菌藥物[3]。 人們一直在探索抗真菌生物學(xué)治療手段,其中基因工程抗體是一種重要的策略[4]。近年來(lái)基因工程抗體技術(shù)的迅猛發(fā)展,為許多疾病的臨床診斷和治療提供了新的思路。嵌合抗體是目前研究較多、也較為成熟的基因工程抗體。它在降低鼠源性單抗的免疫原性的同時(shí),最大限度地保留了親代鼠源單抗的抗原結(jié)合特異性和親和性;在人體內(nèi)半衰期較鼠源單抗長(zhǎng),生物學(xué)功能也較強(qiáng);還可根據(jù)需要更換IgC區(qū)基因的類別、型或亞型,從而改變抗體Fc段的功能。自從1984年Morrison[5]首次成功研制人鼠嵌合抗體以來(lái),現(xiàn)已有多種嵌合抗體用于臨床,取得了良好的效果[6]。眾多臨床試驗(yàn)表明,嵌合抗體應(yīng)用于人體是安全的,且較改型的人源化抗體能更好地保留親本鼠單抗的親和力和特異性[6, 7]。國(guó)外多個(gè)實(shí)驗(yàn)室的研究提示抗白念珠菌抗體具有良好的抗真菌作用,并預(yù)示了良好的臨床應(yīng)用前景[8, 9]。本研究組在前期工作中獲得了單克隆天然抗體3B4,并發(fā)現(xiàn)3B4對(duì)白念珠菌有高特異親和性和明確的抗白念珠菌作用[10]。 本研究采用基因工程技術(shù)對(duì)鼠源性單抗3B4進(jìn)行了人源化改造,將鼠源抗體可變區(qū)與人抗體恒定區(qū)相連接,構(gòu)建了抗白念珠菌人鼠嵌合抗體真核表達(dá)載體,進(jìn)行了真核表達(dá),并初步鑒定了嵌合抗體的生物學(xué)活性。一、抗白念珠菌人鼠嵌合抗體真核表達(dá)載體的構(gòu)建 從含有單克隆天然抗白念珠菌抗體3B4可變區(qū)基因的載體pMDT-V2H和pUC-VL中PCR克隆鼠源單抗3B4的可變區(qū)VH和VL基因,依次插入含有人免疫球蛋白κ輕鏈恒定區(qū)基因和γ1重鏈恒定區(qū)基因的真核表達(dá)載體pMH-CA中,將抗白念珠菌鼠源單抗的可變區(qū)與人IgG1恒定區(qū)連接。經(jīng)酶切和DNA序列測(cè)定正確后,成功構(gòu)建人鼠嵌合基因工程抗體的真核表達(dá)載體PHK。 二、抗白念珠菌人鼠嵌合抗體的真核表達(dá)和生物活性鑒定采用電穿孔方法轉(zhuǎn)染小鼠漿細(xì)胞瘤細(xì)胞系J558L細(xì)胞進(jìn)行嵌合抗體的真核表達(dá),RT-PCR、ELISA方法對(duì)抗體表達(dá)進(jìn)行鑒定。RT-PCR和DNA序列測(cè)定顯示人鼠嵌合抗體重鏈和輕鏈在mRNA水平正確地轉(zhuǎn)錄和剪切,ELISA證實(shí)了抗白念珠菌人鼠嵌合抗體的表達(dá)以及對(duì)角蛋白和白念珠菌芽管的結(jié)合活性。 本研究成功構(gòu)建了抗白念珠菌人鼠嵌合抗體的真核表達(dá)載體,成功轉(zhuǎn)染真核細(xì)胞,并表達(dá)出具有結(jié)合活性的基因工程抗體。為下一步嵌合抗體的功能和臨床應(yīng)用研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 白銀,俞莉章,呂英謙,艾軍魁,周麗君,張春麗,王琰;提高抗人膀胱癌人-鼠嵌合抗體的表達(dá)和抗體的功能鑒定[J];中華醫(yī)學(xué)雜志;2003年04期
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