超大容量人源噬菌体抗体库的构建及禽流感病毒抗体的筛选
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摘要
从20世纪70年代中期杂交瘤技术产生以来,人们一直对利用抗体作为疾病治疗的药物充满了期待。但应用鼠源杂交瘤抗体治疗人类疾病时,结果却令人相当失望。鼠源抗体分子在人体内会作为外来分子被人体免疫系统识别,从而产生人抗鼠抗体反应(human antimurine antibody response,HAM),鼠源抗体在体内引起过敏反应并很快的被人体免疫系统清除掉,从而无法到达治疗的目的。而后一系列的人源抗体技术,为解决这一问题带来了曙光。伴随着鼠抗人源化、人B细胞永生化和抗体基因工程技术的逐渐成熟,不少治疗性抗体也陆续进入临床研究,甚至已经成为上市药物。
噬菌体展示抗体库技术因为操作简单,及筛选高通量,成为最便捷最高效的人源抗体技术之一。但是通过常规技术制备的噬菌体抗体库,由于PCR、连接、转化效率等原因,限制了噬菌体抗体库的容量。此外,由于构建的抗体库不能像抗体基因在体内一样,可以发生重排,抗体库的多样性常常受到限制。缺乏多样性的抗体库经过筛选往往不能获得亲和力高、特异性好的克隆。因此,得到库容量高、多样性丰富的抗体库非常重要。而体外重组系统的建立,有利于增加抗体库的多样性。
本研究利用72对兼并引物扩增了人抗体可变区基因,通过摸索PCR扩增条件、优化制备电转感受态、提高连接物浓度,成功构建了一个具有良好基因多样性的人源天然噬菌体抗体库,库容为1.47×10~7,并通过菌株改造和突变loxP位点的应用,建立了适于抗体库的Cre-loxP重组系统,使得在Cre酶的作用下不同scFv基因间的VH、VL进行重组,抗体轻重链能够自由随机的组合重排,重组过程可以无限重复,每一次重组的产物都可以作为下次重组的底物,便于以后的筛选和亲和力成熟,最后获得8×10~11超大容量的噬菌体单链抗体库,建立了超大容量抗体库抗体库制备人源抗体的技术平台,为以后针对各种病毒、病原菌、毒素等抗原进行筛选,获得特异性人源治疗性单抗奠定了基础。
抗体在阻止病毒入侵宿主细胞,抑制病毒复制方面起着重要的作用。人源抗体技术的发展,也为病毒性疾病的治疗带来了新的亮点。本研究通过固液相及不同H5N1型禽流感病毒株对抗体库进行筛选,经过多毒株三轮淘洗,洗脱下来的噬菌体抗体呈明显增加趋势,表明对病毒HA的噬菌体抗体得到了有效的富集。利用ELISA和HAI等检测手段,获得9株抗体并进行原核表达获得scFv抗体蛋白。其中4株抗体能与H5亚型三株病毒交叉反应,4株中的一株抗体还表现出对H1、H3、H5亚型毒株的强交叉。有望成为禽流感抗体药物,用于高致病性高死亡率流感的治疗。此外,可以用这些抗体寻找HA1上的保守表位,研究流感病毒突变机制,研制通用疫苗。
The therapeutic potential of monoclonal antibodies (mAb) was quickly realised after the hybridoma technique allowed their development in the mid 1970s. But the mouse anitibodies do not fit for the therapy because of HA1MA (human antimurine antibody response) which can lead a unexpectable immune reaction and make the antibodies degrade quickly in vivo. In the following years, the rapid development of human antibody technology make antibodies using as therapeutic drug to be possible. While techniques of humanization, human memorr B cell immortalization and antibody engineering booming, more and more antibodies became performing clinicle researches, and went into the market.
With easily operating and high-throughput screening, phage display antibody library shows to be one of the most useful techniques for gain human antibodies. If the normal process using for construction the library, the capacity would be much smaller than it should be. It's for the reason that a large part of antibody genes would be lost in PCR, ligation and transfromation. In the other side, the antibody light chain and heavy chain genes in library can not recombine freely as in immune system. All of these make gain a good antigen especial reaction antibodies to be difficult. However, after presenting a recombination system, the diversity of library can be enlarged obviously. Additionally, a large library with 10" were gained.
In this research, on the basis of the primers which could amplify almost the whole antibody V-region genes, a normal single-chain phage antibody library has been prepared successfully. Also the Cre/lox recombination system was established. This system ensure the diversity of our scFv library.
Antibodies play important roles in protection against and recovery from virus infection. Techniques of human antibody bring a sparking point for therapy of viral diseases. Herein, the scFv library was sCreened with different H5N1 AIV strains in liquid/solid-phase, while detection with ELISA and HAL Then the positive clones were expressed in E. coli. as scFv proteins. Some clones showed cross-activity to different H5 virus strains. Remarkably, one of them can greatly cross-react with H1, H3 and H5 virus. The results depict that the antibodies against conserved epitops in virus have been gotten. It appears an approachable method for developing therapeutic antibody to influenza viruses who usally change themselves.
噬菌体展示抗体库技术因为操作简单,及筛选高通量,成为最便捷最高效的人源抗体技术之一。但是通过常规技术制备的噬菌体抗体库,由于PCR、连接、转化效率等原因,限制了噬菌体抗体库的容量。此外,由于构建的抗体库不能像抗体基因在体内一样,可以发生重排,抗体库的多样性常常受到限制。缺乏多样性的抗体库经过筛选往往不能获得亲和力高、特异性好的克隆。因此,得到库容量高、多样性丰富的抗体库非常重要。而体外重组系统的建立,有利于增加抗体库的多样性。
本研究利用72对兼并引物扩增了人抗体可变区基因,通过摸索PCR扩增条件、优化制备电转感受态、提高连接物浓度,成功构建了一个具有良好基因多样性的人源天然噬菌体抗体库,库容为1.47×10~7,并通过菌株改造和突变loxP位点的应用,建立了适于抗体库的Cre-loxP重组系统,使得在Cre酶的作用下不同scFv基因间的VH、VL进行重组,抗体轻重链能够自由随机的组合重排,重组过程可以无限重复,每一次重组的产物都可以作为下次重组的底物,便于以后的筛选和亲和力成熟,最后获得8×10~11超大容量的噬菌体单链抗体库,建立了超大容量抗体库抗体库制备人源抗体的技术平台,为以后针对各种病毒、病原菌、毒素等抗原进行筛选,获得特异性人源治疗性单抗奠定了基础。
抗体在阻止病毒入侵宿主细胞,抑制病毒复制方面起着重要的作用。人源抗体技术的发展,也为病毒性疾病的治疗带来了新的亮点。本研究通过固液相及不同H5N1型禽流感病毒株对抗体库进行筛选,经过多毒株三轮淘洗,洗脱下来的噬菌体抗体呈明显增加趋势,表明对病毒HA的噬菌体抗体得到了有效的富集。利用ELISA和HAI等检测手段,获得9株抗体并进行原核表达获得scFv抗体蛋白。其中4株抗体能与H5亚型三株病毒交叉反应,4株中的一株抗体还表现出对H1、H3、H5亚型毒株的强交叉。有望成为禽流感抗体药物,用于高致病性高死亡率流感的治疗。此外,可以用这些抗体寻找HA1上的保守表位,研究流感病毒突变机制,研制通用疫苗。
The therapeutic potential of monoclonal antibodies (mAb) was quickly realised after the hybridoma technique allowed their development in the mid 1970s. But the mouse anitibodies do not fit for the therapy because of HA1MA (human antimurine antibody response) which can lead a unexpectable immune reaction and make the antibodies degrade quickly in vivo. In the following years, the rapid development of human antibody technology make antibodies using as therapeutic drug to be possible. While techniques of humanization, human memorr B cell immortalization and antibody engineering booming, more and more antibodies became performing clinicle researches, and went into the market.
With easily operating and high-throughput screening, phage display antibody library shows to be one of the most useful techniques for gain human antibodies. If the normal process using for construction the library, the capacity would be much smaller than it should be. It's for the reason that a large part of antibody genes would be lost in PCR, ligation and transfromation. In the other side, the antibody light chain and heavy chain genes in library can not recombine freely as in immune system. All of these make gain a good antigen especial reaction antibodies to be difficult. However, after presenting a recombination system, the diversity of library can be enlarged obviously. Additionally, a large library with 10" were gained.
In this research, on the basis of the primers which could amplify almost the whole antibody V-region genes, a normal single-chain phage antibody library has been prepared successfully. Also the Cre/lox recombination system was established. This system ensure the diversity of our scFv library.
Antibodies play important roles in protection against and recovery from virus infection. Techniques of human antibody bring a sparking point for therapy of viral diseases. Herein, the scFv library was sCreened with different H5N1 AIV strains in liquid/solid-phase, while detection with ELISA and HAL Then the positive clones were expressed in E. coli. as scFv proteins. Some clones showed cross-activity to different H5 virus strains. Remarkably, one of them can greatly cross-react with H1, H3 and H5 virus. The results depict that the antibodies against conserved epitops in virus have been gotten. It appears an approachable method for developing therapeutic antibody to influenza viruses who usally change themselves.
引文
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