应用细菌表面展示技术快速筛选抗原表位及研制应急疫苗
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摘要
细菌表面展示技术作为噬菌体表面展示技术的有益补充，已被广泛用于生命科学各个领域。而构建基因工程减毒活疫苗是研究细菌展示系统的最初动因，也是这一系统最活跃的研究方向。但以往研究多是将已知抗原片段或表位展示于细菌表面用作实验性活菌苗。本研究提出了从细菌展示随机肽库筛选获得的抗原表位直接用作应急疫苗的新思路。
本研究首先以针对HBV pres的单克隆抗体为模型，生物淘洗商品化细菌鞭毛展示随机十二肽库，获得该抗体抗原模拟表位，核心序列为R-RG-Y，与HBV preS蛋白的135-140氨基酸同源；将展示模拟表位的菌体克隆直接免疫小鼠，可获得针对HBV preS蛋白的高滴度、高特异性抗体，表明从细菌展示随机肽库中筛选获得的抗原表位直接用于应急疫苗研制是可行的。
为克服常规生物淘洗在多抗表位筛选中的不足，以HBV preS单抗为靶分子利用FACS对模拟文库以及随机肽库FliTrx~(TM)进行筛选，在此基础上建立消减FACS分选HBV preS免疫血清多克隆抗体抗原表位的方法，为今后从病人血清样品中进行抗原表位的筛选奠定了基础。
为更好地获得不同形式抗体的抗原表位，以细菌IgA蛋白酶β结构域为骨架构建一个库容量为5×10~6的线性化随机肽库，经分析库容量与多样性均符合抗原表位筛选要求。该随机肽库是对现有细菌展示系统的有益补充。
总之，本研究以针对HBV-preS蛋白的单克隆抗体和多克隆抗体为模型，建立从细菌表面展示随机肽库中快速获得抗原表位技术，并探讨这些携带模拟表位的细菌克隆直接应用作实验性活菌苗的可行性，探索一条将筛选到的抗原表位直接用于疫苗研制的技术路线，为在发生突发性的或未知类型的生物袭击时，研制应急防护疫苗进行了有益尝试。
Bacterial surface display systems, as an alternative system of phage display, have wide range of biotechnological and industrial applications, in which live bacterial vaccine development is the most promising and attractive. To date, a diverse variety of well-known vaccine candidates (an immunogenic peptide or protein) or mimotopes screened from phage displayed peptide library have been successfully displayed on bacterial surface and have induced higher antibody response to the native antigen. Nevertheless, numerous studies have demonstrated that the insertion of heterologous epitopes into loops of bacterial proteins would potentially change the environment of epitopes and inevitably disturb its structure integrity, which usually leads to the failure of Abs response against the native antigen. Since epitope mapping has also been accomplished by bacterial peptide library, bacteria displayed mimotopes screened from random library should be directly used as recombinant live vaccines.
Firstly, a monoclonal antibody against the HBV preS protein and a commercially available bacterial peptide library were involved as a model system to practice this process. After five successive rounds of biopanning, bacterial clones interacting with the monoclonal antibody 3B9 were greatly enriched. The R-RG-Y motif was identified as mimotopes of HBV preS protein at 135-140 residues from sequence analysis. Strong responses against HBV preS protein were obtained after mice were immunized by i.p injection of live bacteria without any adjuvant. These results imply that bacterially-displayed mimotopes screened from peptide library could be directly used as live vaccine in case of emergency infectious diseases.
To overcome the limitation of conventional biopanning, FACS was subsequently introduced to epitope screening from FliTrx?peptide library. Epitopes of the
monoclonal antibody were successfully identified from the constructed model library or FliTrx?peptide library. Due to more complexity of polyclonal antibody over monoclonal antibody, a subtractive FACS strategy was established, which constitute the foundation for epitope screening from patient serum in the further study.
To complement the existing constrained libraries, a novel bacterial random peptide library, with 5x10 primary bacterial colonies, was constructed using the ?-domain of IgA protease as an anchoring motif. The DNA sequence analysis showed that the library had sufficient complexity and diversity. This non-constrained system provides an alternative tool for further epitope screening.
In conclusion, bacterially-displayed mimotopes screened from bacterial peptide library could be directly used as live vaccines, which proved to be a less expensive, yet more effective and rapid route for vaccine development than the conventional methods. By this strategy, we could also obtain mimotopes for the unrecognized or previously unknown antigens from patient serum. In this context, it is of great significance to develop the effective vaccines for such emerging infectious diseases whose pathogen is still unknown.
本研究首先以针对HBV pres的单克隆抗体为模型，生物淘洗商品化细菌鞭毛展示随机十二肽库，获得该抗体抗原模拟表位，核心序列为R-RG-Y，与HBV preS蛋白的135-140氨基酸同源；将展示模拟表位的菌体克隆直接免疫小鼠，可获得针对HBV preS蛋白的高滴度、高特异性抗体，表明从细菌展示随机肽库中筛选获得的抗原表位直接用于应急疫苗研制是可行的。
为克服常规生物淘洗在多抗表位筛选中的不足，以HBV preS单抗为靶分子利用FACS对模拟文库以及随机肽库FliTrx~(TM)进行筛选，在此基础上建立消减FACS分选HBV preS免疫血清多克隆抗体抗原表位的方法，为今后从病人血清样品中进行抗原表位的筛选奠定了基础。
为更好地获得不同形式抗体的抗原表位，以细菌IgA蛋白酶β结构域为骨架构建一个库容量为5×10~6的线性化随机肽库，经分析库容量与多样性均符合抗原表位筛选要求。该随机肽库是对现有细菌展示系统的有益补充。
总之，本研究以针对HBV-preS蛋白的单克隆抗体和多克隆抗体为模型，建立从细菌表面展示随机肽库中快速获得抗原表位技术，并探讨这些携带模拟表位的细菌克隆直接应用作实验性活菌苗的可行性，探索一条将筛选到的抗原表位直接用于疫苗研制的技术路线，为在发生突发性的或未知类型的生物袭击时，研制应急防护疫苗进行了有益尝试。
Bacterial surface display systems, as an alternative system of phage display, have wide range of biotechnological and industrial applications, in which live bacterial vaccine development is the most promising and attractive. To date, a diverse variety of well-known vaccine candidates (an immunogenic peptide or protein) or mimotopes screened from phage displayed peptide library have been successfully displayed on bacterial surface and have induced higher antibody response to the native antigen. Nevertheless, numerous studies have demonstrated that the insertion of heterologous epitopes into loops of bacterial proteins would potentially change the environment of epitopes and inevitably disturb its structure integrity, which usually leads to the failure of Abs response against the native antigen. Since epitope mapping has also been accomplished by bacterial peptide library, bacteria displayed mimotopes screened from random library should be directly used as recombinant live vaccines.
Firstly, a monoclonal antibody against the HBV preS protein and a commercially available bacterial peptide library were involved as a model system to practice this process. After five successive rounds of biopanning, bacterial clones interacting with the monoclonal antibody 3B9 were greatly enriched. The R-RG-Y motif was identified as mimotopes of HBV preS protein at 135-140 residues from sequence analysis. Strong responses against HBV preS protein were obtained after mice were immunized by i.p injection of live bacteria without any adjuvant. These results imply that bacterially-displayed mimotopes screened from peptide library could be directly used as live vaccine in case of emergency infectious diseases.
To overcome the limitation of conventional biopanning, FACS was subsequently introduced to epitope screening from FliTrx?peptide library. Epitopes of the
monoclonal antibody were successfully identified from the constructed model library or FliTrx?peptide library. Due to more complexity of polyclonal antibody over monoclonal antibody, a subtractive FACS strategy was established, which constitute the foundation for epitope screening from patient serum in the further study.
To complement the existing constrained libraries, a novel bacterial random peptide library, with 5x10 primary bacterial colonies, was constructed using the ?-domain of IgA protease as an anchoring motif. The DNA sequence analysis showed that the library had sufficient complexity and diversity. This non-constrained system provides an alternative tool for further epitope screening.
In conclusion, bacterially-displayed mimotopes screened from bacterial peptide library could be directly used as live vaccines, which proved to be a less expensive, yet more effective and rapid route for vaccine development than the conventional methods. By this strategy, we could also obtain mimotopes for the unrecognized or previously unknown antigens from patient serum. In this context, it is of great significance to develop the effective vaccines for such emerging infectious diseases whose pathogen is still unknown.
引文
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4． Marra MA, et al, The genome sequence of the SARS-associated coronavirus.Science.l May 2003．
5． 董洁,薛沿宁,柳川 等.模拟表位的发现及其在疫苗研究中的应用.细胞与分子免疫学杂 志 2003,19(3) :S4-S6
6． 沈倍奋 主编.分子文库.第一版.北京:科学出版社, 2001
7． Georgiou G, Stathopoulos C, Daugherty PS, et al. Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol 1997,15:29-34．
8． Francisco JA, Campbell R, Iverson BL, et al. Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci U S A 1993,90(22) :10444-8．
9． Irving MB, Pan O, Scott JK. Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics. Curr Opin Chem Biol. 2001,5:314-24
10． Yao ZJ, Chan MC, Kao MC, et al. Linear epitopes of sperm whale myoglobin identified by polyclonal antibody screening of random peptide library. Int J Peptide Protein Res 1996,48:477-485
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