口蹄疫病毒单链抗体构建与表达
发布时间：2018-07-15 16:20
【摘要】：单链抗体（single-chainvariableFragment，scFv）是将抗体的重链可变区（VH）和轻链可变区（VL）通过一段10～25个氨基酸的柔性连接肽（Linker）连接成的小分子抗体。尽管单链抗体去除了抗体的恒定区，并且引入了连接肽，却保留了抗体的特异性。通过噬菌体展示或核糖体展示技术，可以将scFv的抗原结合域以多肽的形式表达。作为一种可替代的抗体，scFv的重链可变区和轻链可变区基因可以从杂交瘤细胞或者B淋巴细胞亚克隆获得。scFv具有许多用途，例如，人工T细胞受体的抗原结合结构域，流式细胞仪，免疫组织化学，并在生物病原体、寄生虫和微生物污染等预防和检测方面有可观的应用前景。 实验利用PCR技术扩增的VH和VL分别属于Balb/c鼠抗体重链和轻链可变区基因，进一步利用SOE-PCR技术，通过（Gly4Ser）3的柔性连接肽构建出库容量为5.74×1013的口蹄疫病毒scFv基因库。以与GenBank中序列相似性为100%，PCR扩增的Balb/c鼠抗体轻链恒定区作为构建核糖体展示文库的间隔序列。在构建scFv条件的基础上，通过一系列PCR和SOE-PCR成功构建出库容量为9.81×1013的口蹄疫病毒scFv核糖体展示文库。经分析，该文库中核糖体展示组件完整，目的序列之间存在多样性，具有可扩增性。将核糖体展示文库进行体外转录和体外翻译，并对翻译后的产物利用SDS-PAGE证实产生了scFv，形成了“mRNA-核糖体-scFv”复合体；并以FMDV抗原和纯化的146S病毒粒子为靶标，利用固相亲和筛选方法筛选该复合体混合物，得到相应的mRNA，经过RT-PCR得到筛选后的DNA文库。通过五轮核糖体展示获得了单链抗体。成功构建了pET-scFv重组表达质粒，并转化BL21（DE3）细胞进行原核表达，经SDS-PAGE分析，在约32KD处有新生表达条带，利用Western-blot进一步证实了scFv确实得到表达。 本研究结合scFv的特性，以纯化FMDV146S病毒粒子免疫Balb/c鼠分离的脾细胞为材料，提取RNA，通过SOE-PCR技术成功构建了FMDVscFv基因库，并进一步构建了FMDVscFv核糖体展示文库，，首次利用核糖体展示技术从文库中筛选出针对FMDV的scFv，并对其进行表达与初步研究，将为scFv用于FMD的研究、预防、治疗提供帮助，也为研制FMD的快速诊断技术奠定基础。
[Abstract]:Single-chain-invincible FragmentscFv is a small molecular antibody that is linked to the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody through a flexible binding peptide (Linker) of 10 ~ 25 amino acids. Although scFv removes the constant region of antibody and introduces ligand peptide, it retains the specificity of antibody. The antigen binding domain of scFv can be expressed as polypeptide by phage display or ribosomal display. The heavy chain variable region and light chain variable region gene can be obtained from hybridoma cells or B lymphocyte subclones as an alternative antibody to scFv, for example, the antigen-binding domain of artificial T cell receptor. Flow cytometry, immunohistochemistry, and the prevention and detection of biological pathogens, parasites and microbial contamination are promising applications. The VH and VL genes of Balb / c mouse antibody were amplified by polymerase chain reaction (PCR), respectively. By using SOE-PCR technique, the FMDV gene library with capacity of 5.74 脳 1013 was constructed by using (Gly4Ser) 3 flexible ligand peptide. The light chain constant region of Balb / c mouse antibody amplified by PCR was used as the spacer sequence to construct the ribosomal display library. On the basis of constructing scFv condition, the library of FMDV scFv ribosome was successfully constructed by a series of PCR and SOE-PCR. The library capacity of FMDV was 9.81 脳 1013. The analysis shows that the ribosomal display module is complete and the target sequences are diverse and scalable. The ribosomal display library was transcribed in vitro and translated in vitro, and the translated product was confirmed by SDS-PAGE to produce scFv-mRNA-ribosomal scFv complex, which was targeted at FMDV antigen and purified 146S virus particles. The complex mixture was screened by solid phase affinity screening method, and the corresponding mRNAs were obtained. The DNA library was screened by reverse transcription-polymerase chain reaction (RT-PCR). Single chain antibodies (scFv) were obtained by five rounds of ribosomal display. The recombinant plasmid pET-scFv was successfully constructed and transformed into BL21 (DE3) cells for prokaryotic expression. After SDS-PAGE analysis, a new expression band was found at about 32KD. The expression of scFv was confirmed by Western-blot. In this study, FMDV scFv gene library was successfully constructed by using purified spleen cells isolated from Balb / c mice immunized with FMDV146S virus particles. FMDVscFv gene library was successfully constructed by SOE-PCR, and FMDVscFv ribosomal display library was further constructed. The scFv for FMDV was screened from the library for the first time using ribosome display technique, and its expression and preliminary study were carried out, which will provide the help for the research, prevention and treatment of FMD, and also lay a foundation for the rapid diagnosis of FMD.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2124664
[Abstract]:Single-chain-invincible FragmentscFv is a small molecular antibody that is linked to the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody through a flexible binding peptide (Linker) of 10 ~ 25 amino acids. Although scFv removes the constant region of antibody and introduces ligand peptide, it retains the specificity of antibody. The antigen binding domain of scFv can be expressed as polypeptide by phage display or ribosomal display. The heavy chain variable region and light chain variable region gene can be obtained from hybridoma cells or B lymphocyte subclones as an alternative antibody to scFv, for example, the antigen-binding domain of artificial T cell receptor. Flow cytometry, immunohistochemistry, and the prevention and detection of biological pathogens, parasites and microbial contamination are promising applications. The VH and VL genes of Balb / c mouse antibody were amplified by polymerase chain reaction (PCR), respectively. By using SOE-PCR technique, the FMDV gene library with capacity of 5.74 脳 1013 was constructed by using (Gly4Ser) 3 flexible ligand peptide. The light chain constant region of Balb / c mouse antibody amplified by PCR was used as the spacer sequence to construct the ribosomal display library. On the basis of constructing scFv condition, the library of FMDV scFv ribosome was successfully constructed by a series of PCR and SOE-PCR. The library capacity of FMDV was 9.81 脳 1013. The analysis shows that the ribosomal display module is complete and the target sequences are diverse and scalable. The ribosomal display library was transcribed in vitro and translated in vitro, and the translated product was confirmed by SDS-PAGE to produce scFv-mRNA-ribosomal scFv complex, which was targeted at FMDV antigen and purified 146S virus particles. The complex mixture was screened by solid phase affinity screening method, and the corresponding mRNAs were obtained. The DNA library was screened by reverse transcription-polymerase chain reaction (RT-PCR). Single chain antibodies (scFv) were obtained by five rounds of ribosomal display. The recombinant plasmid pET-scFv was successfully constructed and transformed into BL21 (DE3) cells for prokaryotic expression. After SDS-PAGE analysis, a new expression band was found at about 32KD. The expression of scFv was confirmed by Western-blot. In this study, FMDV scFv gene library was successfully constructed by using purified spleen cells isolated from Balb / c mice immunized with FMDV146S virus particles. FMDVscFv gene library was successfully constructed by SOE-PCR, and FMDVscFv ribosomal display library was further constructed. The scFv for FMDV was screened from the library for the first time using ribosome display technique, and its expression and preliminary study were carried out, which will provide the help for the research, prevention and treatment of FMD, and also lay a foundation for the rapid diagnosis of FMD.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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