口蹄疫病毒單鏈抗體構(gòu)建與表達(dá)
發(fā)布時(shí)間：2018-07-15 16:20
【摘要】：單鏈抗體（single-chainvariableFragment，scFv）是將抗體的重鏈可變區(qū)（VH）和輕鏈可變區(qū)（VL）通過一段10～25個(gè)氨基酸的柔性連接肽（Linker）連接成的小分子抗體。盡管單鏈抗體去除了抗體的恒定區(qū)，并且引入了連接肽，卻保留了抗體的特異性。通過噬菌體展示或核糖體展示技術(shù)，可以將scFv的抗原結(jié)合域以多肽的形式表達(dá)。作為一種可替代的抗體，scFv的重鏈可變區(qū)和輕鏈可變區(qū)基因可以從雜交瘤細(xì)胞或者B淋巴細(xì)胞亞克隆獲得。scFv具有許多用途，例如，人工T細(xì)胞受體的抗原結(jié)合結(jié)構(gòu)域，流式細(xì)胞儀，免疫組織化學(xué)，并在生物病原體、寄生蟲和微生物污染等預(yù)防和檢測方面有可觀的應(yīng)用前景。 實(shí)驗(yàn)利用PCR技術(shù)擴(kuò)增的VH和VL分別屬于Balb/c鼠抗體重鏈和輕鏈可變區(qū)基因，進(jìn)一步利用SOE-PCR技術(shù)，通過（Gly4Ser）3的柔性連接肽構(gòu)建出庫容量為5.74×1013的口蹄疫病毒scFv基因庫。以與GenBank中序列相似性為100%，PCR擴(kuò)增的Balb/c鼠抗體輕鏈恒定區(qū)作為構(gòu)建核糖體展示文庫的間隔序列。在構(gòu)建scFv條件的基礎(chǔ)上，通過一系列PCR和SOE-PCR成功構(gòu)建出庫容量為9.81×1013的口蹄疫病毒scFv核糖體展示文庫。經(jīng)分析，該文庫中核糖體展示組件完整，目的序列之間存在多樣性，具有可擴(kuò)增性。將核糖體展示文庫進(jìn)行體外轉(zhuǎn)錄和體外翻譯，并對翻譯后的產(chǎn)物利用SDS-PAGE證實(shí)產(chǎn)生了scFv，形成了“mRNA-核糖體-scFv”復(fù)合體；并以FMDV抗原和純化的146S病毒粒子為靶標(biāo)，利用固相親和篩選方法篩選該復(fù)合體混合物，得到相應(yīng)的mRNA，經(jīng)過RT-PCR得到篩選后的DNA文庫。通過五輪核糖體展示獲得了單鏈抗體。成功構(gòu)建了pET-scFv重組表達(dá)質(zhì)粒，并轉(zhuǎn)化BL21（DE3）細(xì)胞進(jìn)行原核表達(dá)，經(jīng)SDS-PAGE分析，在約32KD處有新生表達(dá)條帶，利用Western-blot進(jìn)一步證實(shí)了scFv確實(shí)得到表達(dá)。 本研究結(jié)合scFv的特性，以純化FMDV146S病毒粒子免疫Balb/c鼠分離的脾細(xì)胞為材料，提取RNA，通過SOE-PCR技術(shù)成功構(gòu)建了FMDVscFv基因庫，并進(jìn)一步構(gòu)建了FMDVscFv核糖體展示文庫，，首次利用核糖體展示技術(shù)從文庫中篩選出針對FMDV的scFv，并對其進(jìn)行表達(dá)與初步研究，將為scFv用于FMD的研究、預(yù)防、治療提供幫助，也為研制FMD的快速診斷技術(shù)奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392
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