运用核糖体展示技术筛选SEB抗体的初步研究
发布时间：2018-12-16 12:47
【摘要】： 目的 建立核糖体展示库,利用建立的核糖体展示技术进行筛选SEB抗体的研究。 方法 本试验第一部分为构建原核表达SEB蛋白的重组质粒。选用了已成为在大肠杆菌中蛋白表达的首选的Invitrogen的pET系统,通过SEB基因片段与N端具有6个组氨酸PET 32a载体相连接,使表达产物N端融合6个组氨酸标签,采用金属离子螯合的亲和层析法,利用HiTrap Chelating Ni柱纯化目标蛋白。提供了核糖体展示筛选所需抗原。 本试验第二部分为构建源于小鼠单链可变区基因的核糖体展示库,并利用核糖体展示技术进行筛选SEB抗体的初步研究。通过TRIzol法提取了Balb/c和C57小鼠的总RNA,利用PCR技术扩增小鼠的重链可变区(VH)以及轻链可变区(VL),扩增和连接了核糖体展示所需要的所有元件,构建了包括T7启动子、茎环、核糖体结合位点、单链抗体基因库、间隔序列的核糖体展示的基因摸板。其后对该文库进行了体外转录和体外翻译,以重组SEB为抗原进行了初步筛选。 结果 成功构建了能够表达出SEB蛋白的重组质粒,命名为SEB321。以3mmol/LIPTG诱导,30℃振摇4h,可表达出SEB蛋白,进一步利用HiTrap Chelating Ni柱进行了亲和纯化,再以进行了Western blot鉴定。成功构建了源于小鼠单链可变区基因的核糖体展示库,并且测定出最终库容量为7.33×10~(13)。。其后对该文库进行了体外转录和体外翻译,表明其可以有效地进行体外转录翻译。利用核糖体展示技术以重组SEB蛋白为抗原进行了初步筛选,获得阳性结果。 结论 建立了库容量为7.33×10~(13)的源自非免疫小鼠脾脏的单链抗体可变区基因片段的核糖体展示库。摸索了建库条件,并且利用商业载体PET 32a为原核表达载体成功表达出重组SEB蛋白,以重组SEB蛋白为抗原,进行了初步筛选,筛选SEB抗体结果为阳性。证明了源自非免疫小鼠脾脏的单链抗体可变区基因片段的核糖体展示库能够快速有效筛选抗体,为本课题组以后利用核糖体展示进行其它抗体的筛选奠定基础,也为相关核糖体展示技术的研究提供了借鉴。
[Abstract]:Objective to establish a ribosomal display library and study the screening of SEB antibodies by using the established ribosomal display technique. Methods the first part of this experiment was to construct the recombinant plasmid expressing SEB protein. The pET system of Invitrogen, which has become the first choice for protein expression in Escherichia coli, was selected. The N terminal of the expressed product was fused with 6 histidine tags by connecting the SEB gene fragment with six histidine PET 32a vectors at the N terminal. The target protein was purified by HiTrap Chelating Ni column with affinity chromatography of metal ion chelation. Ribosomal display screening of the required antigen was provided. The second part of this experiment was to construct the ribosomal display library derived from mouse single-stranded variable region gene and to screen SEB antibody using ribosomal display technique. The total RNA, of Balb/c and C57 mice was extracted by TRIzol method. The heavy chain variable region (VH) and the light chain variable region (VL),) of mice were amplified by PCR technique. All the elements needed for ribosomal display were amplified and connected, and the T7 promoter was constructed. Stem ring, ribosomal binding site, single chain antibody gene pool, spacer ribosome display gene palette. Then the library was transcribed and translated in vitro, and the recombinant SEB was selected as antigen. Results the recombinant plasmid expressing SEB protein was successfully constructed and named SEB321.. The SEB protein was induced by 3mmol/LIPTG and shaken at 30 鈩，
本文编号：2382374
[Abstract]:Objective to establish a ribosomal display library and study the screening of SEB antibodies by using the established ribosomal display technique. Methods the first part of this experiment was to construct the recombinant plasmid expressing SEB protein. The pET system of Invitrogen, which has become the first choice for protein expression in Escherichia coli, was selected. The N terminal of the expressed product was fused with 6 histidine tags by connecting the SEB gene fragment with six histidine PET 32a vectors at the N terminal. The target protein was purified by HiTrap Chelating Ni column with affinity chromatography of metal ion chelation. Ribosomal display screening of the required antigen was provided. The second part of this experiment was to construct the ribosomal display library derived from mouse single-stranded variable region gene and to screen SEB antibody using ribosomal display technique. The total RNA, of Balb/c and C57 mice was extracted by TRIzol method. The heavy chain variable region (VH) and the light chain variable region (VL),) of mice were amplified by PCR technique. All the elements needed for ribosomal display were amplified and connected, and the T7 promoter was constructed. Stem ring, ribosomal binding site, single chain antibody gene pool, spacer ribosome display gene palette. Then the library was transcribed and translated in vitro, and the recombinant SEB was selected as antigen. Results the recombinant plasmid expressing SEB protein was successfully constructed and named SEB321.. The SEB protein was induced by 3mmol/LIPTG and shaken at 30 鈩，
本文编号：2382374
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