【摘要】： 目的 建立核糖體展示庫,利用建立的核糖體展示技術(shù)進行篩選SEB抗體的研究。 方法 本試驗第一部分為構(gòu)建原核表達SEB蛋白的重組質(zhì)粒。選用了已成為在大腸桿菌中蛋白表達的首選的Invitrogen的pET系統(tǒng),通過SEB基因片段與N端具有6個組氨酸PET 32a載體相連接,使表達產(chǎn)物N端融合6個組氨酸標(biāo)簽,采用金屬離子螯合的親和層析法,利用HiTrap Chelating Ni柱純化目標(biāo)蛋白。提供了核糖體展示篩選所需抗原。 本試驗第二部分為構(gòu)建源于小鼠單鏈可變區(qū)基因的核糖體展示庫,并利用核糖體展示技術(shù)進行篩選SEB抗體的初步研究。通過TRIzol法提取了Balb/c和C57小鼠的總RNA,利用PCR技術(shù)擴增小鼠的重鏈可變區(qū)(VH)以及輕鏈可變區(qū)(VL),擴增和連接了核糖體展示所需要的所有元件,構(gòu)建了包括T7啟動子、莖環(huán)、核糖體結(jié)合位點、單鏈抗體基因庫、間隔序列的核糖體展示的基因摸板。其后對該文庫進行了體外轉(zhuǎn)錄和體外翻譯,以重組SEB為抗原進行了初步篩選。 結(jié)果 成功構(gòu)建了能夠表達出SEB蛋白的重組質(zhì)粒,命名為SEB321。以3mmol/LIPTG誘導(dǎo),30℃振搖4h,可表達出SEB蛋白,進一步利用HiTrap Chelating Ni柱進行了親和純化,再以進行了Western blot鑒定。成功構(gòu)建了源于小鼠單鏈可變區(qū)基因的核糖體展示庫,并且測定出最終庫容量為7.33×10~(13)。。其后對該文庫進行了體外轉(zhuǎn)錄和體外翻譯,表明其可以有效地進行體外轉(zhuǎn)錄翻譯。利用核糖體展示技術(shù)以重組SEB蛋白為抗原進行了初步篩選,獲得陽性結(jié)果。 結(jié)論 建立了庫容量為7.33×10~(13)的源自非免疫小鼠脾臟的單鏈抗體可變區(qū)基因片段的核糖體展示庫。摸索了建庫條件,并且利用商業(yè)載體PET 32a為原核表達載體成功表達出重組SEB蛋白,以重組SEB蛋白為抗原,進行了初步篩選,篩選SEB抗體結(jié)果為陽性。證明了源自非免疫小鼠脾臟的單鏈抗體可變區(qū)基因片段的核糖體展示庫能夠快速有效篩選抗體,為本課題組以后利用核糖體展示進行其它抗體的篩選奠定基礎(chǔ),也為相關(guān)核糖體展示技術(shù)的研究提供了借鑒。
[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，

本文編號：2382374