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目的:利用酵母展示技术建立筛选scFvs的技术平台,为今后从抗体库中筛选高亲和力抗体奠定基础。方法:改造pYD1载体,将其自身所带的GSLinker上下游两端突变出酶切位点,构建成pYD-x;分别将IL-1β抗体的重链与轻链可变区片段插入pYD-x中GSLinker的上下游,构建出重组质粒pYSD1。从pYSD1上PCR扩增出scFv片段,并将其插入pYD1的MCS区,构建出重组质粒pYSD2。将pYSD1与pYSD2分别转入酿酒酵母EBY100中诱导表达scFv,并用流式细胞术(FCM)检测抗体展示情况。结果:经诱导后,从流式细胞仪检测结果中可以看出EBY100-pYSD1所展示的scFv与抗原结合力很弱,而EBY100-pYSD2则表现出一定强度的结合。结论:本实验证明了pYD-x载体上存在的额外的GSLinker对于抗体展示的必要性,并且成功建立了利用酵母展示载体pYD1进行scFvs筛选的技术平台。
OBJECTIVE: To establish a technology platform for screening scFvs by yeast display technology and lay a foundation for screening high affinity antibodies from antibody library in the future. Methods: The pYD1 vector was transformed and its upstream and downstream ends of GSLinker itself were mutated into pYD-x. The heavy and light chain variable fragments of IL-1β antibody were inserted into pYD-x In the upper and lower GSLinker, to construct a recombinant plasmid pYSD1. The scFv fragment was amplified by PCR from pYSD1 and inserted into the MCS region of pYD1 to construct the recombinant plasmid pYSD2. PYSD1 and pYSD2 were transformed into Saccharomyces cerevisiae EBY100 to induce the expression of scFv, and the antibody expression was detected by flow cytometry (FCM). Results: After induction, the results of flow cytometry showed that scFv exhibited by EBY100-pYSD1 showed weak binding ability with antigen while EBY100-pYSD2 showed a certain degree of binding. Conclusion: This experiment demonstrates the necessity of additional GSLinker on the pYD-x vector for antibody display and has successfully established a technology platform for scFvs screening using the yeast display vector pYD1.