摘要：Camelid single-domain antibody fragments(nanobodies)are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent *** all nanobodies available to date have been obtained by animal immunization,a bottleneck restricting the large-scale application of *** this study,we developed three kinds of gene designatedregion pan-editing(GDP)technologies to introduce multiple mutations in complementarity-determining regions(CDRs)of nanobodies in *** the integration of G-quadruplex fragments in CDRs,which induces the spontaneous multiple mutations in CDRs;however,these mutant sequences are highly similar,resulting in a lack of sequences diversity in the *** also used CDR-targeting traditional gRNA-guided base-editors,which effectively diversify the *** most importantly,we developed the self-assembling gRNAs,which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as *** base-editors guided by self-assembling gRNAs,we can realize the iteratively diversify the *** we believe the last GDP technology is highly promising in immunization-free nanobody library construction,and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.

摘要：旨在研究RNAi沉默STAT3基因对人大细胞肺癌NCI-H460细胞增殖的影响。针对STAT3基因mRNA设计合成5条短发夹DNA,构建重组SiRNA-ST3质粒(命名为SiRNA-ST3-1,2,3,4,N)。用重组质粒分别转染NCI-H460细胞,RT-PCR法检测转染24 h后STAT3 mRNA的表达;Western blotting法检测转染24 h和48 h后STAT3、pSTAT3蛋白表达;MTT法检测转染24 h、48 h、72 h后NCI-H460细胞增殖情况。结果显示,SiRNA-ST3载体构建成功。RT-PCR和Western blotting检测结果表明,NCI-H460细胞转染重组质粒SiRNA-ST3-2和SiRNA-ST3-3后STAT3基因mRNA转录和STAT3、pSTAT3蛋白表达都明显下降(P<0.05)。与未转染组比,SiRNA-ST3-2组和SiRNA-ST3-3组NCI-H460增殖能力24 h、48 h降低明显(P<0.05);与SiRNA-ST3-N组比,SiRNA-ST3-2组和SiRNA-ST3-3组NCI-H460增殖能力48 h降低明显(P<0.05)。由此证实,构建的重组质粒SiRNA-ST3-2、SiRNA-ST3-3能有效靶向沉默STAT3基因,并抑制人大细胞肺癌NCI-H460细胞的增殖能力。