抗c-Myc标签纳米抗体的筛选与应用

目的: 以c-Myc-GST蛋白为靶分子,从纳米抗体噬菌体展示免疫文库中筛选能够特异性识别c-Myc标签(EQKLISEEDL)的纳米抗体。方法: 采用固相淘选技术,筛选出能与c-Myc标签特异性结合的噬菌体,phage-ELISA鉴定阳性克隆并测序,通过基因重组技术将阳性噬菌体编码的纳米抗体基因克隆至原核表达载体pET25b(+),再转化至大肠杆菌Rosetta(DE3),IPTG诱导表达,SDS-PAGE分析重组蛋白表达情况。采用间接ELISA和量子点免疫荧光法验证纳米抗体的结合活性和特异性。结果: 通过4轮固相淘选,具有结合活性的噬菌体克隆得到了有效富集,回收率提高了145倍,阳性率从20.83%提高至85.4%。将phage-ELISA鉴定显色值高的两个纳米抗体A25和A26分别进行了重组表达,SDS-PAGE结果显示均为可溶性表达,表达量为60 mg/L。间接ELISA结果表明重组蛋白A25和A26都能够识别c-Myc标签,量子点免疫荧光法验证得到纳米抗体A25能够对SP2/0细胞内的c-Myc蛋白进行检测。结论: 成功地筛选出与c-Myc标签结合的纳米抗体噬菌体克隆,构建了两个抗c-Myc标签纳米抗体的原核表达载体并实现了可溶性表达,为检测胞内c-Myc蛋白奠定了基础。

Biopanning of Anti c-Myc-tag Nanobodies and Its Application for Bioimaging

Objective: Using c-Myc-GST protein as target molecule, nanobodies that can specifically recognize c-Myc tags (EQKLISEEDL) were screened from phage displayed immune libraries.Methods: Screening of phages specific bingding c-Myc-tag by solid-phase biopanning technology. Positive clones were identified by phage ELISA and then sequenced. The DNA fragment that coded positive nanobody phages were subclone to pET25b(+)vector ,the recombinant expression vector transformed into E.coli Rosetta (DE3) cells for expression under induction of IPTG. SDS-PAGE was used to analysis of recombinant protein expression. Finally, the binding activity and specificity of the nanobodies were confirmed by indirect ELISA and quantum dot immunofluorescence technology. Result: After four cycles solid-phase biopanning, phage with binding activity clones were effectively enriched, the recovery rate was improved by 145 times and positive rate increased from 20.83% to 85.4%. The nanobodies A25 and A26 with high OD₄₅₀ in phage-ELISA were recombinantly expressed , the production yield was 60 mg/L. Indirect ELISA results indicate that the recombinant proteins A25 and A26 can recognize the c-Myc-tag. Quantum dot immunofluorescence technology results showed that A25 could detect c-Myc protein in SP2/0 cells.Conclusion: Anti-c-Myc tagged nanobodies were successfully screened and two prokaryotic expression vector were constructed, the recombinant proteins achieved souble expression. And these laid the foundation for the detection of intracellular c-Myc protein.