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目的寻找能够特异性与前列腺特异性膜抗原(prostate-specific membrane antigen,PSMA)胞外区结合的纳米抗体。方法通过RT-PCR技术在真核细胞HEK-293中重组表达PSMA胞外区。以该重组蛋白为包被抗原,采用固相淘选的方法在天然纳米抗体噬菌体展示库中淘选能够与之特异性结合的噬菌体,以Phage-ELISA方法筛选出具有结合活性的噬菌体克隆。通过细胞ELISA和细胞免疫荧光技术再次验证分子水平淘选到的阳性噬菌体克隆。结果测序证明编码PSMA的重组DNA片段序列正确,Western blot验证该重组蛋白成功表达。4轮固相淘选天然库使具有结合活性的噬菌体克隆得到了有效富集,阳性率从8.3%提高到64.6%。细胞ELISA测值表明PSMA表达阳性细胞明显高于阴性细胞[PSMA+:(0.621±0.043);PSMA-:(0.148±0.014),P<0.01];细胞免疫荧光亦证实分子水平淘选的阳性噬菌体与表达PSMA阳性细胞能发生特异性结合。结论从天然纳米抗体噬菌体库中淘选得到了在分子和细胞水平均能与PSMA胞外区特异性结合的阳性噬菌体克隆。
Objective To find a Nanobody capable of binding specifically to the extracellular domain of prostate-specific membrane antigen (PSMA). Methods Recombinant PSMA extracellular domain was expressed in eukaryotic cells HEK-293 by RT-PCR. Using the recombinant protein as coating antigen, phage clones capable of specific binding with the recombinant protein were panned in a natural nanobody phage display library by using a solid phase panning method. Phage clones with binding activity were screened by Phage-ELISA. The positive phage clones screened at the molecular level were again verified by cell ELISA and immunofluorescence techniques. Results Sequencing showed that the sequence of recombinant DNA encoding PSMA was correct. The recombinant protein was successfully expressed by Western blot. Four rounds of solid phase panning of natural pools enabled the phage clones with binding activity to be effectively enriched, with the positive rate increasing from 8.3% to 64.6%. The results of cell ELISA showed that PSMA positive cells were significantly higher than that of negative cells [PSMA +: (0.621 ± 0.043); PSMA -: (0.148 ± 0.014), P <0.01] PSMA-positive cells express specific binding. Conclusions Positive phage clones capable of specific binding to the extracellular domain of PSMA at both molecular and cellular levels were panned from natural Nanobody phage libraries.