前列腺特异性膜抗原胞外区的真核表达及其纳米抗体的淘选  

Eukaryotic expression of extracellular domain of prostate-specific membrane antigen and biopanning of its nanobodies

作　　者：范校周[1] 郭燕丽[1] 涂追[2] 童海鹏[1] 李浪[1] 王洛夫 黄海韵[1] 许杨[2,4] 

机构地区：[1]第三军医大学西南医院超声科,重庆400038 [2]南昌大学食品科学与技术国家重点实验室,南昌330047 [3]中德联合研究院 [4]第三军医大学大坪医院野战外科研究所泌尿外科,重庆400042

出　　处：《第三军医大学学报》2013年第21期2320-2324,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(30970830);重庆市自然科学基金(CSTC2012jjB0072);江西省教育厅青年科学基金(GJJ12142)~~

摘　　要：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 investigate the nanobodies specifically against the extracellular domain of prostate-specific membrane antigen（PSMA）.Methods The extracellular domain of PSMA was recombined in eukaryotic HEK-293 cells through RT-PCR.With the recombinant protein as coating antigen,the solid phage selection was used in biopanning of nanobody phage display library.And the phage-ELISA was employed to screen out the phage clones with binding activity.Finally,cell ELISA and immunofluorescence technology were applied to verify the above phage clones.Results Sequencing indicated that the recombinant DNA sequence coding extracellular domain of PSMA was correct,and Western blot assay proved the successful expression of the protein.The phage clones with binding activity had got effective enrichment by 4 cycles of biopanning,and the positive ratio was increased from 8.3% to 64.6%.Cell ELISA showed that the absorption of PSMA-positive cells was significantly higher than that of PSMA-negative cells（0.621±0.043 vs 0.148±0.014,P0.01）.Immunofluorescence assay also showed that the phage clone from the above bound specifically to the PSMApositive LNCaP cells.Conclusion Our phage clones derived successfully from the natural nanobody phage library are specially against the extracellular domain of PSMA in molecular and cellular levels.

关 键 词：前列腺特异性膜抗原 纳米抗体 噬菌体展示 

分 类 号：R392.11[医药卫生—免疫学] R392.13[医药卫生—基础医学]