抗前列腺特异抗原单链抗体/人羧肽酶A融合基因的构建及表达

Construction and expression of a single chain anti prostate specific antigen antibody fused to human carboxypeptidase A

目的 构建抗前列腺特异抗原 (PSA)单链抗体(sc Fv) /人羧肽酶 A(h CPA)融合基因 ,为前列腺癌的抗体导向酶 -前体药物疗法奠定基础 .方法 在已克隆抗 PSA sc Fv和 h CPA基因的基础上 ,用加端 PCR分别在 sc Fv基因的 3′端和 h CPA基因的 5′端加上部分连接肽 (linker)基因序列 ,回收后混合 ;应用重叠延伸拼接法 ,将抗 PSA sc Fv基因和 h CPA基因通过 linker序列串联为 sc Fv/h CPA融合基因 ;并将构建的融合基因克隆到融合表达载体 p GEX- 4T- 1中进行表达 .结果 序列测定结果表明 ,构建的抗 PSA sc Fv/h CPA融合基因中 sc Fv和 h CPA序列均正确 ,sc Fv和 h CPA间有 18bp的linker序列 ,推导的氨基酸序列为 GSGGSG,与设计的序列相符 .融合基因经 IPTG诱导表达重组蛋白并以 SDS- PAGE分析 ,在 Mr为 940 0 0左右出现一条新生蛋白带 ,表达量约占菌体总蛋白的 34% .结论 成功构建并原核表达了抗 PSAsc Fv/h

AIM To construct a fusion gene between a single chain variable fragment (scFv) specific for human γ seminoprotein and human carboxypeptidase A (hCPA), which could be used in antibody directed enzyme prodrug therapy for prostatic cancers. METHODS The scFv and hCPA genes were amplified by separate adding on PCR, and a part of linker peptide gene sequence was added on scFv 3′ and hCPA 5′ terminus respectively. The PCR products were mixed and reamplified and the scFv/hCPA fusion gene was spliced by overlap extension. The scFv/hCPA fusion gene was inserted into the prokaryotic fusion protein expression vector pGEX 4T 1 and expressed in E.coli . RESULTS The sequences of scFv, hCPA and linker peptide were all correct. An 18 bp linker sequence was found between scFv and hCPA, and the predicted amino acid sequence of the linker peptide was GSGGSG. The expression of E 4B 7 scFv/hCPA fusion protein was induced by IPTG. A new anticipated 94 000 protein band appeared on SDS PAGE gel and amounted to 34% of the total bacterial protein. E 4B 7 scFv/hCPA fusion protein bound the prostate cancer cells in competitive binding inhibition assay in vitro . CONCLUSION Fusion gene of E 4B 7 scFv/hCPA has been successfully constructed and expressed in E.coli JM109.