肿瘤靶向治疗活性人羧肽酶A1最适片段的分析和克隆

Analysis and cloning of human carboxypetidase A1 active fragment applicated in ADEPT

目的:获得肿瘤靶向治疗用活性人羧肽酶A1(hCPA1)的最适片段。方法:计算机分析hCPA1活性相关氨基酸,PCR扩增选择最适基因片段,克隆入PQE-80载体,转化Rosetta gami2(DE3)进行表达,与hCPA1活性中心片段比较酶活性。结果:PCR成功扩增了hCPA1活性中心和hCPA1活性短片段基因,酶切鉴定和测序均证实重组表达载体构建成功,表达的蛋白以SDS-PAGE分析,分别在约25×103和20×103处出现新生蛋白条带,蛋白质印迹法实验证实了新生蛋白为目的蛋白。Hippuryl-L-Phenylalanin(H-L-P)实验显示两者均具有羧肽酶活性,且hCPA1活性短片段的活性与活性中心相当。MTT和细胞凋亡实验结果表明两者均能有效地水解前体药物MTX-α-Phe,抑制前列腺癌细胞株PC-3增殖,促进PC-3细胞凋亡。结论:成功克隆和表达了人hCPA1活性短片段基因和hCPA1活性中心,获得相对分子质量小而具有相似活性的hCPA1蛋白,为进一步将其应用于前列腺癌的ADEPT导向治疗创造了条件。

OBJECTIVE： To obtain the human carboxypetidase A1（hCPA1） active fragment which is suitable for application in ADEPT（Antibody-directed enzyme prodrug therapy）.METHODS： After analyzing the structure of human carboxypeptidase A1 active center,a short DNA fragment coding the essential amino acids was amplified by PCR.Then it was cloned into vector pQE-80,and transformed into Rosetta gami2（DE3）to induce the expression.Finally,the activity of the short active fragment was compared with the CPA1 active center fragment.RESULTS： The CPA1 active center and short active fragment gene were colned and inserted into the expression vector pQE-80 sucessfully.After IPTG induction,two novel anticipated protein bands of 25×103 and 20×103 appeared on SDS-PAGE gel.Western blot confirmed that they were the expected proteins.Both of them showed the similar carboxypetidase A1 activity in Hippuryl-L-Phenylalanine test.In addition,MTT and apoptotic assay showed that both of these new proteins effectively hydrolyzed the prodrug MTX-α-Phe,inhibited the perliferation of PC-3 protate cancer cell and promoted its apoptosis.CONCLUSIONS： A short active fragment of CPA1 including less few amino acids but the same activity compared to human carboxypeptidase A1 active center is obtained.It will be helpful for further study in ADEPT of prostate cancer.