羧肽酶A1及其活性中心基因的表达及活性分析

目的:克隆羧肽酶A1(carboxypeptidase A1,CPA1)全酶及其活性中心基因,构建重组表达载体进行诱导表达,分析表达产物活性.方法:RT-PCR扩增CPA1全酶及活性中心基因,测序分析后将目的基因克隆于表达载体pGEX-4T-1,测序证实插入方向正确后转化大肠杆菌BL21,IPTG诱导表达目的基因后SDS-PAGE电泳分析.目的蛋白经变性、复性、纯化后MTT法和集落形成试验鉴定其活性.结果:获得了人CPA1全酶及活性中心基因,目的基因诱导表达后约66 kD及46 kD处可见新生蛋白带;活性分析显示CPA1全酶和活性中心蛋白都具有一定的催化水解活性,但后者对肿瘤细胞的杀伤效果较弱.结论:能成功克隆了人CPA1全酶及其活性中心基因,获得了二者的原核表达产物,体外具有一定活性.可望以人羧肽酶A1全酶及其活性中心基因为新起点,继续完善优化羧肽酶A1系统,为抗体靶向治疗前列腺癌的临床应用奠定基础.

The Expression of Human Carboxypeptidase A1 and Its Active Center Gene

Objective: To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPAlactive center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E. coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test. Results: Human carboxypeptidase Al and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells. Conclusions: Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in. vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.