人胰腺激肽释放酶cDNA的克隆、序列分析和原核表达

摘要: 为了研制人激肽释放酶（KLK1）特异单克隆抗体和进行重组酶的鉴定及纯化，根据已发表的人KLK1序列设计引物，用PCR扩增法从人胰腺单链cDNA库中特异地扩增出KLK1 cDNA。将其克隆入pGEM-T载体中,对5个重组质粒进行了序列测定，其中1个cDNA克隆的核苷酸序列与已发表的人肾/胰/唾液腺KLK1 cDNAs序列完全相同或有3个核苷酸差异。将去除信号肽序列的KLK1 cDNA以正确阅读框插入表达载体pGEX-4T-3,构建成重组质粒pGEX-KLK1。此重组质粒转化的E.coli 经IPTG诱导后表达分子量为48 kDGST-KLK1融合蛋白，表达产物主要以不溶性包涵体形式存在，可溶性部分能被Glutathione Sepharose 4B特异吸附，两者都能被GST特异单克隆抗体识别。用SDS-PAGE分离纯化的融合蛋白免疫小鼠，获得的抗血清的ELISA效价为1∶1600。结果表明，克隆的人KLK1 cDNA及其表达产物是正确的，可以用于人KLK1单克隆抗体的制备。

Cloning, Sequence analysis and Prokaryotic Expression of Human Pancreas Kallikrein cDNA

Abstract: To get monoclonal antibodies for human kallikrein (KLK1), a pair of primers were synthesized according to the previously published sequence for human pancreas KLK1 mRNA and used to amplify the double-stranded cDNA from pooled human pancreas single-stranded cDNAs by PCR. Agarose gel electrophoresis of the PCR product showed a single band of 0.8 kb. After cloning into pGEM-T vector, 5 recombinant plasmids were sequenced, one of which was identical to the previously published human renal/pancreas/salivary KLK1 cDNAs or there was only 3 bp difference. The cDNA was excised from the pGEM-T vector by digestion with restriction endonucleases Pvu Ⅱ and Xho Ⅰ to remove its signal peptide sequence and subcloned downstream into the Sma Ⅰ/Xho Ⅰ site of prokaryotic expression vector pGEX-4T-3. The recombinant plasmid was transformed to BL21 E.coli cells for expression by IPTG induction. SDS-PAGE analysis of the transformant showed an expected size of protein band of 48 kD, which was mainly present in the insoluble fraction of the cell lysate. Western blotting of both cell lysate and Glutathione Sepharose 4B-purified expression product showed that the fusion protein was able to be recognized by a monoclonal antibody specific for glutathione-S-transferase. The antiserum, with an ELISA titer of 1∶1600, was obtained by immunizing mice with the SDS-PAGE purified fusion protein. These results demonstrated both cloned cDNA and its expression product were correct and could be used to prepare specific monoclonal antibodies for human KLK1.