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Cloning, Sequence analysis and Prokaryotic Expression of
Human Pancreas Kallikrein cDNA |
| Shi Weiqing Zhang Lei Sun Huaichang** |
| (Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming, Yangzhou University, Yangzhou 225009,China) |
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Abstract 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.
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Received: 01 January 1900
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