Cloning, Expression Of Human Tissue Kallikrein And Its Preliminary Study On Gene Therapy For Hypertention

Human tissue kallikrein(KLK1) proteolizes kininogen to release kinin, which plays important roles in blood pressure regulation, renal function and so on. To develop the recombinant KLK1, KLK1 cDNA was amplified from pooled human pancreas single-stranded cDNAs by high fidelity PCR. Sequence analysis showed that the KLK1 cDNA was identical to or differed by only 3bp from the previously published human renal/pancreas/salivary KLK1 cDNAs. The cDNA was digested by restriction enzymes to remove its signal peptide sequence and then subcloned into the prokaryotic expression vector pGEX-4T-3. The GST-KLK1 fusion protein of about 48kDa was expressed in E.coli by IPTG induction, which was confirmed by Western blotting. The antiserum and monoclonal antibody were prepared by using the prokaryotic expression product as the antigen or by injection of mice with KLK1 cDNA cloned into eukaryotic expression vector pcDNA. The recombinant vector was mixed with branched 25kDa polyethylenimine and then transfected into COS-1 cells. The expression was revealed by indirect fluorescent assay. The systemic blood pressure of spontaneously hypertensive rats (SHR) was decreased after injection of the recombinant vector, from 165 to 125 mmHg, which lasted for 2 to 3 weeks. The KLK1 cDNA was subcloned into a mammary gland-specific expression vector p205C3 and then was injected into the mice via tail vein. The up to 292 U/ml KLK1 activity was detected in the milk of the gene injected mice by spectrophotometry. These data demonstrated the correctness of KLK1 cDNA and usefulness for production of recombinant KLK1 and gene therapy for hypertension.