Experimental Study On The Effects Of Transfer Of Human Tissue Kallikrein Gene Into Goldblatt Hypertensive Rats

Objective To construct and of human tissue kallikrein gene(hKLK) contained eukaryotic expressing vector and to investigate the hypotensive effects and mechanisms of kallikrein (KLK) gene transfer into skeletal muscle on renal hypertensive rats.Methods Amplified the human tissue kallikrein gene by means of PCR from the template of pBluescripe II KSKK (+) ,subclonged into eukaryotic expressing plasmid and formed high efficient eukaryotic expressing plasmid pcDNA3-KLK.After restriction endonuclease digestion analysis, pcDNA3-KLK was sequenced by ABI 377 sequencer. And the recom- binant plasmid was injected into quadriceps femoris muscle of renal hypertensive rats. Six times of electric pulse at 80v, 1Hz, 40ms were given immediately after the injection. The systolic pressure was measured by tail-cuff twice a week. The expression of hKLK gene and it' s protein were assayed by RT-PCR and Western blot.Results A 717 base pairs single DNA fragment was found in the products by 10g/L agarose electrophoresis, the same size expected for the human tissue kallikrein gene.The insertion fragment could be seen after restricton digestion as expected. A single injection of naked KLK plasmid DNA caused a 8-21 mmHg reduction of systolic pressure for at least 4 weeks as compared with controls injected with vector DNA alone (/XO. 01). Excretion on hKLK construct received animals due to the local expression of KLK and it' s secretion into plasma. The expression of mRNA and protein of hKLK gene was detected in the skeletal muscle of rats after transfection.Conclusion: pcDNA3-hKLK eukarytic recombinant has been constrcted successfully. The hKLK gene could be effectively transfected to rats by electroporation and played decrease of blood pressure role in vivo. Thismethod might be a promising gene therapy method for providing therapeutic protein in circulation.