The Preparation And Activity Detection Of Expressed Antihypertensive Peptides From Recombinant E.coli BL21by Genetic Engineering

Heart cerebrovascular disease and its complication caused by Hypertension havebeen thought as the first murderer to the patients nowadays and could be a threat to thehealth of human beings. It is very necessary to look for a safe material which is having thebiological activity and could fall the blood pressure effectively to avoid the side effectsemerging in the traditional medicines, such as the ache, fever and disgusting. Whilecompared to the synthetic medicines some peptide in the food protein have overcome theshortcoming of low activity and have demonstrating its advantages of safety, validity andlow side effect. However, because of the low content of the effective peptide in the naturalprotein, it is difficult for researchers to refine and purify it from proteins. Using the chemicalsynthesis method to get the peptide also has some inevitable problems. All of these havebeing the barriers in the production of the peptide which can fall the blood pressure.Nowadays, genetic engineering technology has been got a rapid and inspiringdevelopment which is also leading a new enlightenment for the peptide synthesis andproduction. The problems in the peptide production such as the limitation of materials,complications of refinement and purification and the long product cycle will be solved bythis technology.This test was based on Bullfrog muscle protein, this gene sequences which can betranslated to Gly-Ala-Ala-Glu-Leu-Pro-Cys-Ser-Ala-Asp-Trp-Trp were connectedwith arginine and then were inserted by histidine, which a new strand DNA（ACEIP）couldbe made as having the function of falling the blood pressure. Then this DNA sequencewas recombined with the plasmid carrier pET-28which could express in both Escherichiacoli.. After being induced by IPTG, it could be detected by electrophoresis of SDS-PAGEor reformed Trincine-SDS-PAGE. If it was well expressed, separate and purify thepeptides with labeled histidine by immobilized metal-chelated affinity chromatography（Ni-NTA）,;and at last the suppressant to ACE of this peptide should be detected.The result shows that the synthetic gene sequence was connected with plasmid carrier forming pET-28-ACEIP, then it was transferred into the Escherichia coli DH5a toget the positive plasmids. After inducing by IPTG and analyzing of SDS-PAGE, a proteinof8.6kDa is got and confirmed that the gene was expressed successfully. While at lastafter the processing of Ultrasonic wave and being separated and purified by Ni-NTA, apeptide of8.6kDa with the function of falling the blood pressure was got,and detecting it’sactivity in vivo and in vitro,the peptides we got are higher activity of falling the bloodpressure.