Cloneing Of Renalase Gene And It's Expression In Escherichia Coli

Background and Objective: In recent years,the number of patients with chronic renal failure (CRF) increased greatly all over the world,and the mortality and cardiovascular system complications has grown rapidly in these patients. To nephrologists, the challenges is slow the deterioration of renal functions and reduce cardiovascular system complications in the patients with CRF. Hypertension is the most common complication in cases of CRF. The renin angiotensin system(RAS) excessively excited and volume excess of body fluid was generally considered the main mechanism why these patients have hypertention.But some patients who undergone well dialysis and retarded the RAS activity still have high blood pressure.More and more research showed that patients with CRF having a high concentration of catecholamine,it was considered why patients with CRF suffer from high blood pressureand cardiovascular system complications.But its mechanism has been perplexing ephrologists.The kidney not only regulates fluid and electrolyte balance but also functions as an endocrine organ. Renalase is a novel flavin adenine dinucleotide–dependent amine oxidase (renalase) that issecreted into the blood by the kidney and metabolizes catecholamines in vitro (renalase metabolizes dopaminemost efficiently, followed by epinephrine, and then norepinephrine). In humans, renalase gene expression is highest in the kidney but is also detectable in the heart, skeletal muscle, and the small intestine. The plasma concentration of renalase is markedly reduced in patients with end-stage renal disease, as compared with healthy subjects. Renalase infusion in rats caused a decrease in cardiac contractility, heart rate, and blood pressure and prevented a compensatory increase in peripheral vascular tone. These results identify renalase as what we believe to be a novel amine oxidase that is secreted by the kidney, circulates in blood, and modulates cardiac function and systemic blood pressure.Renalase is a novel FAD dependent amine oxidase that is secreted into the blood by the kidney. It degrades catecholamines in vitro and lowers blood pressure in vivo by decreasing cardiac contractility and heart rate and preventing a compensatory increase in peripheral vascular tone. The identification of renalase is an important step in developing a more detailed understanding of cardiovascular physiology and perhaps also in the quest for providing optimal treatment for patients with kidney disease.Methods and Results:1. Cloning and analysis of the renalaseAccording to the mRNA sequence and protein sequence of renalase gene from the Genbank,specific primers for PCR were designed by using the software DNAman. The total RNA was extracted from a patient,s kidney organize after surgical operation, and human Arresten gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR)method, PCR production reclaimed and purified in low melting-point agarose and connected with plasmid pMDl8-T. Molecular cloning to prokaryotic expression plasmid pMDl8-T by recombination strategy. The prokaryotic expression plasmid pMDl8-renalase was identified by restriction enzyme digestion and sequenced.2. Expression of the human renalase in the E.coli BL21(DE3) and analysis of the recombinant human renalase proteinTo gererate the recombinant expression vector ,pMD 18-T renalase were digested with two restriction enzymes XhoI and NedI.The purified renalase fragment was inserted into the pET42b(+) digested with the same enzymes.The ligated product was transformed into the non-expression host DH5a.Then the host was spread on LB agar plate containing Kan and incubated overnight at 37℃.The positive colonies were primarily screened using colony PCR,and then incubated in LB medium containing Kan(5Oug/ml)overnight at 37℃in the Orbital Shaker.Using Guanidine Hydrochloride mini-copy recombinant plasmid DNA were isolated ,and digested with XhoI to confirm inserted positive clone.Prepare the expression host ,competent cells BL21(DE3),dilute lμl(about 1ng) recombinant plasmid with dH2O,and then transform it into BL21(DE3),which was spread on LB agar plate containing Kan and incubated overnight at 37℃.Pick one clone and inoculate 5Oml LB liquid culture medium containing Kan(5Oug/ml). The bacterium haroring pET42b(+)-renalase was grown 3 hours to early log phase at 37℃.After IPTG was added,the expression of the recombinant renalase was induced for 1-4 hours.The cells were collected by centrifugation at 4℃,4OOOrpm for 5minutes.The SDS-PAGE and Western Blotting analysis of the bacterium lysate had shown that there was an additional band that seemed to be about 38KD identified with previously reported recombinant renalase expressed in E.coli.The recombinant protein accounts for about 10%of the whole bacterium protein.Conclution:(1) Using recombinant DNA technique,we have successfully cloned human renalase gene ,and its DNA sequence was similar to that reported.(2) The prokaryotic expression recombinant has been constructed ,and it has successfully expressed in E.coli.The recombinant renalase accounts for 10% of the whole protein of E.coli.(3) Optimize the expression of the recombinant renalase...