Construction And Expression Of Urease Gene Engineering Bacteria And Screening Of Application Models

Hyperuricemia(HUA)was the disease of purine metabolism disorder in vivo and characterized by elevating uric acid(UA),which not only causes gout,but many studies have shown that HUA was the independent risk factors of metabolism-related diseases and cardiovascular disease,such as hypertension,dyslipidemia,fatty liver,insulin resistance,chronic kidney disease.The extent of hazard degree in the increase of serum uric acid(SUA)became a major health pitfall that threatened the quality of life in humans.Based on the fundamental research and prevention of HUA,which became one of the hot spots.At present research shows that the main cause of HUA is the reduction of UA excretion.UA was the final product of purine metabolism in vivo.Under normal conditions,Two-thirds of UA in the body was excreted by kidney,approximately one-thirds of UA was excreted in the intestine.Due to the limitation of water solubility of uric acid,which caused UA stones as for the kidney disorder.At present,the drugs of treating HUA and gout induced the major side effects in liver and kidney.Therefore,the research in UA metabolism turned to the intestine,itbelieved that increased the excretion of UA by intestinal tract to provide a new idea in reducing SUA.Due to the nonsense mutation of the uricase gene did not make UA be further decomposed in the process of human evolution,which leaded to the obstruction of UA broken down into allantoin,carbon dioxide,and hydrogen peroxide.In certain pathological conditions,because purine metabolism disordered,and UA excessively accumulated in the blood,leaded to HUA,which caused gout.In this experiment,an engineering strain of carrying the uricase gene was designed to express uricase in vitro,accelerating the decomposition of UA in the intestine,and play an important role in decresing UA.At the same time,different types of rat models in HUA are established to lay the foundation for the application of subsequent engineering strains.1.Construction of recombinant uricase acid gene engineering bacteria.Using the conserved vector pET-28a-suox(contain uricase gene)as a template,the suox was amplified by PCR(signal peptide gene and uricase-encoding gene),which clone into the shuttle vector pMG36 e of escherichia coli-lactic acid bacteria and is named as pMG36e-suox.Simultaneously,the lactic acid bacteria signal peptide(spo)is synthesized and ligate with the uricase gene(uox)and clone into pMG36 e,which is named pMG36e-spo-uox.Sequencing by to identify the two vectors.The result of sequence is consistent with the Gen Bank query.2.Expression of recombinant uricase.After correct expansion of theBL strain containing pMG36e-spo-uox and p MG36e-suox,the expression levels of the two expression vectors in the periplasmic space,protein inclusion body and supernatant at different time points were detected.The two expression vectors were successfully secreted in the supernatant and the maximum expression was obtained at 9 h.3.Establishment of rat model of hyperuricemia: a comparative study by feeding with simple fructose vs potassium oxonate combined with fructose.A total of 60 male SD rats were randomly divided into 6 groups,with 10 animals in each group,that is,5%Fru group(fed with normal chow diet and water containing 5% fructose),5% FPO group(containing 5%fructose and potassium oxonate),10% Fru group(containing 10%fructose),10% FPO group(containing 10% fructose and potassium oxonate),PO group(containing potassium oxonate)and Con group(control group,normal water).The experiment lasted for 14 weeks,and the levels of serum uric acid(SUA),urea nitrogen(BUN)and serum creatinine(SCr)were measured at every weekend.The pathological changes of the liver and kidneys were observed at the end of experiment.5%FO group,10%Fru group,10%FO group could rapidly increase the SUA level(P<0.01).At weeks 10 and 11,the level of SUA was significantly lower in the 10% Fru group than the 5%FO group and 10% FO group(P<0.01).The levels of BUN and SCr were gradually increased since weeks 6 and 7 in the10%Fru group and 10%FO group(P<0.01).Obviously histological featureswere observed in the kidneys of the rats from the 10%Fru group and10%FO group,but the changes were mildly in the kidney of the 5%FO group.There were no significant morphological changes in the liver of the rats from all the groups.Conclusion:Fructose combined with potassium oxonate can successfully establish a stable and highly effective rat model of hyperuricemia,Different doses of fructose show different effects on renal damage,and can be used for the animal models due to different studies.