| This thesis involves the cloning and expression of three proteins which is aldose reductase, aldehyde reductase (associated with diabetes syndrome and inflammation) and a new cholera toxin similar chimeric protein with brain targeting effect. It also involves the screening of related inhibitors using aldose reductase and aldehyde reductase.Aldose reductase(AKR1B1), a member of the Aldo keto reductase family, is a key rate limiting enzyme in the polyol pathway. More and more researches found that AKR1B1 is associated with many diseases, plays a key role in a series of inflammatory disease and has a direct relationship with the production of many inflammatory mediators. It could be a new target for screening inhibitors among non steroidal anti-inflammatory drugs. In order to screen the corresponding enzyme inhibitors, provide references for drug development and clinical application, we constructed the recombinant plasmid pET-22b-AKR1B1-(His)6 and transformed the plasmid into Rosetta(DE3) host bacteria. Through the optimization experiments, the expression conditions which were 0.175 mMIPTQ 14℃,80 rpm and inducing 14h were confirmed. The recombinant proteins were expressed in soluble and precipitate forms. The fusion protein of AKR1B1-(His)6 with 93% purity was purified by affinity purification. Western blotting and protein sequence analyzer were used to analysis the target protein. By using in vitro enzymatic kinetic method, the specific activity of AKR1B1-(His)6 was determined as (9.4±0.23) U/mg. Then this protein was used to establish the screening methods for aldehyde reductase inhibitors. Thirteen kinds of non steroidal anti-inflammatory drugs were screened for their inhibitory activity on AKR1B1-(His)6.The results showed that Nai Pusheng, Bloven, diclofenac and flufenamic acid have a stronger inhibitory effect, suggesting that they can be used together with anti-diabetic complications drugs.Aldehyde reductase (AKR1A1), a member of the aldehyde ketone reductase family, can restore the toxic aldehyde to nontoxic alcohol. To study the inhibitory effect of non steroidal anti-inflammatory drugs on AKR1A1, providing a reference for the side effects and clinical application of non steroidal anti-inflammatory drugs, we constructed the recombinant plasmid pET-22b-(His)6-DDDDK-AKR1A1 and transformed the plasmid into Rosetta(DE3) host bacteria. Through the optimization experiments, the expression conditions which were 0.25 mMIPTG,16℃,80 rpm and inducing 14h were confirmed. The recombinant proteins were expressed in soluble form. The fusion protein of pelB-(His)6-DDDDK-AKR1A1 with 90% purity was purified by affinity purification. The recombinant proteins were specific sheared by intestinal kinase. After Ni-NTA and Sephadex G-75 purification, wild type AKR1A1 protein could be obtained with 98% purity. The western blotting analysis and protein sequence analysis were used for identification of the fusion protein. In vitro enzyme dynamic method was used to determin the activity of the fusion protein. The results showed that the activity of (His)6-DDDDK-AKR1A1 was (41.3±0.1) U/mg and the activity of AKR1A1 was (79.4±0.15) U/mg.The results also showed that Oxaprozin, ketone of ketoprofen, fluorine destroy acid and tolfenamic acid have a strong inhibitory effect on AKR1A1, providing a reference for the anti-inflammatory drug side effects.In order to obtain a new chimeric protein with brain target effect, we designed a new chimeric protein and expressed it by using prokaryotic co-expression system We contructed recombinant plasmids pET-28a-CTB and pET-22b-EGFP-CTA2-TAT res pectively. By using different resistance of them, both plasmids were transformed into Escherichia coli BL21. After optimizing expression conditions, the recombinant chimer ic protein can be expressed in soluble form. After the purification with Sephadex G-75, Ni-NTA, (CTB)5/EGFP-CTA2-TAT chimeric protein could be obtained. This study provided the material basis for further study in drug delivery carrier. |
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