| Exercise contributes to health,but the scientific mechanism is still lacking in understanding.Muscles and bones are the main organs of movement,Under the stimulation of exercise,It has a regulatory effect on the body through self-and paracrine functions.Irisin is a muscle factor secreted by skeletal muscle,Regulates bone remodeling,fat and sugar metabolism,It has attracted attention as a protein hormone with potential therapeutic effects.In this paper,an engineered Escherichia coli strain containing the Irisin coding sequence was reconstructed,And successfully expressed recombinant Irisin.Established a purification process based on chromatography technology,obtain high purity recombinant Irisin,verified the activity of recombinant Irisin,It also preliminarily analyzed the effect of the combined action of mechanical stimulation and Irisin on the expression of bone remodeling genes in bone cells.By establishing a viable Irisin production process,laid the foundation for further scientific research.The main research contents and conclusions are as follows:(1)The recombinant plasmid p MAL-c5X-Irisin was constructed and transformed into E.coli BL21.Induced fermentation and expressed MBP-Irisin fusion protein inclusion body.The optimized fermentation conditions were as follows: the inoculum amount is 2%,the fermentation time is 3 h,and the amount of IPTG added is 0.3 m M.(2)Purified recombinant Irisin.MBP-Irisin was preliminarily purified by Q anion chromatography,The established elution conditions are: 20 m M Tris,8 M urea,0.1%?-mercaptoethanol,200 m M Na Cl,p H 7.5.Establish dilution renaturation as a renaturation method.And optimize the ratio of enzyme digestion per 1mg protein: 2U enzyme.Then use MBP affinity chromatography to separate Irisin from MBP.Finally,it was purified by Q anion chromatography.Through HPLC detection,the purity of recombinant Irisin was determined to be 91%.WB detection confirmed that the obtained product was recombinant Irisin.(3)The activity of recombinant Irisin was analyzed by 3T3-L1(Mouse embryonic fibroblasts)preadipocyte differentiation and maturation experiment.The 3T3-L1 treated with Irisin was stained with Oil Red O.The absorbance value at 492 nm is:The negative control group is 0.443 ± 0.006,<the experimental group is 0.68 ± 0.006,and <the positive control group is 0.75 ± 0.01(n=3).Shows that recombinant Irisin has the biological activity of inducing 3T3-L1 differentiation.Site-directed modification of the N-terminal amino group of Irisin with PEG.After modification,purified by reversed-phase chromatography to obtain 94% pure Irisin-PEG.And verified that its activity to promote the differentiation of 3T3-L1 is 81.3% of Irisin,and its thermal stability is much higher than that of Irisin.(4)To study the effect of Irisin on the expression of bone remodeling genes in a mechanical environment.Co-stimulation of MLO-Y4 cells using Irisin and FSS(Fluid Shear Force),The m RNA expression of bone remodeling factors Sost(osteostosis protein),OPN(osteopontin)and RANKL(nuclear factor k B receptor activator ligand)in MLO-Y4 were detected by RT-PCR.It was found that FSS can promote the increase of OPN m RNA secretion produced by Irisin stimulation;FSS can inhibit the decrease in secretion of RANKL m RNA and Sost m RNA stimulated by Irisin.It shows that Irisin can cooperate with FSS to affect the expression of bone metabolism-related factors of MLO-Y4.In this study,the prokaryotic recombinant expression and Irisin purification process were established;In vitro experiments confirmed that the prepared Irisin has biological activity;PEG site-directed modification can improve stability and maintain certain biological activity. |
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