Screening,Identification Of Streptomyces CL-64 With Inhibitory Activities On Calcineurin And Condition Of Purificating The Active Substance

Calcineurin is the only known Ser/Thr protein phosphatase which is regulated by cellar second messenger Ca²⁺and Calmodulin（CaM）,it’s also known as protein phosphatase 2B.Calcineurin plays an important role in physical activity like T cell activation,cell signal transduction,immune regulation,learning and memory,Alzheimer’s disease.CsA（Cyclosporin A）and Fk506（Tacrolimus）as Calcineurin inhibitor is the most widely used clinical immunotherapy drug.This study has screened more than 3000 strains from soil by a yeast report gene（CDRE::LacZ）based high throughput screening model.Three strains with high calcineurin inhibitory activity,CL-6 is a bacterial,CL-15 and CL-64 are actinomycetes.The fermentation supernatant of CL-64 strain with the highest inhibitory activity,it inhibited yeast calcineurin-dependent gene（CDRE::LacZ）expression with IC₅₀ is 5μL.Based on the test with yeast△mpk1 and△cnb1 synthetic lethal model,we also found that the 3μL fermentation supernatant of CL-64 strain has little effect on calcineurin regulatory subunit deficient strain△cnb1,but it shows synthetic lethal effect on MAP kinase deficient strain△mpk1,therefore,we can think that the molecular target is CN.CL-64 strain were indentified by morphological,physiological and biochemical characteristics and 16S rRNA gene sequence analysis.It demonstrate that CL-64 strain can hydrolyze starch,cellulose and catalase,it can’t produce melanin and H₂S.The evolutionary relationship of CL-64strain is similar to Streptomyces griseus subsp.And it belongs to the Streptomyces spp.Media and culture conditions of CL-64 strain were optimized by three factors and three levels orthogonal test.The optimum fermentation conditions of CL-64 were as follows:glucose 25g/L,peptone 2 g/L,CaCl₂ 0.6 g/L,at initial pH of 7.5,200 r/min,28℃,liquid volume 50mL in 250 mL flask,inoculation size of 2%for 6 d.At the optimum conditions,the calcineurin inhibitory activity of fermentation supernatant of CL-64 increased 15.63 times.The results produced by separation and purification of the active substance of Streptomyces CL-64 demonstrated that it inhibited yeast calcineurin-dependent gene（CDRE::LacZ）expression with IC₅₀ is0.16μL.After the same volume of ethyl acetate extracted 24h,we can get the organic phase to rotary evaporation,the temperature is 40℃,dissolve with 100mL sterile water,IC₅₀ was detected as 0.0145μL.After H103 macroporous resin adsorption,we used 20%,40%,60%,80%,100%ethanol to gradient elute,and 60%ethanol eluantion is 100mL Which is collected to rotary evaporate,dissolved in sterile water with an equal volume.IC₅₀ was detected as 0.113μL.we can get the D293 and 201×7（717）anion exchange resin to ion exchange chromatography with0.5mol/L NaCl as eluent eluate,collecte 13 mL first peak of eluention.IC₅₀ is detected as 5μL.We can select the first peak eluention to isolate and purificate by G-25 sephadex chromatography,collecte 3 mL eluention and IC₅₀ was detected as 20.8μL.High Performance Liquid Chromatography（HPLC）detected sample after separation and purification,The peak time is about 2.7min,with a purity of 87.29%.This study screened three strains with significant inhibitory activity of CN.CL-64 is belongs to Streptomyces with the highest inhibitory activity detected by Saccharomyces cerevisiae reporter gene（CDRE::LacZ）model,we also use Saccharomyces cerevisiae△mpk1 and△cnb1 double synthetic lethal genetic model to verify.This study has developed the method of separation and purification active substance of CL-64 strain,Following experiments canion continue to improve the identification of the strain species and active substances structure,and also can explore the role of the active substance mechanism by a human model kit in vitro.