Sequence Analysis,Modeling And Cloning,Expression Of Hydroxylase Gene From CGMCC5098

Vitamin D3 is just a prohormone in mammals and it must be activated by special hydroxylation reaction to exert its biological activity.Activated vitamin D3 with great medicinal value is involved in various important physiological functions in the human body and plays an important role in treatment of various diseases,such as osteoporosis and rickets clinically.VD is mostly converted to active VD by microorganism at home and abroad currently.Our group has got a strain named Pseudonocardia autotrophica CGMCC5098 by screening which has the ability to convert VD3 to 25-OH-D3 and CGMCC5098 has been applied in industrial production through optimizing the culture medium.But there are still a few problems,such as more by-product,long cycle of the strain growth.Therefore this paper researches:Genomic DNA of P.autotrophica CGMCC5098 was extracted and sequenced.Twenty-nine CYP450 genes were found.There were 9 possible hydroxylase gene in these CYP450 genes using BLAST and the most likely hydroxylase gene was Gene 4849.Gene 4849 was cloned into PET28 vector,achieving PET28-Vdh.Then the vector PET28-Vdh was convert into BL21(DE3)and the recombination strain was named B-Vdh which can express protein we want.Seting up a system of electron transport chain for CYP450 in vitro,the expression protein converted VD3 to 25-OH-D3 successfully.To lay the foundation of reform the structure of enzyme by site-specific mutagenesis,homology modeling and molecular docking simulation were used to study the Gene 4849 and Gene 1440,predicting active site acting on VD2 and VD3.Constructing a system of electron transport chain for CYP450 into E.coli has been tried.Gene 4833 and Gene 4852 as well as Gene 5561 and Gene 5562 were considered as the gene pair for electron transport chain using BLAST.Expression vector pCDFDuet 1-4833 and pCDFDuetl-5561 were constructed successfully.These expression vectors were convert into recombination strain B-Vdh respectively,achieving B-Vdh-4833 and B-Vdh-5561.