Expression And Enzyme Activity Study Of Human 1 Alpha Hydroxylase In Saccharomyces Cerevisiae

Obejective: To further investigate the therapeutic potential of human 1 alpha hydroxylase treatment of chronic kidney disease min-eral and bone metabolism disorders,we constructed a eukaryotic expression vector expressing recombinant protein 1 alpha hydroxylase, inductive expressed in Saccharomyces cerevisiae,studying its expressi-on and enzyme activity.Methods: 1.According to the Gen Bank data known of 1 alpha hydroxylase(AK314953) of gene sequence, by Chang Sha Peng Yuan bio limited for bio synthesis,get the gene fragment of CYP27B1. 2.According to Gen Bank of gene sequence design primer,PCR amplifi-cation, recovery and purification,The target gene CYP27B1 and pl-asmid p YES2/CT were respectively double enzyme cutted, recovery and purification,the recombinant target gene CYP27B1 and plasmid p YES2/CT with co-nnections,constructing into recombinant plasmid p YES2/ CT-CYP27B1.which were transformed into E.coli TOP10,meanwhile screening and identificating the transformant.3. Extract th-e recombination plasmid p YES2/CT-CYP27 B.transforming Saccharomyces cerevisiae INVSc1 by lithium acetate method,screening and identificating the transformant. 4.The recombinant plasmid containing S. cerevisiae INVSc1 joining galactoseinducible recombinant protein CYP27B1 expression,contrast with the control group.Expression products of SDS-PAGE and Western Blot were analyzed and identified. 5.Saccharomyces cerevisiae expression strain INVSc1 with express purpose protein was cultured at a particular concentration of 25 hyd-roxy vitamin D inductive medium for 24 hours,contrast with thecontrol group,and have conversion in vitro experiments to identify the enzyme activity.Results: 1.The 1 alpha hydroxylase gene fragment about 1.527 Kb was amplified by PCR from a preparation of 1 alpha hydroxyla-se gene strain,having the same size with 1 alpha hydroxylase gene fragment in Genbank.2.The recombinant Bacillus coli containing of plasmid p YES2/CT-CYP27 B was digested with Hind III and Eco RI.Getting 1.527 kb 1 alpha hydroxylase gene fragment and having the same sequence with 1 al-pha hydroxylase gene in the Genbank.3.The use of lithium acetate transformation successfully transfor-med plasmid p YES2/CT-CYP27B1 into Saccharomyces cerevisiae INVSc1,the target gene fragment about 1.527 Kb was amplified by P CR havin-g the same size with target gene.4.The molecular weight of the expressed recombinant protein was ab-out 72 KD through the analysis of recombinant Saccharomyces cerevisiae by discontinuous SDS-PAGE,and target protein expression in bacteria in the form of soluble protein expression.Western Blot results showed a specific band at 72 KDa place, consistent with the expected protein with a relative molecular mass.5.Results of in vitro conversion experiment:The concentration of 25 hydroxy vitamin D in the induction medium was significantly decreased after the conversion of the Saccharomyces cerevisiae gene engineering bacteria, compared with the control group.The difference was statistical-ly significant(P<0.05), confirmed that the recombinant protein has 1 hy-droxylase enzyme activity.Conclusion: Construct the expression of engineering strain 1 al-pha hydroxylase and Saccharomyces cerevisiae INVSc1 successfully,which have the enzymes activity.