Studyon The Hydroxylation Of Vitamin D₃ By Cytochrome P450 27A1

CYP27A1 is a cytochrome P450 enzyme mainly existing in the liver and a multifunctional and significant CYP450 enzyme.In human beings,CYP27A1 plays an essential role in C-25 hydroxylation process of vitamin D₃ and bile acid biosynthesis,which is an important enzyme in the metabolism of human life.Up to now,the study about Vitamin Dhydroxylase on human has come of age,while that on poultry is still at the fledgling level.Vitamin D plays an important part in the number of eggs laid,the hardness of eggshell,the strength of bone and the hatching rate.Therefore,it is of great significance to study vitamin D hydroxylase in chickens.According to the research and analysis,there was high homology of CYP27A1 between human beings and chickens.Moreover,CYP27A1 in the chickens is also a key hydroxylase in the metabolism of endogenous and exogenous compounds.Thereby it can be speculated that CYP27A1 in chickens may be involved in the metabolism of vitamin D₃.In this study,CYP27A1 in chickens was selected as the research object to explore whether CYP27A1 in chickens has an effect of hydroxylationon on vitamin D₃.In this study,firstly,bioinformatics software was used to analyze the amino acid sequence of Cyp27a1 code of chicken,which provided references for further studies on the heterologous expression and physicochemical properties of Cyp27a1 in chickens.Then,the ORF sequence of Cyp27a1 gene in chickens was cloned by taking the cDNA sequence of that as a template.Then,the ORF fragment of chicken Cyp27a1 was connected to the vector of pcDNA^TM3.1/myc-His（-）A,in order to construct the eukaryotic expression vector of pcDNA-Cyp27a1 and successfully express in 293T cells.Finally,the analysis of enzyme activity was carried out on the heterogenic expression of recombinational Cyp27a1 gene in the chickens,with constructing a recombination system of extracorporeal membrane components and incubating the albumen of the heterogenic expression of recombinational Cyp27a1 gene in the chickens and vitamin D₃ in vitro.The results of this study could provide data for the follow-up studies of CYP27A1 in chickens based on the effect of hydroxylation to vitamin D₃ given by chicken CYP27A1 preliminarily discussed.The main results are as follows:1.Through sequence alignment,the similarity of chicken CYP27A1 gene and human CYP27A1 was about 57%.Then,various bioinformatics software was used to analyze the amino acid sequence of chicken Cyp27a1 gene.According to the analysis result,there were similar functions of CYP27A1 in chickens and CYP27A1、CYP27B1 in human beings,which is a protein located in the inner lining of mitochondria with the typical structure of P450enzymes indicated by secondary and tertiary structure projections.2.Connect the ORF fragment of chicken Cyp27a1 to the vector of pcDNA^TM3.1/myc-His（-）A to construct the eukaryotic expression vector.After sequencing,the test results were consistent with the size of predicted band.Then,transfect the vector of pcDNA-Cyp27a1 transiently into 293T cells.According to the results detected by Western Blotting,the molecular weight of the expressed protein band was 58.827 kD,which was consistent with the predicted one.Thus,it can be proved that chicken Cyp27a1 was successfully expressed in 293T cells.3.As the test results of enzyme activity showed,the 25（OH）D₃ was not detected in the control group of pcDNA^TM3.1/myc-His（-）A,while it was detected in the experimental group of pcDNA-Cyp27a1.Conclusion:The above results indicated that the CYP27A1 of chicken is commensurate with the CYP27A1of human,which is a Vitamin D 25-hydroxylase that can make Vitamin D₃hydroxylate into 25（OH）D₃.