1.Cloning,expression And Activity Detection Of Vitamin D₃ Hydroxylase Gene 2.The Relationship Between Brachial Ankle Pulse Wave Velocity And The Risk Of Osteoporosis Risk Of Osteoporosis

Part Ⅰ Cloning,expression and activity detection of vitamin D₃ hydroxylase geneVitamin D is an extremely important fat-soluble and non-hydrosoluble steroid hormone,dissolving in many kinds of organic solvents.Vitamin D is especially vital for the human body.The forms of Vitamin D compounds are diversified and among all the different com-pound forms,vitamin D2 and vitamin D3 are the most common ones.Vitamin D3 itself has no physiological activities until the formation of active vitamin D3 takes effect in the body during the hydroxylation of its structure.Active vitamin D3 can regulate calcium and phosphorus metabolism in the body,maintain better bone development.It also can regulate immune response,and at the same time,regulating cell proliferation and apoptosis,playing a role in inhibiting tumor growth,etc.In addition,Vitamin D affects the expression of more than 200 different genes.Hence,active vitamin D3 has made it widely used in many industries such as clinical treatment and pharmaceutical research.Vitamin D3 hydroxylase is a cytochrome P450 enzyme that is involved in biosynthesis and metabolism of many physiologically active substances,such as endogenous and exogenous compounds.Meanwhile,it is also involved in catalyzing a variety of biotransformation and other different biochemical reactions.CYP450 enzyme is involved in and plays an important role in the transformation of sterols.A significant increase in the research on the structure and function of CYP450 creates a research focus on the cloning and expression of CYP450 gene.In this research,the activity of vitamin D3 hydroxylase in actinomycetes NRRL B16348 and NRRL B24484 was preliminarily analyzed,and no obvious hydroxylase activity was detected.The gene engineering bacteria of vitamin D3 hydroxylase was constructed by genetic engineering technology,and its expression was realized in E.coli.The experiments completed genome extraction,in vitro PCR amplification and recovery of target genes SsP450 and SsP450-2,and constructed recombinant plasmids of pJET1.2/blunt-SsP450 and pJET1.2/blunt-SsP450-2;on this basis,constructed the pET28a-SsP450 and pET28a-SsP450-2 recombinant plasmids,which were used to achieve the expression of the vitamin D3 hydroxylase genes SsP450 and SsP450-2 in E.coli.Through SDS-PAGE analysis,a protein band（44kDa）of recombinant vitamin D3 hydroxylase was obtained in recombinant E.coli.Moreover,to enhance the activity of vitamin D3 hydroxylase in the recombinant strain,this study successfully constructed the recombinant plasmid pCDF Duet-l-fdr-fdx gene of the electron transport chain.In addition,the vitamin D3 hydroxylase recombinant plasmid pET28a-SSP450 and pET28a-SSP450-2 and the electron transport chain gene recombinant plasmid pCDF Duet-l-fdr-fdx were transferred into E.coli BL21（DE3）to complete the hydroxylation of vitamin D3 in cells.Finally,the cell transformation products of vitamin D3 were analyzed by LC-MS,and the activity of vitamin D3 hydroxylase was confirmed in the recombinant E.coli.Part Ⅱ discusses the relationship between brachial ankle pulse wave velocity and the risk of osteoporosis Risk of osteoporosis:a cross-sectional evidence from a Chinese Community-based CohortObjectives:The association between atherosclerosis and osteoporosis has been fully demonstrated in the elderly.However,it is not clear whether they have made progress in the early stage through a common mechanism of action.The aim of this study was to assess the relationship between atherosclerosis and osteoporosis by measuring the brachial ankle pulse wave velocity（baPWV）and the Asian osteoporosis self-assessment tool（OSTA）index in healthy Chinese aged 40 and above.We further evaluated whether baPWV can be used as a predictor of osteoporosis.Methods:A total of 3984 adults who lived in Yunyan District of Guiyang City（Guizhou Province,China）for 5 years or more were selected by cluster random sampling.We carefully filled in the questionnaire and measured its basic information,such as weight,height,waist circumference,blood pressure,etc.we all received OSTA and baPWV measurement within one month,and collected the blood samples of the target subjects,including triglyceride,high-density lipoprotein cholesterol,total cholesterol,low-density lipoprotein cholesterol,fasting venous plasma glucose,fasting blood glucose,fasting blood glucose Plasma glucose was measured in the posterior vein.In the end,1407 subjects（1088 females and 319 males）met the inclusion criteria.Results:The average baPWV value of all subjects was 1475±302cm/s（range:766-3459cm/s）.Among all the participants,110 people were found to be at high risk of osteoporosis（OSTA index<-4）,and the baPWV value of this group was higher than that of non high risk group（1733±461 cm/s vs 1447±304cm/s,P<0.001）.By adjusting age,gender,body mass index,waist circumference,diastolic blood pressure and endogenous creatinine clearance rate,OSTA index was negatively correlated with baPWV（p=-0.294,P<0.001）.BaPWV was an independent predictor of high risk of osteoporosis（β=-0.001,P<0.001）,and the results were consistent in age and gender subgroups.The best cut-off point of baPWV was 1693 cm/s,with a sensitivity of 52.8%and a specificity of 83.6%.Conclusions:PWV measurement of atherosclerosis is closely related to the severity of osteoporosis assessed by OSTA;baPWV may be a simple and useful indicator for predicting the risk of osteoporosis and fracture.