【摘要】：維生素D只是一種激素原,而通過特定的羥基化反應(yīng)轉(zhuǎn)化成的活性維生素D�；钚跃S生素D參與了人體中的多種代謝調(diào)控,并在臨床上對各種疾病,如骨質(zhì)疏松癥、佝僂病等具有治療效果,有很大的藥用價值。目前國內(nèi)外大都采用生物轉(zhuǎn)化法轉(zhuǎn)化維生素D成活性維生素D。本組已篩選到一株具有VD羥基化功能的放線菌Pseudonocardia aautotr加pWcaa CGMCC5098。雖然已經(jīng)通過培養(yǎng)條件優(yōu)化,已經(jīng)運用于工業(yè)化生產(chǎn)25-OH-D3,但是為進一步解決副產(chǎn)物多,放線菌生長周期長等問題,本文進行了以下研究:對P.autorophica CGMCC5098進行全基因組提取和測序,從中找到29個CYP450基因。BLAST比對分析得到9個可能的羥基化酶基因,其中Gene 4849可能性最大。對Gene4849進行克隆,構(gòu)建表達載體PET28-Vdh,并轉(zhuǎn)化到BL21(DE3)中。含有表達載體PET28-Vdh的重組大腸桿菌B-Vdh能成功表達目的蛋白。體外構(gòu)建CYP450酶電子傳遞鏈體系,利用表達的目的蛋白成功催化VD3,使之轉(zhuǎn)化成25-OH-D3。采用生物信息學(xué)方法對Gene4849和Gene 1440進行同源建模和分子對接,預(yù)測作用于VD2和VD3的活性位點,為后續(xù)定點突變改造維生素D羥基化酶奠定基礎(chǔ)。嘗試構(gòu)建CYP450酶的電子傳遞鏈體系。通過BLAST比對,找到兩對可能的氧傳電子鏈基因?qū)?Gene 4833和Gene 4852以及Gene 5561和Gene 5562。成功構(gòu)建表達載體pCDFDuet1-4833和pCDFDuet1-5561,且轉(zhuǎn)化到重組菌B-Vdh中,得到重組菌B-Vdh-4833 和 B-Vdh-5561。
[Abstract]:Vitamin D is just a hormone that is converted into active vitamin D through a specific hydroxylation reaction. Active vitamin D is involved in many kinds of metabolic regulation in human body and has great medicinal value in treating various diseases such as osteoporosis rickets and so on. At present, biotransformation of vitamin D into active vitamin D is widely used at home and abroad. An actinomycetes strain Pseudonocardia aautotr and pWcaa CGMCC5098. with VD hydroxylation function has been screened. Although it has been applied to the industrial production of 25-OH-D3 through the optimization of culture conditions, in order to further solve the problems of more by-products and longer growth cycle of actinomycetes, In this paper, the following studies were carried out: 29 CYP450 genes were found by genomic extraction and sequencing of P.autorophica CGMCC5098, and 9 possible hydroxylase genes were obtained by BLAST comparison analysis, among which Gene 4849 was the most likely. Gene4849 was cloned, the expression vector PET28-Vdh, was constructed and transformed into BL21 (DE3). The recombinant Escherichia coli B-Vdh containing the expression vector PET28-Vdh can successfully express the target protein. The CYP450 enzyme electron transport chain system was constructed in vitro, and the expressed target protein was successfully used to catalyze the conversion of VD3, to 25-OH-D3. The bioinformatics method was used to model the homology of Gene4849 and Gene 1440 and to predict the active sites of VD2 and VD3, which laid a foundation for the modification of vitamin D hydroxylase by site-directed mutagenesis. The electron transport chain system of CYP450 enzyme was constructed. By BLAST comparison, two pairs of possible oxygen transfer electron chain gene pairs were found: Gene 4833 and Gene 4852, and Gene 5561 and Gene 5562. The expression vectors pCDFDuet1-4833 and pCDFDuet1-5561, were successfully constructed and transformed into the recombinant strain B-Vdh. The recombinant bacteria B-Vdh-4833 and B-Vdh-5561 were obtained.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R915

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