CGMCC5098菌羟基化基因的分析模拟及克隆表达
发布时间：2018-11-09 09:31
【摘要】：维生素D只是一种激素原,而通过特定的羟基化反应转化成的活性维生素D。活性维生素D参与了人体中的多种代谢调控,并在临床上对各种疾病,如骨质疏松症、佝偻病等具有治疗效果,有很大的药用价值。目前国内外大都采用生物转化法转化维生素D成活性维生素D。本组已筛选到一株具有VD羟基化功能的放线菌Pseudonocardia aautotr加pWcaa CGMCC5098。虽然已经通过培养条件优化,已经运用于工业化生产25-OH-D3,但是为进一步解决副产物多,放线菌生长周期长等问题,本文进行了以下研究:对P.autorophica CGMCC5098进行全基因组提取和测序,从中找到29个CYP450基因。BLAST比对分析得到9个可能的羟基化酶基因,其中Gene 4849可能性最大。对Gene4849进行克隆,构建表达载体PET28-Vdh,并转化到BL21(DE3)中。含有表达载体PET28-Vdh的重组大肠杆菌B-Vdh能成功表达目的蛋白。体外构建CYP450酶电子传递链体系,利用表达的目的蛋白成功催化VD3,使之转化成25-OH-D3。采用生物信息学方法对Gene4849和Gene 1440进行同源建模和分子对接,预测作用于VD2和VD3的活性位点,为后续定点突变改造维生素D羟基化酶奠定基础。尝试构建CYP450酶的电子传递链体系。通过BLAST比对,找到两对可能的氧传电子链基因对:Gene 4833和Gene 4852以及Gene 5561和Gene 5562。成功构建表达载体pCDFDuet1-4833和pCDFDuet1-5561,且转化到重组菌B-Vdh中,得到重组菌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.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915
本文编号：2320030
[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.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915
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