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Genetic engineering preparation method of cytidine triphosphate

A technology of cytidine triphosphate and genetically engineered bacteria, which is applied in the field of preparation of cytidine triphosphate, can solve problems such as long conversion time, impact on conversion rate, and increase in clinical dosage, and achieve easy control of reaction conditions, rapid reaction, and high conversion rate high effect

Inactive Publication Date: 2011-09-28
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this process is unlikely to completely separate all the enzymes in the glycolysis system, the enzyme inactivation rate is high, the conversion rate is affected, and the conversion time is long
In recent years, the demand for CTP has increased, and the amount of clinical use has also increased significantly, which has been unable to meet the market demand.

Method used

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  • Genetic engineering preparation method of cytidine triphosphate
  • Genetic engineering preparation method of cytidine triphosphate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, construction of Escherichia coli with pyrE, pyrF, pyrH, ndk operator on chromosome

[0024] 1.1. Amplification of pyrE, pyrF, pyrH, ndk genes and construction of vectors

[0025] The primers for pyrE gene, pyrF gene, pyrH gene, ndk gene and pyrG gene were designed according to the whole genome sequence of Escherichia coli (genebank accession number: ECK3632).

[0026] The genome of Escherichia coli K12MG1655 was extracted.

[0027] Then, the pyrE, pyrF, pyrH, and ndk genes were respectively amplified using the Escherichia coli K12MG1655 genome as a template. After sequencing, the pyrE fragment was connected to the plasmid pUC18 through the EcoRI and SacI restriction sites to obtain the plasmid pUCpyrE; The pyrF fragment was connected to the plasmid pUCpyrE by the SmaI site to obtain the plasmid pUC-pyrEF; then the pyrH fragment was connected to pUC-pyrEF via the SmaI and XbaI sites to obtain the plasmid pUCpyrEFH; finally the ndk fragment was connected by...

Embodiment 2

[0042] Embodiment 2, pyrG gene expression vector construction

[0043] According to the known nucleic acid sequence of the pyrG gene of Escherichia coli K12MG1655, the full-length pyrG gene is amplified by using the chromosome of Escherichia coli as a template.

[0044] The genomic DNA of Escherichia coli strain DH5α preserved in our laboratory was extracted by conventional bacterial DNA extraction method.

[0045] The pfu enzyme with good fidelity was used for amplification, and after adding A tail, it was connected to pUC19-T vector, and the positive clones were picked and sent to the sequencing company, and the sequence confirmed by sequencing was used in the next step.

[0046] The pyrG gene connected to the pUC19-T vector was digested from the vector, the restriction enzymes used were Sma I and BamH I, and the vector pUC18 was digested with restriction enzymes Nru I and BamH I, After recovering the digested product of the vector, perform an enzyme ligation reaction with ...

Embodiment 3

[0047] Embodiment 3, the construction of the genetic engineering bacterium that produces cytidine triphosphate

[0048] Prepare the recombinant K1-E obtained in Example 1 as a competent cell, and then transform the plasmid pUCG constructed in Example 2 into K1-E, and name it K1-E / pUCG, namely Genetically engineered bacteria capable of catalyzing the synthesis of cytidine triphosphate, such as orotate and ammonium chloride, are stored in a -80°C refrigerator with glycerin.

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Abstract

The invention discloses a genetic engineering preparation method of cytidine triphosphate. In the method, a culture or a treatment substance of single genetic engineering strain is used as an enzyme source; during catalysis, ammonium chloride, saratin and the like are used for reaction, so that the cytidine triphosphate is generated in a reaction solution and accumulated and is extracted from the reaction solution. The invention has the benefits that: (1) the reaction is easier to control due to the adoption of the microbial catalytic reaction ; (2) the cost of a substrate is lower and the cytidine triphosphate can be produced at low cost; and (3) the reaction is quick and the conversion rate is higher. The genetic engineering preparation method can be widely applied to preparation of the cytidine triphosphat.

Description

technical field [0001] The present invention relates to a kind of preparation method of cytidine triphosphate, specifically relate to in Escherichia coli simultaneously highly express orotidine pyrophosphorylase (pyrE), orotidine decarboxylase ( The invention relates to pyrF), uridine kinase (pyrH), nucleoside diphosphate kinase (ndk) and CTP ligase (pyrG) genes, belonging to the field of biotechnology. Background technique [0002] Cytidine triphosphate (CTP), also known as cytidine nucleoside triphosphate, CTP is catalyzed by uridine 5′-triphosphate (UTP), and is a kind of ribose-5 in cytidine. A nucleotide with three molecules of phosphate attached to the '-OH group. Cytidine triphosphate is a high-energy phosphate compound that participates in the synthesis of nucleic acids and phospholipids, promotes protein synthesis, and provides energy. It is one of the direct precursors of RNA biosynthesis and participates in the synthesis of polysaccharides. CTP has the function ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N15/55C12N15/60C12N15/54C12N15/52C12N15/63C12R1/19
Inventor 汪仁李晓丹夏冰江玉梅贺佳王忠
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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