A kind of pyrimidine nucleoside phosphorylase gene and its application

A pyrimidine nucleoside phosphorylase gene and a pyrimidine nucleoside phosphorylase technology are applied to the pyrimidine nucleoside phosphorylase gene and its application fields, and can solve the problems of small amount of enzyme and the like

Active Publication Date: 2018-04-03
JIANGSU ALPHA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of natural strains containing nucleoside phosphorylase to synthesize nucleoside drugs has the disadvantage of a small amount of enzymes, and the construction of genetically engineered bacteria can solve the problem of enzyme amounts

Method used

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  • A kind of pyrimidine nucleoside phosphorylase gene and its application
  • A kind of pyrimidine nucleoside phosphorylase gene and its application
  • A kind of pyrimidine nucleoside phosphorylase gene and its application

Examples

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Embodiment 1

[0033] The establishment of embodiment 1 genetically engineered bacteria

[0034]According to the Escherichia coli pyrimidine nucleoside phosphorylase gene (Genbank: NC_000913.3) announced by Genebank, the pyrimidine nucleoside phosphorylase gene fragment was artificially synthesized, and the gene fragment was used as a template to expand the fragment by PCR amplification (on both sides of the fragment) BamH I and EcoR I endonuclease fragments), its nucleotide sequence is shown in SEQ ID NO.3 (Genbank: NC_000913.3). And the gene was inserted into the pET-28a(+) plasmid by using the HindⅢ and BamH I endonuclease sites, and the ligated vector was transferred into Escherichia coli BL21(DE3) to establish the pyrimidine nucleoside phosphorylase genetically engineered bacteria. The primers for PCR amplification of pyrimidine nucleoside phosphorylase gene are: the upstream primer is 5'-GTCTGATGTTTTTCATCTCG-3' (SEQ ID NO.4), and the downstream primer is 5'-CCCGTTTTCCCGCTGGCTTT-3' (SEQ...

Embodiment 3

[0044] The shaking flask culture of embodiment 3 recombinant escherichia coli

[0045] The recombinant Escherichia coli E.Coli BL21 NP1506 obtained in Example 2 was inoculated into 50 mL of LB medium containing kanamycin (30 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L , pH 7.0), cultured at 37°C for 6 hours in a shaker at 200 rpm. Transfer 3% bacterial cell culture solution to a 250 mL shake flask equipped with LB medium (containing 30 μg / mL kanamycin), and culture under the same conditions with shaking. When the OD600 value of the bacterial solution reaches about 0.6, add the inducer isopropyl βD-thiogalactopyranoside (IPTG) to a final concentration of 0.1mmol / L, and place the culture solution in a shaker at 28°C and 200rpm to induce Express for 6 hours. After expression, the cells were collected by centrifugation (8000rpm, 10min, 4°C), washed twice with phosphate buffer (pH7.5, 10mmol / L), dispersed in the same pre-cooled buffer, and ultrasonicated in an ice-wa...

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Abstract

The invention relates to the fields of biotechnology and biological medicine, in particular to a pyrimidine nucleoside phosphorylase gene and the application thereof. The nucleotide sequence of the pyrimidine nucleoside phosphorylase gene is shown in SEQ ID NO.1, and the amino acid sequence of pyrimidine nucleoside phosphorylase coded by the gene is shown in SEQ ID NO.2. A genetically engineered bacterium containing the gene is obtained by transferring the gene into escherichia coli. By culturing the genetically engineered bacterium and optimizing the fermentation technology, large-scale production of recombined pyrimidine nucleoside phosphorylase is achieved, and the recombined pyrimidine nucleoside phosphorylase can be used for catalyzed synthesis of Sofosbuvir midbody (2'R)-2'-deoxygenation-2'-fluorine-2'-methyl uridine and production of other nucleoside bulk pharmaceutical chemicals and medical intermediates.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biomedicine, and relates to a pyrimidine nucleoside phosphorylase gene and its application, in particular to a class of pyrimidine nucleoside phosphorylase and its gene, and a recombination containing the pyrimidine nucleoside phosphorylase gene Expression vector and genetic engineering bacteria, and used to catalyze the synthesis of sofosbuvir intermediate (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, and other nucleoside APIs and medicines Production of intermediates. Background technique [0002] Sofosbuvir (trade name Sovaldi) is an oral drug for the treatment of hepatitis C (HCV) developed by Gilead Sciences in the United States. It was approved for marketing by FDA in December 2013. Sofosbuvir is the first drug approved for the full oral treatment of hepatitis C, and the world's first marketed HCV polymerase nucleoside inhibitor, which interferes with the viral genetic material RNA by acting ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N1/21C12P19/38C12R1/19
CPCC12N9/1077C12P19/385C12Y204/02002
Inventor 陈峻青石利平尹晓龙
Owner JIANGSU ALPHA PHARM CO LTD
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