Nucleoside phosphorylase, encoding gene and high-producing strain and application of nucleoside phosphorylase

A nucleoside phosphorylase high-yield, nucleoside phosphorylase technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low conversion rate and high cost of nucleoside drugs, and achieve easy purification and good application. Potential, high substrate conversion effect

Active Publication Date: 2016-06-15
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conversion rate of existing nucleoside phosphorylases is low, and the cost of preparing nucleoside drugs is relatively high

Method used

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  • Nucleoside phosphorylase, encoding gene and high-producing strain and application of nucleoside phosphorylase
  • Nucleoside phosphorylase, encoding gene and high-producing strain and application of nucleoside phosphorylase
  • Nucleoside phosphorylase, encoding gene and high-producing strain and application of nucleoside phosphorylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] This example illustrates the biological properties and identification of Brevibacillus borstelensis LK01 producing nucleoside phosphorylase.

[0026] A nucleoside phosphorylase-producing strain LK01 was obtained from the organic solvent-resistant bacterial library in our laboratory.

[0027] Biological properties of the strain LK01: the strain is a Gram-positive bacterium, the morphological characteristics of the bacteria are rod-shaped, the cell size is (0.3-0.4) μm*(2-7) μm, there are obvious depressions, and there are flagella around. Mesozoic spores, movement. The colonies are round, milky white in color, smooth, opaque, and not concave. Oxidase reaction was negative and gelatin reaction was positive. The starch hydrolysis test was negative, the nitrate reaction was positive, the VP reaction was negative, the glucose reaction was positive, the citric acid reaction was positive, the arabinose reaction was negative, and the xylose reaction was negative.

[0028] Af...

Embodiment 2

[0030] The nucleoside phosphorylase produced by Brevibacillus borstelensis LK01 is named as nucleoside phosphorylase PyNP.

[0031] This example illustrates the isolation and cloning procedure of the gene encoding nucleoside phosphorylase PyNP.

[0032] Total DNA was extracted by the phenol-chloroform method. According to the analysis of the whole gene sequencing results of Brevibacillus borstelensis (NCBI Reference Sequence: I532_RS05705), a gene encoding pyrimidine nucleoside phosphorylase was obtained, and primers SF and SR were designed according to the gene sequence.

[0033] The sequence of SF (SEQ ID NO: 3) is: ACGGAGCTCGAATTC G GATCC ATGCGCATGGTCGATATCATTG.

[0034] The sequence of SR (SEQ ID NO: 4) is: CGCGGCAGCCATATG G CTAGC CTATTCGGTTATGATTTTGTAAATTAATGG.

[0035] Among them, the underlined part of the primer SF is the NheI restriction site, and the underlined part of the primer SR is the BamhI restriction site.

[0036] Using the genome of Brevibacillus bo...

Embodiment 3

[0038] This example illustrates the preparation of recombinant expression vectors and recombinant bacteria.

[0039] The nucleoside phosphorylase PyNP gene fragment obtained in Example 2 was double-digested with restriction endonucleases BamHI and NheI at 37°C for 12 hours, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit . Under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pET28a digested by BamHI and NheI overnight at 16°C to obtain the recombinant expression plasmid pET-PyNP.

[0040] Transform the recombinant expression plasmid pET-PyNP into Escherichia coli E.coliBL21(DE3) competent cells, screen the positive recombinants on the resistance plate containing kanamycin, select single clones, and verify the colonies by PCR Positive clone means obtaining positive recombinant bacteria E. coli BL21(DE3) / pET-PyNP.

[0041] Inoculate the recombinant strain E.coliBL21(DE3) / pET-PyN...

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Abstract

The invention provides nucleoside phosphorylase, an encoding gene and a high-producing strain and application of the nucleoside phosphorylase and relates to the technical field of biological pharmacy.The high-producing strain of the nucleoside phosphorylase is classified and named Potsdam brevibacillus borstelensis LK01, and the preservation number is CCTCC NO:M2015685.The nucleoside phosphorylase generated by the strain and the encoding gene of the nucleoside phosphorylase are further provided.The invention further provides a gene containing recombinant expression vector or recombinant bacteria and application thereof.The nucleoside phosphorylase is used for preparing nucleoside medicine, the reaction conditions are mild, the product ingredient is single, no by-product is generated, the substrate conversion rate is high, the product is easy to purify, the problems existing in existing chemical method galactosylated modification of nucleoside medicine are solved, and good application potential is achieved.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a nucleoside phosphorylase, a coding gene, a high-yield strain and applications thereof. Background technique [0002] Natural nucleosides are formed by ribose or deoxyribose and natural bases. Due to the diversity of natural bases, there are various types of ribonucleosides or deoxyribonucleosides. The bases or sugar groups in natural nucleosides are chemically modified to form new nucleosides, and such synthetic nucleosides are nucleoside analogs. Nucleoside analogs are similar to nucleotides in chemical structure, and can be mixed into the process of DNA synthesis, but because they do not have the function of nucleotides, the synthesized nucleic acid chain loses its normal function. Nucleoside drugs can be obtained by modifying the pyrimidines and purines of the normal bases through chemical modification methods such as aza, deaza, and substitution. Nucleoside ana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/10C12N15/54C12P19/38C12R1/01
CPCC12N9/1077C12P19/385C12N1/205C12R2001/01
Inventor 何冰芳刘柯周有治张劲松储建林
Owner NANJING TECH UNIV
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