Prokaryotic expression method of streptomyces murinus AMP (Adenylate) deaminase gene and application of expression product of gene

A prokaryotic expression technology of Streptomyces grinotis, applied in the field of molecular biology, to achieve the effects of stable enzyme activity, easy purification, and good thermal stability

Inactive Publication Date: 2014-05-07
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Now the research on the recombinant expression of AMP deaminase gene in China has never been reported, and the research on the recombinant expression of this enzyme in foreign countries is less, only in the patent "AMP deaminase derived from actinomycetes and Its application" (Applicant: Amano Enzyme Co., Ltd.) reported once

Method used

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  • Prokaryotic expression method of streptomyces murinus AMP (Adenylate) deaminase gene and application of expression product of gene
  • Prokaryotic expression method of streptomyces murinus AMP (Adenylate) deaminase gene and application of expression product of gene
  • Prokaryotic expression method of streptomyces murinus AMP (Adenylate) deaminase gene and application of expression product of gene

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Experimental program
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Effect test

Embodiment 1

[0029] Cloning of embodiment 1 Streptomyces griseus AMP deaminase gene

[0030] Using the genome of Streptomyces griseis MCCC1A01641 as a template, the primer pair AMPDF1 / AMPDR1 was designed to specifically amplify the AMP deaminase gene of Streptomyces griseis, and EcoRI and Not I restriction sites (indicated by underlines) were added to both ends of the primers. The sequence for AMPDF1 / AMPDR1 is as follows: AMPDF1: 5'-GCG GAATTC GCGCCGCCGCCCCGGCAG-3' (SEQ ID NO: 1); 5'-GCC GCGGCCGC TCACCCCCGGGCGTGCGCCC-3' (SEQ ID NO: 2).

[0031] The PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 70°C for 1 min, extension at 72°C for 1.5 min, and extension at 72°C for 10 min after 30 cycles.

[0032] The resulting PCR product was purified using a gel purification kit.

Embodiment 2

[0033] Example 2 Construction of recombinant expression vector pET28-AMPD

[0034] Both the expression vector pET-28a and the PCR product obtained in Example 1 were digested with EcoRI / Not I at 37°C for 2 hours, and the digested product was purified. The purified product was ligated overnight at 16°C and transformed into E.coli JM109, and then, AMPDF1 Use / AMPDR1 as a primer to perform colony PCR to screen positive clones; extract the plasmids of the above positive clones, transform them into E.coli BL21, and screen pET28a-AMPD-JM109 positive strains; extract recombinant plasmids, identify them by double enzyme digestion, see figure 1 .

Embodiment 3

[0035] Example 3 Transformation of recombinant expression vector pET28-AMPD into E.coli BL21

[0036] The pET28a-AMPD-JM109 positive strain plasmid obtained in Example 2 was extracted, transformed into E.coli BL21, and positive clone transformants were screened.

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Abstract

The invention discloses a prokaryotic expression method of a streptomyces murinus AMP (Adenylate) deaminase gene. The method comprises the following steps: amplifying AMP deaminase gene sequence by designing a specific primer by taking a streptomyces murinus genome as a template, adding cleavage sites at two ends of the sequence, performing subcloning to an expression carrier to construct recombinant plasmids, further converting the recombinant plasmid into escherichia coli competence, and finally performing IPTG (Iisopropyl-beta-d-Thiogalactoside) inducible expression and purifying to obtain a final AMP deaminase expression product. According to the method, AMP deaminase from the streptomyces murinus is successfully expressed in escherichia coli for the first time, the experimental result shows that a recombinant enzyme is high in enzyme activity, stable in enzyme activity and easy to purify, can be applied to the fields of foods and medicines, and lays the foundation for industrial production of the AMP deaminase.

Description

technical field [0001] The invention relates to the prokaryotic expression of AMP deaminase gene, in particular to a prokaryotic expression method of AMP deaminase gene derived from Streptomyces grinotis and the application of the expression product, belonging to the technical field of molecular biology. Background technique [0002] AMP deaminase, English name AMP deaminase, abbreviated as AMPD, is an aminohydrolase, which can catalyze the deamination of AMP to generate inosinic acid (IMP) and NH 3 , IMP has important applications in the fields of food and pharmaceuticals. In addition, AMP deaminase is one of the three main enzymes in the metabolic cycle of purine nucleotides, which plays an important role in maintaining the body's immunity and adenylate energy charge. Therefore, AMP deaminase is an important enzyme in industrial production. [0003] AMP deaminase widely exists in various organisms. It was first prepared from rat skeletal muscle, and then from human red ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/78C12R1/465
Inventor 张梁石贵阳方炜丁重阳顾正华
Owner JIANGNAN UNIV
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