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Adenylate cyclase, and coding gene, vector, bacterial strain and application thereof

An adenylate cyclase and coding technology, applied in vectors, strains and applications, adenylate cyclase, the coding gene field, can solve the problem of unclear high-efficiency expression mechanism of key enzyme adenylate cyclase, ionic The relationship between the environment and cell activity and the regulatory mechanism need to be further studied, and the yield is low, so as to achieve the effect of short cycle, mild conditions and few by-products

Active Publication Date: 2011-10-12
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also limitations in the production of cAMP by fermentation using Brevibacterium liquefaction, Bacillus liquefaction, Corynebacterium, Arthrobacter, etc., such as low yield, unclear high-efficiency expression mechanism of key enzyme adenylate cyclase, and the relationship between ionic environment and cell activity. The relationship and the regulating mechanism need to be further studied

Method used

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  • Adenylate cyclase, and coding gene, vector, bacterial strain and application thereof
  • Adenylate cyclase, and coding gene, vector, bacterial strain and application thereof
  • Adenylate cyclase, and coding gene, vector, bacterial strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Screening of Arthrobacter A302

[0031] Taking the cyclic adenosine monophosphate-producing strain isolated from the soil as the starting strain, the N + Ion beam mutagenesis (the vacuum degree in the target chamber of the low-energy ion implanter is 2×10 -4 , the injection energy is 20kev, and the dose is 5×10 13 ions cm -2 .s -1) after the spore suspension was diluted 100 times with sterile water, got 0.1ml and spread it on the beef extract plate medium and cultivated it for 3 days at 30°C, from which a single colony was selected and cultivated on the beef extract slant for 2 days. Transfer one loop to a 500mL shake flask containing 30mL liquid seed medium (glucose 10g / L, peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, NaCl 3g / L), at 30°C Incubate on a shaker at 250rpm for 20 hours. Then transfer 4mL to 40mL liquid fermentation medium (glucose 50g / L, K 2 HPO 4 10g / L, KH 2 PO 4 10g / L, MgSO 4 1g / L, urea 5g / L, peptone 5g / L) in a 500mL shak...

Embodiment 2

[0033] Example 2 Cloning of Arthrobacter adenylate cyclase gene cya

[0034] 1. Extraction of Arthrobacter genome

[0035] Arthrobacter (Arthrobacter) A302 was inoculated in 30 mL medium (medium composition (g.L -1 ) is as follows: glucose 10g, peptone 10g, acid hydrolyzed casein 10g, yeast extract 5g, beef extract 10g, sodium chloride 3g, add distilled water to 1L), culture at 30°C until logarithmic growth phase, use bacterial genomic DNA extraction reagent The genome was extracted using a cassette (purchased from Beijing Gaining Jinnuo Biotechnology Co., Ltd.).

[0036] 2. Amplify the conserved sequence

[0037] According to the amino acid sequence of adenylate cyclase in the NCBI database, the degenerate primers for amplifying the conserved sequence were designed as follows:

[0038] S220: 5'-GCTCTCCGCCCGGAA(A / G)(A / C / T)T(A / G / C / T)TGG(A / C)G-3'

[0039] AS1: 5'-CAGCCGGGCGGC(A / G / C / T)A(A / G / T)(A / G)TT(A / G / C / T)AC-3'

[0040] The PCR reaction system is as follows:

[0041] ...

Embodiment 3

[0082] Example 3 Construction of cya gene expression vector

[0083] Design expression primers according to the nucleotide sequence of the obtained cya gene, respectively introduce NdeI and EcoRI restriction sites (see the horizontal line) at the 5' end and 3' end of the primers, and the primer sequences are as follows:

[0084] sAC: 5'-TTC CATATG ATGAACGATGAGGACCAGCA-3'

[0085] asAC:5'-CCG GAATTC TTAGTCCAGCACAAGCCCCT-3'

[0086] The PCR reaction system is as follows:

[0087]

[0088]

[0089] The PCR reaction conditions are as follows:

[0090] Denaturation at 94°C for 5 min; cycle 30 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 53°C for 30 s, extension at 72°C for 80 s, and finally extension at 72°C for 10 min.

[0091] The PCR product of about 1100bp was separated and purified by gel electrophoresis, and the gel was recovered. The gel recovery product was connected to PMD18T-simple to construct the PMD18T-cya re...

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PUM

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Abstract

The invention provides adenylate cyclase, and a coding gene, a vector, a bacterial strain and application thereof. A DNA (Deoxyribose Nucleic Acid) sequence for coding the adenylate cyclase provided by the invention has a base sequence represented by SEQ ID NO.:1. Furthermore, the invention further provides the adenylate cyclase coded by the DAN sequence, a recombinant vector including the DAN sequence, a host cell including the recombinant vector, and applications of all in production of the adenylate cyclase. The genetic engineering strain of colibacillus, disclosed by the invention, can beused for expressing the adenylate cyclase with high efficiency. The inducible enzyme activation thereof can achieve 7 U / mg or 10 U / mg. The bacterial strain is used for producing cyclic adenosine monophosphate and has the advantages of simple process, moderate condition, short cycle, few by-products and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to an adenylate cyclase, its coding gene, carrier, bacterial strain and application. Background technique [0002] Cyclic adenosine monophosphate (cAMP) is a physiologically active substance that widely exists in the human body. As a second messenger in cells, it plays an important role in glucose metabolism, fat metabolism, nucleic acid synthesis, protein synthesis, etc. important regulatory role. Clinically, cAMP is used to treat chronic congestive heart failure, pulmonary heart disease, myocardial infarction, myocarditis and cardiogenic shock; it has a certain effect on improving symptoms such as palpitations, shortness of breath, and chest tightness in rheumatic heart disease; it can improve the efficacy of acute leukemia combined with chemotherapy. It can also be used to induce remission of acute leukemia; in addition, it also has certain curative effect on elderly chr...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/63C12N1/21C12P19/32C12R1/19
Inventor 应汉杰何颖柏建新谢婧婧陈晓春熊健陈勇吴菁岚李楠
Owner NANJING UNIV OF TECH
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