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Novel non-ribosomal peptide synthetase gene and cloning and expression of adenylylation structural domains thereof

An adenylation structure, peptide synthase technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., to achieve the effect of great application prospects

Inactive Publication Date: 2012-10-10
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although its synthesis mode and enzyme catalytic method are very conservative, due to the abundance of available amino acid substrates and the diversity of non-ribosomal peptide synthetase's own structure, its types far exceed those synthesized by ribosomes. kind

Method used

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  • Novel non-ribosomal peptide synthetase gene and cloning and expression of adenylylation structural domains thereof
  • Novel non-ribosomal peptide synthetase gene and cloning and expression of adenylylation structural domains thereof
  • Novel non-ribosomal peptide synthetase gene and cloning and expression of adenylylation structural domains thereof

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Embodiment 1

[0038] 1. Acquisition of a novel non-ribosomal peptide synthetase gene NRPS114

[0039] Using the method of sequence screening with degenerate primers, seven clones containing NRPS genes were obtained from the South China Sea deep-sea sediment library, and one clone was selected for full-length sequencing. The sequencing was completed by Beijing Huada Gene Co., Ltd., and then bioinformatics software was used. Analyze the resulting sequence, as attached figure 1 And attached figure 2 shown. The sequence contains a complete non-ribosomal peptide synthetase gene of 17202bp, named NRPS114.

[0040] 2. Design primers A1F / A1R, A2F / A2R, A3F / A3R, A4F / A4R, A5F / A5R according to the sequence of the adenylation domain.

[0041] 3. PCR amplification

[0042]

[0043] 4. The PCR program is as follows:

[0044]

[0045] 5. Gel recovery of adenylylation domain gene amplification products

[0046] Follow the steps in the E.Z.N.A gel extraction kit manual for gel recovery.

[0047...

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Abstract

The invention discloses a novel non-ribosomal peptide synthetase gene named NRPS114. The nucleotide sequence of the non-ribosomal peptide synthetase gene is shown in SEQ ID NO 1. Besides, the invention further discloses a preparation method of the non-ribosomal peptide synthetase gene, a plasmid pCC1FOS<TM> Fosmid-NRPS114 containing the non-ribosomal peptide synthetase gene and an expression vector of a recombinant plasmid pET28a-A-2his of five adenylylation structural domains in the non-ribosomal peptide synthetase gene. Additionally, the invention further discloses the five adenylylation structural domains and a preparation method thereof. Recombinant adenylylation structural domains are obtained by means of over-expression of the adenylylation structural domains A1, A2, A3, A4 and A5 in an escherichia coli prokaryotic expression system, and the non-ribosomal peptide synthetase gene has high activity enabling amino acid to undergo adenylylation, provides more genetic manipulation materials for study of combinatorial biology and has a huge application prospect in research and development of new drugs.

Description

technical field [0001] The present invention relates to a novel non-ribosomal peptide synthetase gene and five adenylylation domain genes contained therein, especially a novel non-ribosomal peptide synthetase gene derived from South China Sea deep-sea sediment microorganisms and its contained Five adenylation domain genes. Background technique [0002] Non-ribosomal peptides are a large class of biologically active natural products synthesized by bacterial or fungal non-ribosomal peptide synthetases, and have good resistance to enzymatic and chemical degradation in vivo. Its structure is very complex and varied. There are many non-ribosomal peptides used in clinical practice, such as antibiotics (daptomycin), antitumor (bleomycin), antifungal drugs or immunosuppressants (cyclosporine). With the frequent emergence of bacterial resistance, the reserve of antibiotics is becoming less and less, and the demand for new antibiotics is becoming more and more urgent. Therefore, it ...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/10C12N9/90C12N15/63C12N1/21C12R1/19
Inventor 陆勇军周世宁刘莉璇黄雅莉何磊
Owner SUN YAT SEN UNIV
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