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Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof

A technology for recombining Escherichia coli and exogenous genes, applied in microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of low conversion rate of whole cells, toxic substances, and unsuitable large-scale preparation of recombinant catalysts.

Inactive Publication Date: 2009-12-16
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0025] In view of the above existing catalytic synthesis of N-acetylneuraminic acid, the recombinant catalyst is not suitable for large-scale preparation, and the defects of toxic substances will be introduced during the preparation process, and the conversion rate of whole cells is low, the cost is high, post-processing is troublesome, and it is difficult to scale up. Insufficient scale production, the problem to be solved in the present invention is to provide a strain of recombinant Escherichia coli, that is, a strain of recombinant Escherichia coli that co-expresses foreign genes under temperature control and its application in the preparation of N-acetylneuraminic acid

Method used

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  • Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof
  • Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof
  • Recombinant escherichia coli of temperature-control coexpression exogenous gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Recombinant genetic engineering strain Escherichia coli DT26 (pBVNsS) CCTCCNO: M 209018 construction

[0076] 1. Cloning of N-acetylneuraminic acid aldolase gene (nanA)

[0077] The genomic DNA of strain Escherichia coli K12 ATCC 25404 was prepared by conventional methods. For this process, please refer to the method of small-scale preparation of bacterial genome in the "Refined Molecular Biology Guide" published by Science Press; use synthetic primers from Escherichia coli K12 The N-acetylneuraminidase gene nanA was obtained by PCR amplification from the genomic DNA. The fragment was digested with EcoRI and PstI and recovered, and then ligated to the plasmid pBV220 treated with the same enzyme and recovered (see Acta Virology, Vol. 6, No. 2, pages 111-116).

[0078] Among them, the above-mentioned Escherichia coli K12ATCC 25404 is used as the source strain of the nanA gene, and the primer is designed according to the sequenced genome sequence of the strain: the ...

Embodiment 2

[0116] Example 2: Application of whole-cell catalyst in the synthesis of N-acetylneuraminic acid

[0117](1) Plate culture: Streak the above-mentioned Escherichia coli DT26(pBVNsS)CCTCC NO:M 209018 strain onto an LB plate containing 1.5% agar by mass and volume and containing 100μg / ml of ampicillin, culture at 37℃ 12 hours.

[0118] (2) First-level seeds: Under aseptic conditions, use a sterile toothpick to pick a single colony on the plate in step (1), and then inoculate it into 5ml of liquid medium containing 100μg / ml ampicillin, 30 Incubate with shaking at ℃ for 12 hours.

[0119] (3) Shake flask culture: Under aseptic conditions, take the culture solution cultured in step (2) at a volume ratio of 1% and inoculate 30ml of LB liquid medium containing 100μg / ml of ampicillin. Incubate for 2 hours on a rotary shaker at 30°C at 200 rpm.

[0120] (4) Induction: Move the shaker to a 45°C shaker and induce at 180 rpm for 4 hours.

[0121] Wherein: the LB medium formula described in the...

Embodiment 3

[0131] Example 3: Application of whole cell catalyst in the synthesis of N-acetylneuraminic acid

[0132] (1) Plate culture: Streak Escherichia coli DT26 (pBVNsS) CCTCC NO:M 209018 strain onto an LB plate containing 1.5% agar by mass and volume and containing 100 μg / mL ampicillin, culture at 37°C for 12 hour.

[0133] (2) First-grade seeds: Under aseptic conditions, use a sterile toothpick to pick a single colony on the plate in step (1), and then inoculate it into 5ml of liquid medium containing 100μg / ml ampicillin, 37 Incubate with shaking at ℃ for 12 hours.

[0134] (3)Secondary seed: Under aseptic conditions, take the culture solution cultivated in step (2) with a volume ratio of 1%, and inoculate 500ml of LB inorganic salt liquid medium containing 100μg / ml of ampicillin Incubate with shaking at 30°C for 12 hours.

[0135] (4) Fermenter culture: Under aseptic conditions, take the culture solution obtained in step (3) to inoculate 5 L of the above LB inorganic salt liquid mediu...

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Abstract

The invention discloses recombinant escherichia coli of a temperature-control coexpression exogenous gene, which is named as Escherichia coli DT26 (pBVNsS) CCTCC NO:M 209018. The genotype of the recombinant escherichia coli is delta nagE::FRT; and the recombinant escherichia coli contain a temperature-control coexpression carrier pBVNsS. Simultaneously, the invention also discloses the application of the recombinant escherichia coli used as a catalyst for the complete cell catalytic preparation of N-acetylneuraminic acid. In the process of the large-scale catalytic production of the N-acetylneuraminic acid, the recombinant escherichia coli of the temperature-control coexpression exogenous gene has the characteristics of safe and convenient preparation of the catalyst, simple operation of the catalytic process, cheap price, higher efficiency, easy extraction and the like.

Description

Technical field [0001] The present invention relates to a strain of genetically recombinant Escherichia coli and its application, in particular to a strain of recombinant Escherichia coli that co-expresses foreign genes under temperature control and its application in preparing N-acetylneuraminic acid. Background technique [0002] Sialic acid is the general term for neuraminic acid and its derivatives. It is a class of nine-carbon amino sugars containing carboxyl groups. Its biological functions are closely related to important physiological and pathological phenomena, and have a wide range of application prospects [Traveling , 1998; Schauer, 2000]. N-acetyl-D-neuraminic acid (N-acetyl-D-neuraminic acid, Neu5Ac) is the most widely distributed type of sialic acid, and it itself and its analogs have important medicinal prospects. Among them, the structural analog of N-acetylneuraminic acid, Zanamivir (Zanamivir) has been approved for commercial anti-influenza virus drugs in 1995 [...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/26C12R1/19
Inventor 许平张奕南马翠卿
Owner SHANDONG UNIV
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