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Escherichia coli expression strain and method thereof for producing N-acetyl-D-neuraminic acid

A technology of Escherichia coli and neuraminic acid, applied in the field of genetic engineering, can solve the problems of reduced gene activity, non-uniform strain, inconvenient experimental process, etc., and achieves the effects of high yield, simple and fast operation, and easy gene operation.

Inactive Publication Date: 2015-05-20
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method of cloning and expressing on the vector is more effective, but there are some inherent disadvantages, such as the two genes need to be cloned on their respective vectors, transformed into two strains, and then separated to induce the expression of the enzyme; antibiotics are required To maintain the Xunzi of the plasmid, but the use of antibiotics increases the difficulty of separation and purification of the enzyme and the price of the product, the gene cloned on the plasmid has heterogeneous strains, and the activity of the gene will decrease after long-term use, etc.
These are likely to be detrimental to gene expression and enzyme activity
In addition, the experimental process of manipulating the genome of the host bacteria to improve the transformation efficiency is not easy

Method used

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  • Escherichia coli expression strain and method thereof for producing N-acetyl-D-neuraminic acid
  • Escherichia coli expression strain and method thereof for producing N-acetyl-D-neuraminic acid
  • Escherichia coli expression strain and method thereof for producing N-acetyl-D-neuraminic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction of isomerase gene BT0453 and aldolase gene nanA coupling plasmid

[0041] Design primer R133: 5'-GGG GGATCCATATG GATTTTAAGAAACTAGCG-3', (SEQ ID NO.1), BamHI and NcoI restriction sites are underlined; R134: 5'-GGG GTCGAC CTATAGCGGTTCTAATAC-3', (SEQ ID NO.2), the SaII restriction site is underlined. Using Bacteroides thetaiotaomicron VPI-5482 genomic DNA (from Professor Jeffrey Gordon of Washington University, St.Louis, USA) as a template, R133-R134 PCR obtained 1.2kb BT0453 gene, and the 1.2kb fragment was cloned into TA vector pMD18 - TSimple, obtained recombinant clone LS1842.

[0042] Design primer R135: 5'-GGG ACTAGTCATATG GCAACGAATTTACGTGGC-3', (SEQ ID NO.3), SpeI and NdeI restriction sites are underlined; R136: 5'-GGA CTCGAG

[0043] TCACCCGCGCTCTTGCATCAAC-3', (SEQ ID NO.4), the XhoI restriction site is underlined. Using Escherichia coli MG1655 genomic DNA (professor John Cronan from the University of Illinois at Urbana-Champaign, ...

Embodiment 2

[0058] Embodiment 2. Recombineering method constructs the genetic engineering strain that isomerase gene BT0453 and aldolase gene nanA are integrated into BL21 (DE3) genome

[0059] 1. Preparation and electroporation of electroporation-competent cells of strains expressing recombinant enzymes induced by isopropyl-β-D-thiogalactoside (IPTG).

[0060]Firstly, the plasmid pTKRed was transformed into Escherichia coli BL21(DE3), and selected with 100 μg / ml spectinomycin at 30°C. A single colony of the obtained strain was inoculated into LB liquid culture containing 100 μg / ml spectinomycin, cultivated overnight at 30°C, transferred to 100ml of the same medium at a volume ratio of 1:00, cultured with shaking at 30°C, until OD600 At about 0.2, add 2mM IPTG for induction culture, continue to cultivate until OD600 is about 0.4, pour the bacterial solution into a pre-cooled centrifuge tube, ice bath for 10 minutes, centrifuge at 4°C, 7000rpm for 5 minutes, and discard the supernatant. B...

Embodiment 3

[0065] Example 3. Protein expression of genetically engineered strains

[0066] After the LS2402 single-colony LB medium was cultured overnight, 1:50 was transferred to 10ml expansion culture. When the OD600 reached about ~0.8, 1mM IPTG was added to induce expression at 30°C for 6h. The results of detecting protein expression by SDS-PAGE are shown in the appendix Figure II . It can be seen from the figure that the size of the protein expressed by BT0453 is 45.7KD, and the size on SDS-PAGE is slightly lower than the molecular weight standard of 45.0KD. This is due to the fact that BT0453 belongs to the glycoprotein type, and the slightly faster migration of glycoproteins on SDS-PAGE is an inherent biological property. The protein size expressed by NanA is 32.6KD, which is in line with the expectation.

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Abstract

The invention relates to a method for producing an N-acetyl-D-neuraminic acid based on genomes through enzymatic synthesis. The method is implemented through integrating an isomerase gene BT0453 and an aldolase gene nanA to a malE gene region so as to obtain a recombinational genetically engineered bacterium, wherein the isomerase gene BT0453 and the aldolase gene nanA are respectively placed under a T7 strong promoter, the isomerase gene BT0453 is derived from a polymorphic bacteroidetes VPI-5482 genome, and the aldolase gene nanA is derived from escherichia coli; enabling a strain to express the isomerase BT0453 and the aldolase NanA under the inducing of isopropyl-beta-D-isopropyl thiogalactoside, through taking a whole cell as a catalyst and n-acetyl-D-glucosamine and sodium pyruvate as substrates, preparing a N-acetyl-D-neuraminic acid through catalytic synthesis, wherein the yield can reach 36.3%. The efficient N-acetyl-D-neuraminic acid expression system has a potential of becoming an N-acetyl-D-neuraminic acid producing strain.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a genome-based method for enzymatically synthesizing N-acetyl-D-neuraminic acid. Background technique [0002] Seasonal influenza and highly pathogenic avian influenza are enemies that threaten human health. Vaccines and primers are two ways to treat influenza. Because influenza viruses are constantly changing, vaccines against influenza viruses are often difficult to have specific curative effects, and highly pathogenic avian influenza is often sudden, and it takes at least several months to develop a vaccine. Time, so anti-influenza drugs are an essential treatment. [0003] There are relatively few types of anti-influenza drugs, only the neuraminidase inhibitors ostamivir, zanamivir and peramivir. Ostamivir has the most obvious clinical effect and is most widely used. But Osmiway also has some shortcomings. For example, Ostamivir has various side effects such as mental d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/19
Inventor 尚广东李玲严振亚石牡丹
Owner NANJING NORMAL UNIVERSITY
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