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Escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application

A technology of Escherichia coli and combined vaccine, applied in the field of synthetic biology, can solve the problem of less vaccine development

Active Publication Date: 2018-11-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are few vaccines developed against neonatal meningitis-causing Escherichia coli

Method used

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  • Escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application
  • Escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application
  • Escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Gene acquisition

[0113] In the present embodiment, the acquisition derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) from Escherichia coli codon-optimized exotoxin A gene (exotoxin A, GI: 877850); and from Campylobacter jejuni ( Campylobacter jejuni ) and the codon-optimized N-glycosyltransferase gene (undecaprenyl-diphosphooligosaccharide--protein glycotransferase, GI: 905417) of Escherichia coli.

Embodiment 2

[0115] Design of gene deletion primers

[0116] In this example, the λRed recombination system is used to knock out two genes of JM109. In this method, resistance is eliminated every time a gene is knocked out. Below to waaL Taking the gene as an example, the steps of gene knockout are explained in detail. wecA Gene deletion primer design is the same.

[0117] Find the nucleotide sequence of JM109waaL, design primers for deletion and identification of waaL. The deletion primer of waaL is waaL-FRT-chl-FRT-F / R, and the identification primer is S-waaL-F / R. Nucleotide sequences are shown in Table 1-8.

Embodiment 3

[0119] JM109Δ waaL build

[0120] 3.1 Transformation of plasmid pSim

[0121] Wild-type JM109 frozen at -80°C was picked and streaked on a non-resistant LB plate, and cultured overnight at 37°C. The next day, a single clone was picked, inoculated into 5 mL LB medium, and cultured overnight at 37° C., 220 rpm. The next day, transfer to 200ml LB medium according to the inoculum volume of 1%. 37°C, 220rpm, cultivate to OD 600 About 0.6-0.8, ice bath for 20min, 5500rpm, 5min, collect the bacteria in a sterilized 50ml centrifuge tube, 4℃, 5500rpm, centrifuge for 5min, discard the supernatant, and use 50ml ice-bathed sterile 10% Resuspend the bacteria in glycerol, 4°C, 5500rpm, and centrifuge for 5 minutes. Repeat the above operation 3 times. For the last time, use the residual liquid when the supernatant was discarded to resuspend the bacteria, and transfer 80 μL to a new sterile EP tube. Freeze at -80°C.

[0122] Thaw the competent cells frozen at -80°C in ice for 10 minutes...

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Abstract

The invention discloses an escherichia coli engineering bacterium for synthesizing glycoprotein conjugate vaccines for neonatal meningitis escherichia coli and application, and relates to a method forconstructing a cell factory for synthesizing O1 serum type glycoprotein conjugate vaccines for neonatal meningitis escherichia coli. O1 antigens are constructed based on a DNA Assembler method by utilizing efficient homologous recombination efficiency of saccharomyces cerevisiae so as to synthesize gene cluster plasmids; the O1 antigens are converted in escherichia coli JM109 to synthesize gene cluster shuttle plasmids, and the plasmids are identified by virtue of lipopolysaccharide extraction, gel electrophoresis and silver staining; waaL and wecA in the JM109 are deleted with the aid of FLP-FRT so as to eliminate the interference of original incomplete O antigens; and pET28a(+) plasmids are reconstructed and induced to synthesize the glycoprotein conjugate vaccines, the glycoprotein ispurified by virtue of an AKTA Primeplus protein purification workstation, and the glycoprotein is identified by virtue of western-blotting. The constructed recombinant escherichia coli provides a novel idea for synthesizing the glycoprotein conjugate vaccines by a biological method.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and relates to a method for synthesizing a neonatal meningitis-causing E. coli glycoprotein-conjugated vaccine by using recombinant Escherichia coli. More specifically, it relates to an Escherichia coli engineering bacterium for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its application. Background technique [0002] Neonatal meningitigenic Escherichia coli (NMEC) belongs to a large group of extraintestinal pathogenic Escherichia coli, mainly infecting newborn infants and people with low immunity, and is an opportunistic pathogen. After colonization through the gastrointestinal tract or respiratory tract mucosa, NMEC can invade the blood circulation system and multiply in large numbers, forming high-concentration bacteremia. After the concentration of bacteremia reaches a certain threshold, NMEC will enter the link of brain infection. B...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70A61K39/108A61P31/04C12R1/19
CPCA61K39/0258A61P31/04C07K14/245C12N15/70Y02A50/30
Inventor 王磊黄笛江小龙
Owner NANKAI UNIV
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