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Escherichia coli engineering bacteria for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its 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: 2022-03-25
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 bacteria for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its application
  • Escherichia coli engineering bacteria for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its application
  • Escherichia coli engineering bacteria for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] gene acquisition

[0113] In this example, the obtained source from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) codon-optimized exotoxin A gene from E. coli (exotoxin A, GI: 877850); and from Campylobacter jejuni ( Campylobacter jejuni ) and an E. coli codon-optimized N-glycosyltransferase encoding gene (undecaprenyl-diphosphooligosaccharide--protein glycotransferase, GI: 905417).

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, and this method eliminates the resistance of each gene knocked out. Below with waaL Take gene as an example, elaborate the steps of gene knockout, wecA The gene deletion primers are designed the same.

[0117] Find the nucleotide sequence of JM109waaL, design waaL deletion primers and identification primers. 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 Tables 1-8.

Embodiment 3

[0119] JM109Δ waaL build

[0120] 3.1 Transformation of plasmid pSim

[0121] Pick wild-type JM109 cryopreserved at -80°C, streak on non-resistant LB plates, and culture at 37°C overnight. The next day, single clones were picked and inoculated into 5 mL of LB medium at 37° C., 220 rpm, and cultured overnight. The next day, according to 1% of the inoculum, it was transferred to 200ml of LB medium. 37°C, 220rpm, incubate 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, use 50ml ice-bathed sterile 10% Resuspend the bacterial cells in glycerol, 4°C, 5500 rpm, and centrifuge again for 5 min. Repeat the above operation 3 times. For the last time, use the residual liquid from discarding the supernatant to resuspend the bacterial cells, and take 80 μL into a new sterile EP tube. Freeze at -80°C.

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

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Abstract

The invention discloses an Escherichia coli engineering bacterium for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugated vaccine and its application. It relates to a method for constructing a cell factory for synthesizing neonatal meningitis-causing Escherichia coli O1 serotype glycoprotein-binding vaccine. It uses the highly efficient homologous recombination efficiency of Saccharomyces cerevisiae to construct the O1 antigen synthesis gene cluster plasmid based on the method of DNA Assembler. The O1 antigen synthesis gene cluster shuttle plasmid was transformed in Escherichia coli JM109, and the plasmid was identified by lipopolysaccharide extraction, gel electrophoresis and silver staining; the deletion of JM109 in JM109 was assisted by FLP‑FRT waaL and wecA , to exclude the interference of the original incomplete O antigen. The pET28a(+) plasmid was transformed and induced to synthesize a glycoprotein-conjugated vaccine. The glycoprotein was purified by AKTA Primeplus protein purification workstation and identified by western-blotting. The recombinant Escherichia coli constructed by the invention provides a new idea for biosynthesizing glycoprotein-conjugated vaccines.

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 Escherichia coli glycoprotein conjugate vaccine by using recombinant Escherichia coli. More specifically, it is an Escherichia coli engineering bacteria for synthesizing neonatal meningitis-causing Escherichia coli glycoprotein conjugate vaccine and its application. Background technique [0002] Neonatal meningitis-causing Escherichia coli (NMEC) is a large group of extraintestinal pathogenic Escherichia coli. After colonizing the mucosa of the gastrointestinal tract or respiratory tract, NMEC can invade the blood circulatory system and multiply, resulting in high concentrations of bacteremia. After the bacteremia concentration reaches a certain threshold, NMEC will enter the brain infection link. By expressing a variety of specific virulence factors to resist the host immune system attack, NMEC can cross the human...

Claims

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Application Information

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