Construction and application of genetically engineered escherichia coli of group of extraintestinal pathogenic escherichia coli glycoprotein conjugate vaccines

A technology of Escherichia coli and conjugated vaccines, applied in the field of synthetic biology, can solve problems such as time-consuming, affecting the stability between batches, and the position of product coupling is not clear

Pending Publication Date: 2021-03-16
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considerable consumption occurs at each step, and due to the random nature of chemical coupling, the position of final product coupling is unclear, affecting b

Method used

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  • Construction and application of genetically engineered escherichia coli of group of extraintestinal pathogenic escherichia coli glycoprotein conjugate vaccines
  • Construction and application of genetically engineered escherichia coli of group of extraintestinal pathogenic escherichia coli glycoprotein conjugate vaccines
  • Construction and application of genetically engineered escherichia coli of group of extraintestinal pathogenic escherichia coli glycoprotein conjugate vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] gene acquisition

[0127] In this example, the obtained source from Vibrio cholerae ( Vibbrio cholerae ) codon-optimized cholera toxin B from E. coli (CTB, GI: 877850); and from Neisseria meningitidis ( Campylobacter jejuni ) and an E. coli codon-optimized O-glycosyltransferase encoding gene (undecaprenyl-diphosphooligosaccharide--protein glycotransferase, GI: 905417).

Embodiment 2

[0129] Design of Gene Deletion Primers

[0130] This example uses the CRISPR-cas9 λRed recombination system to delete E.coli The gene of K-12 MG1655 starts with waaL Taking gene as an example, the steps of gene knockout are explained in detail, and the design principles of other gene deletion primers are the same.

[0131] find E.coli K-12 MG1655 waaL nucleotide sequence, designed waaL deletion primers and identification primers. The deletion primers for waaL are GwaaL-F, HwaaL-GwaaL-R, waaL-up-F, waaL-up-Rm, waaL-down-Fm, waaL-down-R, HwaaL-pTarget-backbone-F, GwaaL-pTarget -backbone-R identified primer as S2-waaL-F / R. Nucleotide sequences are shown in Tables 1-8.

Embodiment 3

[0133] 3.1 E.coli K-12 MG1655 Δ waaL build

[0134] (1) Extract pCas plasmid

[0135] The pCas plasmid was extracted with a Sanitary Column Plasmid Mini-Extraction Kit, and the specific steps were shown in the kit instructions.

[0136] (2) Preparation E.coli K-12 MG1655 competent cells

[0137] (3) E.coli Acquisition of K-12 MG1655 / pCas Strain

[0138] The extracted pCas plasmid was transformed into E.coli K-12 MG1655 competent cells were then transferred to recovery medium for about half an hour, coated with kanamycin-resistant 2YT plates, and cultured overnight in a 30°C incubator. The next day, it can be seen that the single colony growing on the plate is the E.coli K-12 MG1655 strain containing the pCas plasmid.

[0139] 3.2 Construction of pTarget-waaL plasmid

[0140] (1) Principles of gRNA design: Open http: / / www.rgenome.net / cas-designer / PAM Type to select SpCas9 from Streptococcus pyogenes: 5'-NGG-3' (because Cas9 protein comes from Streptococcus py...

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Abstract

The invention discloses construction and application of genetically engineered escherichia coli of a group of extraintestinal pathogenic escherichia coli glycoprotein conjugate vaccines On the basis of CRISPR-cas9, zwf, pfkB, lacZ, manA and pykA genes in an E.coli K-12 MG1655 strain are deleted, and strains capable of synergistically utilizing glucose and glycerol are constructed and are respectively used for synthesizing O antigen polysaccharides and maintaining physiological metabolism of bacteria. Further, waaL, enterobacterial common antigen(ECA) and O16 gene clusters are deleted, and a chassis cell is constructed as OSLA. O5 and O7 antigen synthesis gene clusters are respectively introduced, and besides, expression glycosyltransferase and carrier protein are introduced to synthesize various serotype glycoprotein conjugate vaccines. Animal immune experiments prove that purified glycoprotein can stimulate mice to generate antibodies with a protective effect, and the protection ratecan reach 90% or above. The recombinant escherichia coli glycoprotein conjugate vaccines constructed by the invention provide a new choice for immunotherapy against extraintestinal pathogenic escherichia coli infection.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology, and relates to the construction and application of genetically engineered Escherichia coli for designing a group of extraintestinal pathogenic Escherichia coli O5 and O7 glycoprotein conjugate vaccines. Background technique [0002] Extraintestinal pathogenic Escherichia coli (ExPEC) are zoonotic pathogens that typically occupy specific niches in the human (and other animal) gut microbiota, and then drain the gut from this "water storage". pools" migrate to other sites and cause extra-intestinal infections. ExPEC has a complex evolutionary lineage, contains numerous virulence factors, and has great genomic plasticity. These pathogenic strains not only infect the urinary system, but also cause bacteremia or sepsis. According to the difference in infection site, ExPEC can be simply divided into avian pathogenic Escherichia coli (APEC), uropathogenic Escherichia coli (UPEC) and neonatal ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C07K14/245A61K39/108A61P31/04C12R1/19
CPCC12N15/70C12N15/52C07K14/245C07K14/28C12N9/93C12N9/1081C12N9/2471C12N9/90C12N9/1205C12N9/0006A61K39/0258A61P31/04C12Y302/01023C12Y204/99019C12Y503/01008C12Y101/01049C12Y207/0104C12Y204/99018C12N2800/22C07K2319/02C07K2319/21
Inventor 王磊冯露黄笛刘斌江小龙
Owner NANKAI UNIV
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