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Recombinant escherichia coli capable of synthesizing bordetella pertussis oligosaccharide antigen

A technology of recombinant Escherichia coli and Escherichia coli, applied in the fields of genetic engineering and synthetic biology, can solve problems such as hidden dangers of safe production and slow growth

Pending Publication Date: 2022-01-04
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is a bottleneck in the production of this novel antigen from B. pertussis
Firstly, the lipooligosaccharides naturally synthesized by B. pertussis only contain a single terminal trisaccharide unit; secondly, B. pertussis is a pathogenic bacterium, which poses safety hazards
Defects in the glycolytic pathway and TCA cycle in B. pertussis cells result in slow growth

Method used

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  • Recombinant escherichia coli capable of synthesizing bordetella pertussis oligosaccharide antigen
  • Recombinant escherichia coli capable of synthesizing bordetella pertussis oligosaccharide antigen
  • Recombinant escherichia coli capable of synthesizing bordetella pertussis oligosaccharide antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Construction of embodiment 1 knockout plasmid

[0063] Using the CRISPR / Cas9 knockout system to knock out four LPS-related gene clusters and a binding transcriptional repressor gene metJ in E. coli requires the construction of five knockout plasmids: pTargetF-wbbL-galF, pTargetF-rfaD-waaQ, pTar getF-wza-wcaM, pTargetF-rfe-rffM, pTargetF-metJ, the construction process of these plasmids is as follows:

[0064] (1) Select 20nt N complementary to the target sequence of the target gene 20 sequence, specific N 20 The sequences are respectively the underlined sequences of primers pTargetF-wbbL-galF-F, pTargetF-rfaD-waaQ-F, pTargetF-wza-wcaM-F, pTargetF-rfe-rffM-F, pTargetF-metJ-F, and these sequences are modified into The 5' end of the plasmid pTargetF forward primer was used to obtain forward primers pTargetF-wbbL-galF-F, pTargetF-rfaD-waaQ-F, pTargetF-wza-wcaM-F, pTargetF-rfe-rffM-F, pTargetF-metJ -F.

[0065] Using the plasmid pTargetF as a template, the forward primers...

Embodiment 2

[0069] Example 2 Construction of LPS streamlined strain MDCO020

[0070] The four LPS-related gene clusters and the metJ gene of wild-type E. coli MG1655 were knocked out by the CRISPR / Cas9 knockout system, and the LPS-stripped strain MDCO020 was obtained. The specific knockout process is as follows ( figure 2 ):

[0071] (1) Preparation of Escherichia coli electroporation knockout competent cells MG1655 / pCas

[0072] The plasmid pCas is transformed into Escherichia coli MG1655 to obtain recombinant Escherichia coli MG1655 / pCas containing the pCas plasmid, and the Escherichia coli MG1655 / pCas is activated on the LB solid plate adding 30mg / L Kanamycin (Kan), and inoculated into LB (Kan+) test tube was cultivated overnight to obtain seed liquid; the seed liquid was transferred to 25mL LB (Kan+) medium at 1% (v / v), and cultivated to OD at 30°C and 200rpm 600 =0.2, add 500 μL L-arabinose solution to induce, continue to culture to OD 600 =0.5, ice bath for 30min; 4°C, centrifu...

Embodiment 3

[0081] Construction of embodiment 3 expression plasmid pW, pB and pD5

[0082] (1) Extraction of Bacillus pertussis CS strain and Pseudomonas aeruginosa PAO1 genome

[0083] The extraction of B. pertussis CS strain and Pseudomonas aeruginosa PAO1 genome does not require lysozyme lysis, and the specific operation is completed according to the product instructions of the bacterial genomic DNA extraction kit and plasmid DNA extraction kit; the extracted genomic DNA passes 0.8% Agarose gel electrophoresis detection.

[0084] (2) Construction of plasmid pW:

[0085] In the first step, a 1008bp gene fragment was amplified by PCR with primers U-(BPTD_RS00475)-F and D-(BPTD_RS00475)-R using the genome of B. pertussis CS strain as a template, and verified by electrophoresis and purified; Carry out enzyme digestion reaction on the vector pWSK29 with the endonuclease, the reaction temperature is 37°C, the time is 30min, and then the enzyme digestion product is recovered and verified by...

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Abstract

The invention discloses recombinant escherichia coli capable of synthesizing a bordetella pertussis oligosaccharide antigen, and belongs to the field of genetic engineering and synthetic biology. An O-antigen gene cluster wbbL-galF, a core sugar gene cluster rfaD-waaQ, an intestinal common antigen gene cluster rfe-rffM, a claic acid gene cluster wcaM-wza and a transcription binding factor metJ gene on an escherichia coli MG1655 genome are knocked out, core sugar of a pertussis CS strain, trisaccharide of the pertussis CS strain and a gene cluster for controlling the length of a trisaccharide unit are over-expressed, and a recombinant bacterium MDCO020 / pWpBpD5 is obtained. Under the same fermentation condition, the synthetic cell dry weight of the MDCO020 / pWpBpD5 strain is 1.63 times that of the CS strain; and the yield of the oligosaccharide antigen of the MDCO020 / pWpBpD5 strain is two times that of the CS strain.

Description

technical field [0001] The invention relates to a recombinant escherichia coli capable of synthesizing bacillus pertussis oligosaccharide antigen, which belongs to the field of genetic engineering and synthetic biology. Background technique [0002] Lipooligosaccharide (LOS) of B. pertussis is composed of lipid A and oligosaccharides composed of twelve monosaccharides, and the distal end has a unique trisaccharide structural unit, including two rare sugars 2,3-acetylamino-2, 3-dideoxy-mannuronic acid and 2-acetylamino-4-N-methyl-2,4-dideoxy-fucose. The LOS structure of various B. pertussis strains is highly conserved, and the LOS terminal trisaccharide structural unit has not changed in clinical isolates before and after vaccination, indicating that the LOS terminal trisaccharide structural unit is an ideal component of a broad-spectrum anti-pertussis vaccine. Sera from patients with pertussis contain various anti-B pertussis antibodies, but only antibodies against LOS have...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/31C12P19/00A61K45/00A61P31/04C12R1/19
CPCC12N15/70C07K14/245C07K14/195C07K14/21C12P19/00A61K45/00A61P31/04
Inventor 王小元王震范锋锋汪宸卉胡浩
Owner JIANGNAN UNIV
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