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Recombinant escherichia coli, and application of recombinant escherichia coli to preparation of anti-Shigella glycoprotein vaccine

A technology for recombining Escherichia coli and Shigella dysenteriae, which is applied in the field of glycoprotein vaccines, can solve problems such as difficult transformation, late transformation, and too large gene clusters

Active Publication Date: 2017-06-06
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the gene cluster is too large and it is not easy to transform and modify later
[0005] After retrieval, relying on the synthesis pathway of Wzy, a partial gene cluster containing S. dysenteriae 1rfbR (rhamnosyltransferase), rfbQ (rhamnosyltransferase), orf9 (galactosyltransferase) gene and rfp (galactosyltransferase) gene were introduced into E.coli K-12W3110 to realize the The literature or patent of heterologous expression of S.dysenteriae 1LPS has not been published

Method used

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  • Recombinant escherichia coli, and application of recombinant escherichia coli to preparation of anti-Shigella glycoprotein vaccine
  • Recombinant escherichia coli, and application of recombinant escherichia coli to preparation of anti-Shigella glycoprotein vaccine
  • Recombinant escherichia coli, and application of recombinant escherichia coli to preparation of anti-Shigella glycoprotein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the construction of O-Ag gene cluster expression vector

[0036] Primers were designed according to the genome sequence of Shigella dysenteriae type 1 pathogen published by NCBI:

[0037] S.d-F-SmalI: 5'-TCCCCCGGGATGAATAAATATTGTATCTTAGTA-3'

[0038] S.d-R-XbaI: 5'-GCTCTAGATCACATTAATGCTACCAAAAAAGAGT-3'

[0039] Using the genome of Shigella dysenteriae type 1 as a template, part of the O-Ag gene cluster was cloned by PCR, which contained three genes rfbR, rfbQ and orf9. PCR reaction system is as follows: (primer concentration is 20 μ mol / L)

[0040]

[0041] PCR reaction conditions: pre-denaturation at 95°C for 3min, denaturation at 95°C for 30s, annealing at 50°C for 30s, extension at 72°C for 2.5min, extension at 72°C for 10min after 30 cycles, and storage at 16°C.

[0042] The cloned O-Ag gene cluster fragments were respectively digested with endonucleases SmalI and XbaI, and the plasmid vector pACT3 was also digested with endonucleases SmalI and Xb...

Embodiment 2

[0049] The construction of embodiment 2, rfp gene expression vector

[0050] Primers were designed according to the genome sequence of Shigella dysenteriae type 1 pathogen published by NCBI:

[0051] 15b-F-NdeI: 5'-GGAATTCCATATGATGAAGATCTCAATAATAGGGAA-3'

[0052] 15b-R-BamHI: 5'-CGGGATCCTTAATCAGGAATCCCTAGTA-3'

[0053] The rfp gene was cloned by PCR using the genome of Shigella dysenteriae type 1 as a template. PCR reaction system is as follows: (primer concentration is 20 μ mol / L)

[0054]

[0055] PCR reaction conditions: pre-denaturation at 95°C for 3min, denaturation at 95°C for 30s, annealing at 50°C for 30s, extension at 72°C for 2.5min, extension at 72°C for 10min after 30 cycles, and storage at 16°C.

[0056] The cloned rfp gene fragment was digested with endonucleases NdeI and BamHI respectively, and the plasmid vector pET15b was also digested with endonucleases NdeI and BamHI at the same time. The digested rfp fragment and pET15b plasmid vector were recovered ...

Embodiment 3

[0063] Embodiment 3, the construction of recombinant escherichia coli strain

[0064] (1) Preparation of Competent E.coli K-12W3110

[0065] (1) Bacteria E.coli K-12W3110 was cultured overnight at 37°C 200r / min in a 5mL test tube;

[0066] (II) Inoculate the above bacterial solution into 50mL LB medium with 1% inoculation amount, culture at 37°C and 200r / min for 2-3h, until OD600=0.3-0.4 (0.36 is the best, generally not more than 0.4);

[0067] (III) Transfer the bacterial solution to a 50mL centrifuge tube (the centrifuge tube is sterile and cooled at 4°C), bathe in ice for 20min, let the cells stop growing, and centrifuge at 4000r / min at 4°C for 10min;

[0068] (IV) precipitation with an appropriate amount of pre-cooled CaCl 2 Resuspend, centrifuge at 4000r / min at 4°C for 5-10min, discard the supernatant;

[0069] (V) Precipitate and then use an appropriate amount of pre-cooled CaCl 2 Resuspend, ice-bath for 1h, centrifuge at 4000r / min at 4°C for 10min, discard the super...

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Abstract

The invention discloses a lipopolysaccharide synthetase gene cluster recombinant escherichia coli capable of expressing Shigella dysenteriae 1 type pathogenic bacteria. The escherichia coli contains rfbR (rhamnosyltransferase), rfbQ (rhamnosyltransferase) and orf9 (galactosyltransferase) in Shigella dysenteriae 1 type pathogenic bacteria rfb gene cluster, and the genotype is W3110 delta wbbl / p-rfp+p-0-Ag. The invention also discloses application of the escherichia coli to preparation of Shigella1 type pathogenic bacterium resistant glycoprotein vaccine. The recombinant escherichia coli can produce lipopolysaccharide structure of Shigella dysenteriae 1 type pathogenic bacteria; the escherichia coli is used as a platform, so that the Shigella1 type pathogenic bacterium resistant glycoprotein vaccine can be further efficiently produced at low cost; good industrial development and application prospects are realized.

Description

technical field [0001] The present invention relates to a strain of recombinant Escherichia coli and its construction method and application, in particular to a recombinant Escherichia coli capable of expressing the lipopolysaccharide synthase gene cluster of Shigella dysenteriae type 1 pathogen and its use in the preparation of Shigella dysenteriae 1 Application of glycoprotein vaccines against pathogenic bacteria. It belongs to the fields of biotechnology, genetic engineering and microbial fermentation. Background technique [0002] Bacillary dysentery is an intestinal infectious disease caused by Shigella (Shigella), characterized by severe colonic inflammation accompanied by symptoms of systemic toxemia, severe cases can cause septic shock and / or poisoning encephalopathy. Worldwide, there are about 165 million cases of bacillary dysentery each year, causing more than 1.1 million deaths, and about 70% of the onset symptoms are manifested in children under the age of 5. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70A61K39/112A61P31/04C12R1/19
CPCA61K39/0283A61K2039/523C12N9/1051Y02A50/30
Inventor 陈敏孔蕴曲亚军
Owner SHANDONG UNIV
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