Fusion of multiple enterotoxin genes of escherichia coli and application thereof

A technology of Escherichia coli and enterotoxin, which is applied in gene therapy, medical preparations containing active ingredients, and hybrid peptides, etc. It can solve the problems of narrow protection range and inability to induce heat-resistant enterotoxin.

Active Publication Date: 2010-12-15
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] The technical problem to be solved by the present invention is: to overcome the shortcomings of the existing enterotoxigenic Escherichia coli (ETEC) vaccine which has a narrow protection range and cannot induce high levels of anti-heat-stable enterotoxin (STa and STb) antibodies, and provide a multi- Valence fusion enterotoxin immunogen, can be used as a vaccine to control diarrhea caused by bacteria such as ETEC, to achieve the effect of expanding the scope of protection and improving the protection rate

Method used

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  • Fusion of multiple enterotoxin genes of escherichia coli and application thereof
  • Fusion of multiple enterotoxin genes of escherichia coli and application thereof
  • Fusion of multiple enterotoxin genes of escherichia coli and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Preparation of trivalent enterotoxin DNA vaccine and protein vaccine

[0030] (1) Amplification of enterotoxin gene STa

[0031] Directly use the following primers for conventional PCR amplification without using a template. Conditions: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, 25 cycles, and finally extension at 72°C for 5 minutes.

[0032] STa-F1: 5'-agggaattcaccatgaacacattctactgctgcgagctgtgctgcaat-3'

[0033] STa-R: 5'-tagcaggtgggtagcagccagcggcggcgggattgcagcacagc-3'

[0034] (2) Amplification of enterotoxin genes LTB and STb

[0035] The lysate of the K88ac strain (C83902, purchased from the China Veterinary Drug Administration) was used as the template, and the following primers were used for conventional PCR amplification. Extend at 72°C for 40 seconds, 30 cycles, and finally extend at 72°C for 5 minutes.

[0036] LTB-F: 5'-gctacccacctgctagcccagctccccagactattacag-3'

[0037] LTB-R: 5'-tg...

Embodiment 2

[0056] Example 2. Construction of a trivalent enterotoxin DNA vaccine containing chicken insulin signal peptide

[0057] pCI-ins-STa-LTB-STb

[0058] (1) Amplification of chicken insulin signal peptide gene ins

[0059] Directly use the following primers for conventional PCR amplification without using a template. The conditions are: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, 25 cycles, and finally extension at 72°C for 5 minutes.

[0060] insF: 5'-aac accatggctctctggatccgatcactgcctcttctggctctccttgt-3', contains EcoRI site

[0061] insR: 5'-gcagtagaatgtgtttgcatagctggttccagggccagaaaagacaaggagagccag-3'

[0062] (2) Amplification of heat-stable enterotoxin gene STa

[0063] Directly use the following primers for conventional PCR amplification without using a template, and the reaction conditions are the same as in step (1).

[0064] STa-F: 5'-aacacattctactgctgcgagctgtgctgcaatccc-3'

[0065] STa-R: 5'-tagcaggtggg...

Embodiment 3

[0074] Example 3. Construction of trivalent enterotoxin DNA vaccine pCI-upa-STa-LTB-STb containing chicken urokinase-type plasminogen activator (upa) signal peptide

[0075](1) Amplify the signal peptide gene upa of chicken upa

[0076] Directly use the following primers for conventional PCR amplification without using a template. The conditions are: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, 25 cycles, and finally extension at 72°C for 5 minutes.

[0077] upaF: 5'-aac accatgaagttaatcatctttctcacagtaactctctgcac-3', containing EcoRI site upaR: 5'-gcagtagaatgtgttagaatcaagtcctgtgacaagtgtgcagagagttactg-3'

[0078] (2) Amplification of enterotoxin gene STa

[0079] The preparation method is the same as step (2) of Example 2.

[0080] (3) Connect upa and STa to get upa-STa

[0081] The template uses the mixture of the PCR products upa and STa in the first two steps, the primers use upaF and STa-R (see Example 2), and ...

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Abstract

The invention relates to an escherichia coli (E. coli) trivalent enterotoxin fusion gene and application thereof, and belongs to the field of genetic engineering subunit vaccines. The enterotoxigenic E. coli (ETEC) is a main pathogen causing baby-animal and infantile diarrhea. Aiming at the defects that the conventional TEC vaccine has narrow protection range and cannot induce high-level antitoxin, the invention designs a trivalent E. coli enterotoxin fusion with the fusion mode of 5'-STa-LT-STb-3' or 5'-STb-LT-STa-3'. After the multivalent enterotoxin gene or protein immunizes animals, high-level STa, STb and LT resistant antibodies can be induced to be generated. Therefore, the escherichia coli (E. coli) trivalent enterotoxin fusion gene can neutralize the toxicity of a key pathogenic factor enterotoxin, improve the immunity protection rate and widen the protection range without being limited by E. coli pilus types so as to effectively control ETEC diarrhea.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a genetic engineering subunit vaccine, in particular to an enterotoxin-producing Escherichia coli (ETEC) multivalent enterotoxin immunogen, which is used for preventing and treating diarrhea caused by ETEC and other bacteria. Background technique [0002] Enterotoxigenic Escherichia coli (ETEC) can cause acute and infectious diarrhea in young animals (including piglets, calves, lambs, etc.), seriously endangering the healthy development of animal husbandry and causing huge economic losses. ETEC is also the main pathogen of diarrhea among infants and tourists in developing countries. Every year, ETEC-infected diarrhea cases are as high as 650 million people worldwide, resulting in the death of about 380,000 children under the age of 5 (Sizemore et al., 2004, Expert Rev. Vaccines). [0003] The mechanism of ETEC-induced diarrhea is that the bacteria colonize the epithelial cells of the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00A61K48/00A61K39/116A61P31/04A61P1/12A61K39/108
Inventor 徐永平尤建嵩金礼吉李晓宇马永生王林会李化强吴菲菲
Owner DALIAN UNIV OF TECH
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