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Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof

A genetically engineered vaccine, Escherichia coli technology, applied in antibacterial drugs, pharmaceutical formulations, medical preparations containing active ingredients, etc. Easy separation and purification, good immune protection effect

Inactive Publication Date: 2007-10-31
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Inoculating the body with whole pathogenic bacteria or lysates is a relatively traditional vaccinological method. However, due to the complex antigen composition of EHEC O157:H7 bacteria, it contains a variety of pathogenic factors, and has a large number of common antigens with common E. coli, which is easy to cause adverse reactions. Response, so that the research and application of EHEC O157:H7 bacterial vaccines are limited
At present, the most advanced EHEC O157:H7 vaccine research in foreign countries is the combination vaccine of O157 lipopolysaccharide and Pseudomonas aeruginosa protein A developed by Konadu E Y, which entered Phase I clinical trials in 1998, but the progress was slow, and some articles pointed out that O157 lipopolysaccharide The induced antibody has the effect of prompting O157 bacteria to release lethal Shiga toxin similar to antibiotics, so the possibility of success of this route is small (1. Konadu EY and TranHT. Infect Dis, 1998, 177 (2): 383; 2. Konadu E and Donohue-Rolfe A, Calderwood SB. Infect Immun, 1999, 67(11): 6191; 3. Ahmed A and Li J, Shiloach Y. Infect Dis, 2006, 193(4): 515)

Method used

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  • Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof
  • Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof
  • Genetic engineering vaccine of enterohemorrhagic escherichia coli 0157:H7 and the preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Construction of EspA-Intimin-C300 Fusion Gene of Enterohaemorrhagic Escherichia coli O157:H7

[0081] 1. Primer design and PCR amplification

[0082] 1) The primers were designed as follows: according to the genes encoding EspA and Intimin-C300 of O157:H7 (SaKai standard strain) found from GenBank, the primers were designed and analyzed using the software Primer Premier 5.0. The linker (the part in the box) sequence is designed at the 3' end of the upstream gene (EspA) and introduced into the BamHI site, and the two genes can be connected correctly with the sticky end digested with BamHI. Primers were synthesized by Shanghai Yingjun Company.

[0083] EspA:

[0084] Upstream primer P1:

[0085] 5′- CCATGG ATACATCAAATGCAAC-3′(NcoI)

[0086] Downstream primer P2:

[0087] 5′- TTACCAAGGGATATT-3'(BamHI) Intimin-C300:

[0088] Upstream primer P3:

[0089] 5′- ACTTCAGCACTTA-3' (BamHI)

[0090] Downstream primer P4:

[0091] 5′- CTCGAG TTCTACACAAACCGCATA-3' (Xho...

Embodiment 2

[0102] Construction and expression of EspA-Intimin-C300-Stx2B fusion protein of Escherichia coli O157:H7

[0103] 1. Primer design and PCR amplification

[0104] 1) The primers are designed as follows: according to the gene encoding Stx2B of O157:H7 (SaKai standard strain) found in GenBank and the fusion gene EspA-Intimin-C300 sequence constructed in Example 1, the primers are designed using the software Primer Premier5.0 and analysis. The sequence of the linker (the part in the box) was designed at the 3' end of the upstream fusion gene (EspA-Intimin-C300), and the fusion gene EspA-Intimin-C300 and the gene encoding Stx2B were connected by overlapping extension PCR method. Primers were synthesized by Shanghai Yingjun Company.

[0105] EspA-Intimin-C300:

[0106] Upstream primer P1:

[0107] 5′- CCATGG ATACATCAAATGCAAC-3′(NcoI)

[0108] Downstream primer P2:

[0109] 5′- TTCTACACAAACCG-3′

[0110] Stx2B:

[0111] Upstream primer P3:

[0112] 5′- AAGAAGATGTTTAT-3′...

Embodiment 3

[0132] Animal immunity and antibody detection

[0133] The target protein EspA-Intimin-C300-Stx2B was purified and immunized 4-5 weeks old Balb / c mice, 100ug / mouse / time, 100μL antigen was mixed with the same amount of Freund's complete adjuvant, and injected subcutaneously in the abdomen and groin of mice immunity. Once a week, blood was collected 4 days after the 3rd and 4th immunizations, and ELISA was used to detect the change of serum specific antibody titer.

[0134] Results: The antibody-positive rate of EspA-Intimin-C300-Stx2B was 90% after 3 times of immunization, and 98% after 4 times of immunization.

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Abstract

The invention discloses a poly valency fuse type enterorrhagia property bacillus coli O157:H7 gene engineering vaccine, which comprises the following steps: adopting enterorrhagia property bacillus coli O157:H7 Vi antigen Shiga's toxin II; combining subunit, compact sticking element and III type secretory protein A; constructing fuse engineering bacteria through gene retooling method; proceeding high density ferment; proceeding a series of purity course; getting fuse protein molecule vaccine with high purity. This invention possesses simple craft, good repeatability and higher protein purity.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a multivalent fusion type enterohaemorrhagic Escherichia coli O157:H7 genetic engineering vaccine and a preparation method thereof. Background technique [0002] Enterohemorrhagic Escherichia coli (EHEC) is the main pathogen of hemorrhagic enteritis, and its main serotype is O157:H7. The bacterium has been identified as a pathogenic bacterium for more than 20 years, and there have been outbreaks of different scales all over the world, including China. EHEC O157:H7 infection can cause diarrhea, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic platelets in 5% to 10% of cases Reduce severe complications such as thrombotic thrombocytopenic purpura (TTP), which can lead to death in severe cases. EHEC O157:H7 infection has become a global public health problem because of its outbreak tendency, strong pathogenicity and lethality, and antibiotic treatment ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/108C12N15/62A61P31/04
Inventor 毛旭虎邹全明刘艳青王庆旭易勇顾江余抒
Owner ARMY MEDICAL UNIV
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