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Enterorrhagia colibacillus 0157:H7 Shiga toxin 2A1 subunit active segment Stx2a1 recombination protein, expression method and application

A technology of Escherichia coli and O157, applied in the field of genetic engineering, to achieve the effect of good specificity, efficient immune response, immune prevention and protection, and good immunogenicity

Inactive Publication Date: 2012-05-23
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no Stx2A at home and abroad 1 Related research reports on full-length sequence construction and expression

Method used

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  • Enterorrhagia colibacillus 0157:H7 Shiga toxin 2A1 subunit active segment Stx2a1 recombination protein, expression method and application
  • Enterorrhagia colibacillus 0157:H7 Shiga toxin 2A1 subunit active segment Stx2a1 recombination protein, expression method and application
  • Enterorrhagia colibacillus 0157:H7 Shiga toxin 2A1 subunit active segment Stx2a1 recombination protein, expression method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Enterohaemorrhagic Escherichia coli O157:H7 Stx2A 1 subunit active fragment Stx2a 1 clone

[0059] 1. Strains and reagents

[0060] Escherichia coli 99A021 was provided by the Jiangsu Provincial Center for Disease Control,

[0061] Plasmid pET-22b and Escherichia coli BL21, DH5α are preserved by the applicant's laboratory,

[0062] ExTaq DNA polymerase, restriction endonucleases Xho I and Nde I, T 4 Both ligase and DNAmarker are products of Dalian TaKaRa Company;

[0063] The plasmid extraction kit is a product of OMEGA;

[0064] The gel recovery kit is a product of BBI;

[0065] Bacterial Genomic DNA Extraction Kit is a product of Tiangen Company.

[0066] 2. According to EHEC O157:H7 Stx2A 1 The full-length gene sequence of the subunit is shown in SEQ ID NIO:3.

[0067] Application of DNAstar software analysis: see attachment figure 2 illustrate.

[0068] 3. Design and synthesis of primers (underlined enzyme cleavage sites)

[0069] According t...

Embodiment 2

[0101] Example 2: EHEC O157:H7 Stx2A 1 subunit active fragment Stx2a 1 Expression and purification in E. coli

[0102] 1. Induced expression of the target gene:

[0103] The correct active fragment Stx2a identified by Nde I, Xho I double digestion and sequencing 1 The recombinant strains and pET-22b vector / BL21 bacterial liquid were inoculated into the LB liquid culture liquid containing ampicillin, and the expression was induced according to the conventional method, (37°C shaker culture to A 600 0.6-0.8, adding SDS-PAGE loading buffer to each sample for freezing, adding IPTG to a final concentration of 0.1mmol), shaking at 37°C for 1 hour, 3 hours, and 5 hours, sampling and adding SDS-PAGE loading buffer Frozen storage, boiled in boiling water for 5 minutes, followed by conventional SDS-PAGE gel electrophoresis analysis (see attached Figure 5 ).

[0104] The results showed that compared with pET-22b empty vector, recombinant Stx2a 1 The protein has a distinct band arou...

Embodiment 3

[0114] Embodiment 3: Enzyme-linked immunosorbent assay (ELISA) method detects the production of the antibody of EHEC O157:H7 toxoid immunized mice:

[0115] 1. Preparation of EHEC O157:H7 toxoid:

[0116] The natural EHEC O157:H7 toxin protein purified in our laboratory was inactivated by formalin inactivation by adding formaldehyde at a final concentration of 1%, and inactivated at 37°C for 5 days.

[0117] 2. Immunization program:

[0118] Immunize five Balb / c mice aged 5-6 weeks, 100μg / mouse, 100μl inactivated natural toxin, add the same amount of immunogen Freund's adjuvant for the first immunization, and then add incomplete Freund's adjuvant to inject the mouse abdomen and Inguinal subcutaneous immunization. Two weeks after the first immunization, the second immunization was carried out, and then once a week, and the blood supernatant of the mice was collected on the seventh day after the fourth immunization to detect the antibodies produced.

[0119] 3. Using EHEC O15...

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Abstract

The invention discloses a enterohemorrhagic escherichia coli O157:H7 shiga toxin 2A1 subunit active fragment Stx2a1 recombinant protein, including 1) enterohemorrhagic escherichia coli O157:H7 shiga toxin 2A1 subunit active fragment Stx2a1 and expression vector fragment; or 2) enterohemorrhagic escherichia coli O157:H7 shiga toxin 2A1 subunitactive fragment Stx2a1 protein carboxyl terminal acquires protein and expression vector fragment with similar function by losing and adding one or more amino terminals. The invention also discloses a preparation method of aforementioned enterohemorrhagic escherichia coli O157:H7 shiga toxin 2A1 subunit active fragment Stx2a1 recombinant protein, application thereof in preparation of a test kit and preparation of subunit vaccine for prevention and cure infection of EHEC O157:H7.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to an enterohemorrhagic Escherichia coli O157:H7 Shiga toxin 2A 1 subunit active fragment Stx2a 1 Protein expression methods and applications. Background technique [0002] Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was first isolated in 1975, because it can produce lethal Shiga toxin (Stx), and was recognized as a serious zoonotic pathogen in 1982. Its infection has the characteristics of outbreak trend, strong lethality, and the risk of exacerbating the disease by antibiotic treatment. It has become a global public health problem and poses a huge threat to human health. At the same time, EHEC O157:H7 is easy to cultivate, has strong infectivity, and has various transmission routes, making it very likely to be used as a bacterial weapon and bioterrorism agent in future military warfare; the potent pathogenic factor of EHEC O157:H7 may also be used for genetic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/265C12N15/31C12N15/70G01N33/68A61K39/108A61P31/04
Inventor 曾浩刘璐邹全明罗萍张卫军
Owner ARMY MEDICAL UNIV
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