Fusion protein SAmB as well as coding gene and applications thereof

A fusion protein and protein technology, applied in gene therapy, fusion polypeptide, fusion with toxin, etc., can solve problems such as limitations, technical difficulties, and protein instability

Active Publication Date: 2011-01-19
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this recombinant toxoid mimics the natural toxin structure (1A:5B), including 1 A subunit and 5 B subunits. Because the recombinant expression process requires structural assembly, the preparation is technically difficult, the process is not easy to scale up, and the protein is not stable. Stable and not easy to store, which brings limitations to its further application

Method used

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  • Fusion protein SAmB as well as coding gene and applications thereof
  • Fusion protein SAmB as well as coding gene and applications thereof
  • Fusion protein SAmB as well as coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, construction, expression and purification of recombinant fusion protein SAmB

[0031] The strains and plasmids used in the present invention are shown in Table 1 below; the primers used in the present invention are shown in Table 2 below. Ni affinity chromatography column HisTRAP FFTM and The FPLC purification system is a GE product.

[0032] Table 1. Bacterial strains and plasmids used in the experiments

[0033]

[0034] Table 2. Primer sequences used in experiments

[0035]

[0036] a The underlined part is a restriction endonuclease site.

[0037] b Linked sequences are underlined in bold font.

[0038] 1. Construction of recombinant expression vector

[0039] 1. Acquisition of the gene encoding the fusion protein

[0040] Use primers 1 and 2 to PCR amplify the A subunit protein gene (Stx2A gene) of type II Shiga toxin from the genome of EHEC O157:H7EDL933 (see Table 1 for the characteristics and origin of the strain) (shown in sequence ...

Embodiment 2

[0056] Example 2, the immunogenicity of the fusion protein SAmB and the in vitro anti-adhesion and anti-toxin activity of the immune serum

[0057] Reagents used in this example: Sorbitol MacConkey basic agar, MTT, and Gimsa dyes were all purchased from Kejunzhou; DMEM medium was purchased from GIBCO; standard fetal bovine serum was purchased from Biochrom; double antibodies were purchased from Hyclone; Cefixime granules were products of Guangzhou Baiyunshan General Pharmaceutical Factory, and 1% potassium tellurite was purchased from Beijing Luqiao Technology Co., Ltd. Experimental animals were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from SIGMA.

[0058] 2S protein is used as a positive control in the present invention to compare and evaluate the immunogenicity and protective effect of SAmB. 2S protein is a kind of fusion protein that is connected by...

Embodiment 3

[0070] Embodiment 3, the immunoprotective effect of fusion protein SAmB anti-EHEC O157:H7 infection

[0071] Animal immunization steps are the same as in Example 2.

[0072] Stx1 and Stx2, as two toxin proteins, are used in the present invention to challenge SAmB-immunized mice. Its preparation method sees with embodiment 2.

[0073] 1. Observation of the protective effect of the mice in the immunized group against the lethal dose of EHEC O157:H7 lytic bacteria challenge

[0074] Carry out 10LD50 lethal dose O157: H7 lytic bacteria challenge test (1LD50=10 through SAmB immune three times) 7 CFU), every group of 15 mice, 21 days statistical survival rate, the result 93.3% mice all showed to be protected (14 / 15), and placebo immunization group (PBS) 15 mice all died (0 / 15) ( Figure 5 A), Only 26.7% of mice survived in the 2S immunized group (4 / 15).

[0075] 2. Observation of the protective effect of the mice in the immunized group against the lethal dose of Stx toxin challe...

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Abstract

The invention discloses a fusion protein SAmB as well as a coding gene and applications thereof. The fusion protein provided by the invention is named as SAmB, and comprises an A-subunit mutant protein of N-end II-type shiga toxin and a B-subunit protein of C-end I-type shiga toxin, wherein, the A-subunit mutant protein is a protein which mutates the active site amino acid residue of the A-subunit protein of the N-end II-type shiga toxin. The fusion protein SAmB provided by the invention can be used as a drug used for preventing and/or treating the shiga toxin pathogenic bacterial infection or infection complication.

Description

technical field [0001] The present invention relates to the fusion protein SAmB related to the prevention and / or treatment of Shiga toxin-producing pathogenic bacteria infection, its coding gene and application. Background technique [0002] Enterohemorrhagic E.coli (EHEC) O157:H7 infection is a new food-borne infectious disease and a worldwide public health problem. EHEC O157:H7 infection mainly causes serious complications such as infantile diarrhea, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). symptoms, which can lead to death in severe cases. Since the first outbreak of EHEC O157:H7 in the United States in 1982, there have been repeated outbreaks in Japan, the United States, the United Kingdom, Canada, Australia and other countries, especially between May and August 1996. EHEC O157 was first reported in Xuzhou in my country since 1986: Since H7 infection, EHEC O157:H7 Escherichia coli has been isolated from hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/108A61K48/00A61P31/04
CPCC07K2319/00C07K2319/55A61K38/00C07K14/25A61K39/0283A61K2039/55566A61P31/04
Inventor 王慧蔡昆李涛侯晓军王琴高翔
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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