Food-poisoning fungus concatenated fusion mycinamicin and use thereof

A food-poisoning bacteria and toxin technology, applied in the biological field, can solve the problems of lengthy, lack of extensive multiple fusion toxin kits, complex food testing process, etc.

A food-poisoning bacteria and toxin technology, applied in the biological field, can solve the problems of lengthy, lack of extensive multiple fusion toxin kits, complex food testing process, etc.

CN1818066AInactive Publication Date: 2006-08-16JILIN UNIV
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  • Food-poisoning fungus concatenated fusion mycinamicin and use thereof
  • Food-poisoning fungus concatenated fusion mycinamicin and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Preparation of the Nucleotide Sequence of the Food Poisoning Bacteria Concatenated Fusion Toxin of the Present Invention

[0014] Materials and methods

[0015] 1.1 Strains

[0016] Staphylococcus aureus A\B (26072, 26075) Staphylococcus aureus A\B (26072, 26075), Clostridium botulinum A (62A) Clostridium botulinum A (62A), Escherichia coli O157:H7E.coli O157:H7; other strains: Pseudomonasaerugionsa, Staphylococcus aureus C (C 1 , C 2 ) Staphylococcus aureus C (C 1 , C 2 ), Staphylococcus aureus E, Listeria monocytogene, Yersinia enterocolitica (52207), Yersinia frederiksenii, Yersinia intermedia , Yersinia kristensenii, Yersiniapseutotuberculosis, Yersinia pestis, Vibrioparahaemolyticus, Salmonella arizonae, Salmonella paratyphi A, Salmonella paratyphi C , Salmonella choleraesuis, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, Salmonella Dublin, Salmonella London, Salmonella aberdeen, Salmonella newport, Bacillus cereus cereus, Ba...

Embodiment 2

[0035] Sequencing showed that the recombinant toxin genes were original genes or partial gene sequences. Example 2 Expression and purification of the recombinant toxin of the present invention

[0036] Hc-VT 1B -SEA-VT 2B - SEB gene, hereinafter referred to as HVSVS, pET-22b expression vector, expressed in host strain E.coli DH5α at 37°C or 25°C for 1-6h, or overnight, induced by 1mmol / L IPTG. After the soluble protein expressed by the pET-22b expression vector was subjected to SDS-PAGE electrophoresis, the target protein band was excised and recovered as an immunogen. See figure 2 .

[0037] The expression forms of HVSVS in pET-22b at 37°C for 4 hours were soluble (5.29%) and inclusion body (4.69%), induced by 1mmol / L IPTG. At 25°C, the expressed protein was almost entirely soluble (9.9%). The molecular weight of the expressed protein is 112.33kDa. Most of the soluble protein is located in the cytoplasm, and a small part is located in the periplasm.

[0038] The amin...

Embodiment 3

[0039] Use TE (pH 8.0) buffer to suspend the expressed bacterial cell pellet, break the expressed bacterial cell with ultrasonic waves, centrifuge, and remove the precipitate; put the supernatant into a dialysis bag, dialyze and concentrate with PEG20000 for 2 hours, and concentrate the supernatant Cut out the target protein band after SDS-PAGE electrophoresis, slurry it in a homogenizer, mix with 2 times TE (pH 8.0) buffer, place at 4°C for 12 hours, and shake 3 to 4 times during the period , and then centrifuged at 10000r / min for 20min, the supernatant is the fusion toxin HVSVS expression protein. Protein concentration was measured with a UV spectrophotometer. Use 2 mg of purified soluble expressed protein per time plus an equal volume of complete Freund's adjuvant to mix or fully emulsify with a syringe to immunize rabbits for the first time, and take the back subcutaneous multi-point injection method. Afterwards, rabbits were immunized 4 times with the same amount of prot...

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Abstract

The invention is about the recombined toxin of the bacteria which can lead the food poisoning. The Hc-VT1B-SEA-VT2B-SEB linked by the linker sequence is from the gene Hc of the botulin A, the A gene: SEA and the B gene: SEB of Staphylococcus aureus, the VT1B and VT2B of E.Colli O 157. The serum antibody can anti above five bacteria toxin. So it can be used to produce the reagent box of recombined toxin to detect the food.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically includes concatenated fusion bacterial toxin and its establishment method, and becomes the beginning of a broad-spectrum immunological detection method. Background technique [0002] Food poisoning bacteria is a problem often encountered in food hygiene, and bacterial (toxin) food poisoning is one of the most common forms. The conventional method for the inspection of bacteria in food is to identify each bacterium (toxin) and its type during the inspection. The advantage is that it is scientific and accurate, but this process is complicated and lengthy. Toxin-producing strains are isolated and then determined whether they are Toxin producing strain or what kind of toxin. The rapid detection methods currently used are difficult to meet the requirements of wide detection coverage. In terms of the detection methodology of pathogens or toxins in food, one is to find accurate detection tech...

Claims

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Application Information

Patent Timeline
16 Aug 2006
Publication
CN1818066A
IPC
C12N15/31; C12N15/62; C07K14/195; C07K19/00; C07K16/12; G01N33/53
Inventors
柳增善; 于师宇