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Salmonella bacteriophage tail receptor binding protein RBP-55 and application thereof in detection of salmonella

A technology of RBP-55, receptor binding, applied in the direction of phage, virus/phage, application, etc., to achieve the effect of expanding the scope of application

Pending Publication Date: 2022-06-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]2) The other is an immunological detection method, which mainly relies on the specific reaction of antigen and antibody for detection. A common detection technology is enzyme-linked immunosorbent immunoassay Adsorption method (ELISA), which has the advantages of specificity and sensitivity, but it is difficult to obtain high-affinity antibodies
Ordinary methods will greatly increase the detection time while achieving enrichment and enrichment

Method used

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  • Salmonella bacteriophage tail receptor binding protein RBP-55 and application thereof in detection of salmonella
  • Salmonella bacteriophage tail receptor binding protein RBP-55 and application thereof in detection of salmonella
  • Salmonella bacteriophage tail receptor binding protein RBP-55 and application thereof in detection of salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Extraction of Salmonella phage STP55 genome

[0066] Pick a single colony of Salmonella (ATCC 14028) from the solid medium, shake and culture at 37°C for 3h to OD 600nm 0.8-1.0, add 5mL of phage STP55 suspension, shake and culture at 37°C for 4h-6h until the culture medium changes from turbid to clear, take 1mL of high-titer phage suspension, add deoxyribonuclease and ribonuclease RNase in sequence A, incubate at 37°C for 40 min; add 20 μL 2mol / L ZnCl 2 , incubate at 37°C for 7 minutes; after centrifugation, dissolve the pellet in 500 μL TES buffer, and place in a water bath at 65°C for 15 minutes; digest with proteinase k (20 mg / mL) and cool, add 60 μL pre-cooled 3 mol / L potassium acetate, and place on ice for 15 minutes After centrifugation, the supernatant was extracted with phenol / chloroform / isoamyl alcohol (25:24:1) to extract DNA, washed with 70% ethanol and then dissolved in TE buffer to obtain phage genome samples. -Generation Sequencing, NGS) to mea...

Embodiment 2

[0068] The construction of embodiment 2 recombinant plasmids

[0069] Add the predicted orf 55 with a His tag at the N-terminus, design primers to introduce Xho I and NdeI restriction endonuclease sequences on both sides of orf 55, and combine the fragment with the empty vector pET28b (+) with Xho I and Nde I 37 Double enzyme digestion at ℃ overnight, the digestion product was recovered after verification on agarose gel electrophoresis, orf 55 was mixed with the double digestion product of pET28b(+), ligated with T4 DNA ligase overnight at 16 °C, and the ligation product Transformed into Escherichia coli T7E competent cells by heat shock method. Pick a single colony in LB medium, culture it overnight and send it for sequencing, and determine the correct sequence as a positive transformant.

Embodiment 3

[0070] Example 3 Induced Expression and Purification of Recombinant Phage Receptor Binding Protein

[0071] The transformed competent cells were evenly spread on LB agar medium containing kanamycin (50 μg / mL), and cultured overnight at 37°C. Pick a single colony containing the recombinant plasmid and inoculate it in 5 mL LB medium containing kanamycin (50 μg / mL), and culture overnight at 37°C. Transfer 200μL to 20mL LB medium containing resistance, culture at 37℃ to OD 600nm =0.6-0.8, add IPTG (final concentration 1mmol / L) and culture at 37°C for 4h. The cells were resuspended in PBS, the cells were disrupted by ultrasonication, and samples were taken for SDS-PAGE to verify the expression status. Equilibrate the nickel column to room temperature, equilibrate with the washing solution, pass the collected supernatant sample through the column, wash the nickel column with a buffer containing 20mM imidazole, elute the target protein with an eluent of 250mM imidazole, and dialyze...

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Abstract

The invention discloses a salmonella phage tail receptor binding protein RBP-55 and an application of the salmonella phage tail receptor binding protein RBP-55 in salmonella detection. The amino acid sequence of the salmonella bacteriophage receptor binding protein RBP-55 is SED ID No. 1; the bacteriophage receptor binding protein RBP-55 disclosed by the invention can recognize and bind a receptor on the surface of salmonella, and is coupled with an activated water-soluble carboxyl quantum dot to form a salmonella bacteriophage receptor binding protein-quantum dot conjugate RBP-55-QDs; the probe is a bifunctional detection probe which specifically recognizes and binds salmonella and generates a fluorescence signal. The salmonella rapid fluorescence detection kit is used for qualitative and quantitative detection of a to-be-detected sample, the detection time is about 1 h, and the detection limit reaches 1 CFU / mL. The application range of the phage receptor binding protein in food safety detection is expanded.

Description

technical field [0001] The invention relates to the field of food biotechnology, in particular to a Salmonella phage tail receptor binding protein RBP-55 and its application in detecting Salmonella. Background technique [0002] Salmonella spp. is one of the important foodborne pathogens, which can cause various clinical symptoms, including nausea, vomiting, abdominal pain, diarrhea, fever and headache, posing a huge threat to human and animal health. Salmonella is widely found in nature, and is commonly found in human or animal intestines. Feces and intestinal contents are important sources of pollution. Most agricultural products will be exposed to pollutants during the production process, thus contaminating Salmonella, livestock and poultry meat, eggs, Dairy foods are most susceptible to Salmonella contamination. According to the European Food Safety Authority (EFSA), among the diseases caused by Salmonella, the main sources are eggs and egg products, followed by cheese,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C12N15/33C12N15/70C12N1/21G01N33/569G01N33/58C12R1/19
CPCC07K14/005C12N15/70G01N33/56916G01N33/588C12N2795/10122G01N2333/255
Inventor 王小红丁一峰王佳朱文娟邵彦春黄晨曦
Owner HUAZHONG AGRI UNIV
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