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Penicillin-binding protein and application thereof

A technology for binding protein and penicillin, applied in penicillin binding protein and its application field, can solve the problems of high technical level requirements of analysts, time-consuming, uncomfortable and rapid detection of β-lactam antibiotic residues, etc. The sample pretreatment process is simple, and the effect is suitable for on-site rapid detection

Active Publication Date: 2017-04-26
WUHAN POLYTECHNIC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods have good selectivity, high sensitivity, high accuracy, low detection limit, and can detect multiple elements or compounds at the same time, they require expensive instruments and equipment, and the sample pretreatment process is cumbersome and time-consuming. High level requirements, not suitable for rapid detection of β-lactam antibiotic residues in food enterprises

Method used

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  • Penicillin-binding protein and application thereof
  • Penicillin-binding protein and application thereof
  • Penicillin-binding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0022] The preparation of embodiment 1Bt-PBP3 protein

[0023] 1. Total DNA extraction of Bacillus thuringiensis

[0024] (1) Inoculate the Bacillus thuringiensis strain BMB171 in a 5ml PA bottle for overnight activation at 28°C, transfer to LB medium in 50ml liquid medium at 1% inoculation amount the next day, and cultivate to logarithmic at 28°C and 200r / min During the growth period, the cells were collected by centrifugation and washed once with STE.

[0025] (2) Add 5.75ml Solution I (25mM Tris-HCl (pH8.0), 10mM EDTA, 50mM glucose) and 250μL lysozyme (200mg / mL) to ice overnight,

[0026] (3) Add 3ml of 10% SDS in a water bath at 50-60°C for 30min; then add 3.6ml of 5MNaCl, put on ice for 10min and then directly add phenol: chloroform: isoamyl alcohol (25:24:1) to remove protein; centrifuge at 12000r / min for 15min Then take the supernatant, add 2 times the volume of absolute ethanol to precipitate. Wash once with 70% ethanol.

[0027] (4) Dry the precipitate with 200 μL...

Embodiment 2B

[0047] Example 2 Determination of Bt-PBP3 Protein Conjugating Ability with Penicillin

[0048] 1. Preparation of penicillin G standard samples

[0049] Penicillin was dissolved in PBS buffer solution to prepare a standard solution with a concentration of 1 μg / mL, and this solution was added to known antibiotic-free reconstituted milk to prepare a standard sample with a certain concentration of penicillin. Subsequently, the standard samples were placed on a shaker for 15 minutes and thoroughly mixed.

[0050] 2. Bt-PBP3 coated microtiter plate

[0051] Add 100 μL of penicillin-binding protein Bt-PBP3 solution (4 μg / mL) to the microwells of the microtiter plate, and place at 4°C overnight to immobilize the Bt-PBP3 protein on the surface of the microwells. Pour off the liquid in the well, and add PBS buffer solution (1.47g / LNa 2 HPO 4 , 0.43g / L KH 2 PO 4 and 6.79g / L NaCl; pH7.2) 300μL, washed 3 times, and patted dry on filter paper.

[0052] 3. Sealing of the microtiter pl...

Embodiment 3

[0062] Example 3 Application of Bt-PBP3 Protein Determination of Penicillin G Residues in Food

[0063] 1. Pretreatment of test samples

[0064] The test sample is milk: skimmed milk samples are directly loaded for testing, pure milk samples are centrifuged at 5000 rpm for 5 minutes, and directly tested after skimming.

[0065] Test samples are meat, fish, shrimp and eggs: Weigh 10g of the sample, add 20mL of 10% acetonitrile aqueous solution (volume ratio), mix vigorously for 15 minutes, centrifuge at 8000rpm for 5 minutes, take the supernatant, dry it with nitrogen, and use 1mL Dissolved in deionized water for detection.

[0066] The test sample is honey: Weigh 10g sample, add 20mL deionized water to dissolve, enrich penicillin through Oasis solid-phase extraction column, wash the column with 5mL deionized water, dry it under negative pressure, then elute with 5mL methanol, collect and elute Blow dry with liquid nitrogen, dissolve with 1mL deionized water, and use for dete...

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Abstract

The invention provides a novel penicillin-binding protein Bt-PBP3 and encoding genes thereof. The protein contains an amino acid sequence shown in SEQ ID No.2 or the protein is obtained by substitution, deletion and / or addition of one or more amino acids on the amino acid sequence shown in the SEQ ID No.2 and has the equal activity. The protein can be used for preparing a penicillin detection reagent, and used for penicillin detection, no expensive instrument equipment is needed, the sample pretreatment process is simple, and the requirement for the technological level of analysis personnel is low, and the protein is suitable for rapid on-site detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a penicillin-binding protein and its application. Background technique [0002] With the improvement of people's living standards in our country, people pay more and more attention to food safety, and the problem of antibiotic residues in food has attracted widespread attention. β-lactam antibiotics refer to a large class of antibiotics containing a β-lactam ring in their molecular structure, including penicillins and their derivatives, cephalosporins, monoamide rings, carbapenems, and penem-like enzyme inhibitors Agents, etc., as well as newly developed cephamycins, thiamycins, monocyclic β-lactams and other atypical β-lactam antibiotics. It is the most widely used class of antibiotics in existence. Long-term intake of food with β-lactam antibiotic residues will seriously endanger people's health. Antibiotic residues can destroy the balance of gastrointestinal flora, cause long-term...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12N15/70C12N1/21C12Q1/37C12R1/19
CPCC12N9/485C12Q1/37C12Y304/16004
Inventor 鞠守勇陈其国周娅敏尹喆徐顺高朱虹翼袁昌宁
Owner WUHAN POLYTECHNIC
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