Penicillin-binding protein Bt-pbp2X and application thereof

A technology of penicillin and protein, which is applied in penicillin-binding protein and its application field, can solve the problems of high precision, low accuracy, difficulty in screening sensitive strains, inability to adapt to the detection of antibiotic residues in milk, etc., to achieve convenient and rapid detection, technology and The effect of low knowledge level requirements and simple and convenient sample pretreatment process

Active Publication Date: 2016-11-09
岩璟生物科技武汉有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the microbial method is widely used in grassroots units, the accuracy and accuracy of this method are not high enough, and the detection process is cumbersome and tim

Method used

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  • Penicillin-binding protein Bt-pbp2X and application thereof
  • Penicillin-binding protein Bt-pbp2X and application thereof
  • Penicillin-binding protein Bt-pbp2X and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of Bt-pbp2X protein

[0025] 1. Total DNA extraction of Bacillus thuringiensis

[0026] (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.

[0027] (2) Add 5.75ml Solution I (25mM Tris-HCl (pH8.0), 10mM EDTA, 50mM glucose) and 250μL lysozyme (200mg / mL) to ice overnight,

[0028] (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.

[0029] (4) Dry the precipitate with 200 μL ddH ...

Embodiment 2

[0049] Example 2 Determination of Bt-pbp2X Determination of Penicillin Interaction by Microthermal Mobility Apparatus (MST, NanoTemper Technologies)

[0050] Microthermophoresis (MST) is based on the following principle: the protein is fluorescently labeled, and an infrared laser light source is used to generate a temperature field. After the small molecule compound binds to the fluorescently labeled protein, the charge and conformation of the protein change, and the fluorescence The thermophoretic movement speed of the labeled protein in the temperature field changes, and this change is reflected in the change of the fluorescence value. Dilute the small molecule to a certain gradient concentration. If the small molecule binds to the fluorescently-labeled protein, the thermophoretic movement of the fluorescently-labeled protein also shows a certain regularity. From this, the binding curve of the two can be fitted. The dissociation constants (Kd values) of small molecules and p...

Embodiment 3

[0057] Example 3 Drawing of Bt-pbp2X Protein Detection Penicillin Standard Curve

[0058] 1. Preparation of penicillin standard samples

[0059] 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 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.

[0060] 2. Bt-pbp2X coated microtiter plate

[0061] Add 100 μL of penicillin-binding protein Bt-pbp2X solution (4 μg / mL) to the microwells of the microtiter plate and place at 4°C overnight to immobilize the Bt-pbp2X protein on the surface of the microwells. Pour off the liquid in the wells, add PBS buffer solution (300 μL) to each well, wash 3 times, and pat dry on filter paper.

[0062] 3. Sealing of the microtiter plate

[0063] Add 150 μL of 2% BSA blocking solution to each space, incubate at...

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Abstract

The invention provides a novel penicillin-binding protein Bt-pbp2X and encoding genes thereof. The protein has an amino acid sequence shown in SEQ ID No.2, or has the amino acid sequence obtained through substitution, deletion and/or increase of one or more amino acids from the amino acid sequence shown in SEQ ID No.2 with equal activity. The protein can be used for preparing a penicillin antibiotic detection reagent and can be applied to rapid detection of penicillin antibiotic residues. A complex instrument is not needed, the sample pretreatment process is very simple, specific requirements are made for technological and knowledge level of detection people, and the protein is very suitable for spot rapid 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 continuous improvement of people's living standards in our country, people pay more and more attention to food safety, and the problem of excessive antibiotic residues in food has aroused people's strong repercussions. 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 added to food. Long-term intake of food with excessive residues of β-lactam antibiotics will seriously cause people's health. Excessive residues of antibiotics can destroy ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12Q1/48
CPCC12N9/104C12N9/1048C12Q1/48
Inventor 鞠守勇陈其国朱虹翼徐顺高王超
Owner 岩璟生物科技武汉有限公司
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