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Indirect ELISA (enzyme linked immunosorbent assay) detection method for escherichia coli OmpT (Outer-membrane protease T) antibody

A technology of Escherichia coli, detection method, applied in the field of biotechnology detection, to achieve a highly specific effect

Inactive Publication Date: 2012-10-03
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no ELISA detection method using OmpT recombinant protein to detect Escherichia coli

Method used

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  • Indirect ELISA (enzyme linked immunosorbent assay) detection method for escherichia coli OmpT (Outer-membrane protease T) antibody
  • Indirect ELISA (enzyme linked immunosorbent assay) detection method for escherichia coli OmpT (Outer-membrane protease T) antibody
  • Indirect ELISA (enzyme linked immunosorbent assay) detection method for escherichia coli OmpT (Outer-membrane protease T) antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] This embodiment is used to illustrate the acquisition of the ompT gene and the construction of the expression vector. The specific steps are as follows:

[0054] 1. According to the ompT gene sequence of Escherichia coli (GenBank Accession No. AE014075), use Oligo6.0 software to analyze, design a pair of primers by yourself, and add restriction enzyme sites BamH I and Sal I at both ends of the primers (square box) and protective bases. Analyzed by computer BLAST software, it has good specificity. The nucleotide sequences of the primers are:

[0055] ompT-T1: 5'-CG ATGCGGGCGAAACTTCT-3' (SEQ ID No. 1)

[0056] ompT-T2: 5'-GGC TTAAAAGGTGTACTTAAGACCAGC-3' (SEQ ID No. 2)

[0057] 2. Use the extracted E.coli 2002-1 strain DNA as a template for PCR amplification. The amplification conditions are: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 45s, annealing at 60.5°C for 45s, extension at 72°C for 90s, and 30 seconds cycle, with a final extension at 7...

Embodiment 2

[0064] This example illustrates the induced expression and purification of recombinant proteins. The specific steps are as follows:

[0065] 1. For colonies with the correct sequence, pick a single colony and inoculate it in liquid LB medium containing Amp+, shake overnight at 37°C. Take the overnight culture and inoculate 2% of the liquid LB medium containing Amp+, culture with shaking at 37°C until the A600 value is 0.5-1.0, add IPTG to a final concentration of 1 mM, continue shaking at 37°C, and in the cultured At different times (2, 3, 4, 5, 6 h), take 1 mL of the bacterial solution from the culture and transfer it to a centrifuge tube, centrifuge at 10,000 r / min for 5 min, collect the bacterial pellet, add 90 μL 2× loading buffer and 10 μL Suspend and mix with 1mol / L DTT, boil for 5min, centrifuge at 10,000r / min for 5min and use immediately, or store at -20°C and heat at 100°C for 5min before use.

[0066] 2. SDS-PAGE gel electrophoresis: Assemble the electrophoresis equ...

Embodiment 3

[0069] This example illustrates the antigenicity and specificity verification of OmpT protein.

[0070] 1. Carry out according to the "semi-dry method": transfer the protein in the gel after SDS-PAGE to the NC membrane, use the whole cell immune serum and protein immune serum as the primary antibody respectively, and HRP-labeled goat anti-mouse IgG as the primary antibody. Secondary antibodies were used for western-blotting to detect expressed proteins. Results: A clearly visible band ( image 3 ), indicating that the expressed OmpT protein has good antigenicity and conservation, and high purity.

[0071] 2. Use a micro-sampler to add the diluent (except the first well) to the 96-well polystyrene microwell reaction plate (elisa plate) at 25 μL per well. Add 50 μL of MpT purified protein immunized mouse serum to the first well, take 25 μL of the serum from the first well and add it to the second well, mix 5 times by pipetting with a sampler, and then use a micro-sampler to dr...

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Abstract

The invention provides an indirect ELISA (enzyme linked immunosorbent assay) detection method for an escherichia coli OmpT (Outer-membrane protease T) antibody, and a detection antigen on an ELISA plate in the ELISA is OmpT recombinant protein. The detection method comprises the following steps that after being diluted according to the ratio of 1:640, the serum to be detected is incubated with the OmpT recombinant protein on the ELISA plate at the temperature of 37 DEG C for 1.5 h, and the plate is washed three times; rabbit anti-bovine IgG marked by horse radish peroxidase is added to be incubated with the serum to be detected at the temperature of 37 DEG C for 1.5 h; a TMB (tetramethylbenzidine) substrate is added for developing for 10 min; and OD450 is detected after the reaction is ended, the antibody is positive if the OD450 is larger than or equal to 0.335, and the antibody is negative if the OD450 is smaller than 0.335. The recombinant protein prepared from the symbolic gene OmpT of the escherichia coli is used as the detection antigen for ELISA, the recombinant antigen can be produced on a large scale in a standardizing mode, has strong specificity, and is suitable for the antibody detection after the infection of all serotype bacteria of the escherichia coli.

Description

technical field [0001] The invention belongs to the field of biotechnology detection and relates to an indirect ELISA detection method for Escherichia coli OmpT antibody. Background technique [0002] Escherichia coli (E.coli) is the most important member of the 12 genera of Enterobacteriaceae. It exists widely in nature and is an important zoonotic pathogen that can cause co-infection of humans and animals. According to its pathogenic mechanism, it can be divided into three categories: symbiotic, intestinal pathogenic and extraintestinal pathogenic. In the past, there were many studies on enteropathogenic E.coli, but in recent years, extraintestinal pathogenic E.coli has attracted much attention. However, the surface structure of E.coli bacteria is complex, and there are many types of serotypes. The serotypes of E.coli that are prevalent in different regions are also different, and there are cross-reactions with other 11 genera of Enterobacteriaceae. It is the serum of E.c...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531
Inventor 崔玉东佟春玉马金柱朱战波
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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