KR20100101288A - Specific protein domain for the detection of salmonella contamination and its antibody manufacture method and also their utilization - Google Patents

Abstract

According to the present invention, food poisoning accidents caused by food hazard microorganisms are frequently occurring as imported foods are opened in Korea, and group meals such as factories and schools increase. Salmonella causes salmonellosis in various animals and infectious food poisoning from contaminated food in humans. Due to its pathogenicity in animals and humans, it is known to cause diseases such as enteric fever, gastroenteritis and septicemia. Invasion (HilA) and Flagellin (FliC) were selected as specific proteins to detect Salmonella, and overexpressed after cloning Salmonella's HilA (65kda) and FliC (55kda) -sized protein genes for antibody production. Protein was isolated and purified. Antibodies capable of detecting Salmonella were prepared using purified HilA and FliC proteins. The produced antibody was immobilized on a gold chip to detect Salmonella through the SPR method, which is a non-labeling method. Salmonella HilA rabbit polyclonal antibody and Salmonella FliC rabbit polyclonal antibody in this study can be applied to various biosensors and can detect the presence of pathogenic microorganisms quickly.

Description

Specific Protein Sites for Salmonella Contamination Detection and Antibody Preparation and Application thereof. {Specific Protein Domain for the Detection of Salmonella Contamination and its Antibody Manufacture Method and also their utilization}

The present invention measured the rapid response and high specificity of the antibody produced to detect Salmonella genus causing human pathogenicity due to food poisoning and intestinal infection through SPR, an analysis and measurement method of an unlabeled immunobio sensor chip, and more quickly and accurately Salmonella The present invention was completed by confirming the ability to detect genus bacteria.

For the detection of existing microorganisms, cultures were selected using a selective medium, followed by observation of bacterial growth and morphology to determine whether the pathogenic microorganisms were infected. Pre-enrichment media, selective enrichment media, and isolation on selective media were used. Presumptive evidence was confirmed by serological methods. However, since these methods take a long time to analyze, polynucleotide-based antigen-antibody reaction experiments for the detection of Salmonella in food and the establishment of a faster and more rapid detection method for pathogens. Many methods have also been developed, including DNA hybridization assays using oligonucleotide probes and DNA detection through polymerase chain reaction (PCR). In particular, methods for detecting and amplifying specific genes based on PCR have been reported to have high specificity in the search for pathogenic bacteria. However, methods using PCR also have higher detection specificity than conventional detection methods, but it takes 2 to 4 hours to amplify a specific region of the gene. In addition, many immunoassay technologies such as radio-immunoassay, fluorescence labeled antibody assay and enzyme-link immunosorbent assays (ELISA) have been developed, but the detection cost is expensive and time-consuming, and the pretreatment process is complicated (Oh Byung-Keun. 2004 Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium.Biosensors and Bioelectronics. 19: 1497-1504.)

Surface plasmon resonance (SPR) technology is well known for measuring the interaction between biomolecules, and has the advantage of being able to easily search samples of biological media with high specificity and sensitivity in a short time. Listeria monocytogenes (Paul Leonard. 2004.A generic approach for the detection of whole Listeria monocytogenes cells in contaminated samples using surface plasmon resonance.Biosensors and Bioelectronics.19: 1331-1335.), Vibrio cholerae O1 cholerae O1 using surface plasmon resonance.Biosensors and Bioelectronics. 21: 2315-319.), Salmonella It is used to detect a variety of pathogenic bacteria, such as).

Biological detection of Salmonella genus takes a long time for biological methods, serological methods, DNA hybridization assays, PCR, etc., and radio-immunoassay, fluorescence labeled antibody assay, enzyme-link immunosorbent assays (ELISA), etc. There are problems such as long time required, complexity of the pretreatment process, and the need for skilled personnel. In addition, the state-of-the-art immune biosensor is lacking.

HilA prediction 7 domains (15 mer peptides) and antibodies thereto, FliC prediction 4 domains (15 mer peptides) and antibodies thereto were prepared.

Salmonella HilA rabbit polyclonal antibody, Salmonella FliC rabbit polyclonal antibody, particularly antibodies of Salmonella carried out in the present invention can be applied to various biosensors and will be able to quickly detect the presence of pathogenic microorganisms.

In order to achieve the above object, the present invention provides for the rapid detection of the presence of pathogenic microorganisms by making an antibody showing a specific response to Salmonella harming humans.

Peptides with high specific antigenicity were selected for domain search for peptide sequences of Salmonella's Invasion (HilA) and Flagellin (FliC) proteins. The hydrophobicity and antigenecity of selected peptide regions were examined. In addition, the present invention was immunized by binding the carrier protein to increase the antibody titer because Peptide antigen is difficult to rise antigen titer. KLH (Keyhole Limpet Haemocyanin) was used as a carrier and bound using BSA. Emulsions were prepared using peptides conjugated to carriers and immunized. Subcutaneous injection was performed with 500 µg / 0.5 ml of emulsion per rabbit, and the primary and secondary tertiary boosting for the measurement of antigenic immunity were the same antigens at 4, 6 and 8 weeks, respectively, using IFA (Incomplete Freund's adjuvant, Difco, Rockford) was mixed in the same manner and injected by subcutaneous 200μg / 0.5ml per rabbit. A small amount of blood was collected from each boosting step to obtain serum, and antibody production and titer were measured. One week after the third boosting, blood was collected. When a small amount of blood is collected, the tail tip is cut and the blood is collected.

The immune response induced by injection of antigen was determined by ELISA to determine the degree of antibody present in rabbit serum. Antibody purification was performed by attaching the antigen to affinity-gel to prepare a column, separating the antibody, and using a column method using protein A. Finally, dialysis was performed. As a result, 15 ml (1 mg / ml) of HilA and 15 ml (10 mg / ml) of FliC were harvested. The prepared antibody was dissolved in PBS buffer containing 50% glycerol and stored at -20 ° C, and used as Ab that specifically reacts with Salmonella.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1> gene amplification by PCR

Genomic DNA extracted from Salmonella strain was used as template DNA, and PCR amplification was performed using Gene Cycle ^TM (BIO-RAD Co.). For PCR reaction solution, add 3µl of each prepared primer (10pmol), add 5µl of template DNA (10ng Genomic DNA), 39µl of sterile distilled water to PCR Mixer (Bioneer Co. Korea), and add mineral oil. It was. PCR reaction conditions were 1 cycle of denaturation at 94 ° C for 4 min, 1 cycle of denaturation at 94 ° C for 1 min, 30 cycles of annealing at 60 ° C for 1 min, 72 ° C for 1min, 30sec, and final extension at 72 ° C for 10min. Amplified DNA was confirmed by electrophoresis with 0.8% agarose gel. The results are shown in FIG. 1 (HilA) and FIG. 2 (FilC).

1-1 primer

Flic-Up: (BamHⅠ / NdeⅠ): 5'-gatcggatcccatatggcacaatgcattaatacaaac -3 '

Flic-down: (XhoⅠ / SalⅠ): 5'-gatcctcgaggtcgacacgcagtaaagagaggacgttttg-3 '

HilA-Up: (EcoRⅠ / NdeⅠ): 5'-gatcgaattccatatgccacattttaatcctgttcct-3 '

HilA-down: (XhoⅠ / SalⅠ): 5'-gatcctcgaggtcgacccgtaatttaatcaagcggggatc-3 '

< Example  2> of antigenic protein Cloning  And overexpression

Genomic DNA of Salmonella was extracted and amplified by PCR, and gene fragments of about hilA (1.6kb), fliC (1.5kb) and size were obtained. The obtained gene fragment was cloned into pBliscriptSK (+) and transformed into DH5α host, which is E. coli. The transformed host cell was cultured and the plasmid was extracted and recloned into overexpression vector pET22b (+). It was confirmed that the hilA and fliC genes were cloned, and transformed into BL21 (DE3) Lys competent cells to induce overexpression by adding IPTG to the host cell, and overexpressed the antigen protein for 6 hours. As a result, the HilA protein was overexpressed at 65kDa and the FliC protein at 55kDa. The results are shown in FIG. 3 (HilA) and FIG. 4 (FliC).

2-1 Nucleotide sequence and amino acid sequence of the Salmonella .

hilA gene .

2-2 Nucleotide sequence and amino acid sequence of the Salmonella .

flic gene .

< Example  3> His - Tag Using HilA , FliC Separation and purification of

Using the pET expression system, Salmonell sp. After overexpressing the proteins HilA and FliC, they were separated and purified using the Ni-NTA spin kit (Qiagen Co.). SDS-PAGE was performed to isolate and purify 65 kDa HilA protein and 55 kDa FliC protein. The results are shown in FIG. 5 (HilA) and FIG. 6 (FliC).

< Example  4> peptide Made of

The peptide sequences of HilA and FliC determined as specific proteins for the genus Salmonella were analyzed to find the most suitable peptide domain for antibody formation.

In the domain search for the amino acid sequences of Salmonella's HilA and FliC, when the peptide antibody was made with a specific domain region with high similarity, other nonspecific proteins could be detected, so the general domain region was excluded. In addition, the region containing the glycosylation site and myristoylation site was excluded because the synthesized peptide and the expressed protein were not the same, so the probability of detection by the antibody made with the synthetic peptide was very low. Areas with low hydrophobicity and high antigenicity are generally more likely to produce specific antibodies, and the hydrophobicity and antigenicity of the HilA whole protein and FliC whole protein were selected to select the areas with high potential for external exposure in the tertiary structure. , HilA had the best prediction 7 site and FliC had the best 4 prediction site. Prediction of the peptide region by homology search for other proteins revealed that FliC prediction 4 and HilA prediction 7 were good positions for peptide antibody production, high antigenicity and hydrophobicity, and low homology in other species. As it was determined that there was no reactivity, HilA determined prediction 7 and FliC determined prediction 4 to synthesize the peptide. The results are shown in FIG. 7 (HilA) and FIG. 8 (FliC).

A. HilA

 B. FliC

< Example  5> Immobilization of Antibodies

SPR sensor chip CM5 is coated with gold leaf on glass plate with 50nm thickness and dextran is applied on the gold leaf part. One of the test materials is used fixed to the dextran surface. The dextran surface is in contact with the flow-cell portion of the microchannel device and reacts with the sample flowing through the channel device. Depending on the type of surface of the sensor chip, it contacts the cells of two or four independent flow path devices, and if necessary, various experiments can be performed using different immobilized ligands for each cell. In this experiment, the carboxyl group has a negative charge on the surface of the sensor chip that is commonly used in contact with the solution. Dextran is a linear polymer of glucose, which has almost no nonspecific binding to the biological material, and has a very high concentration of the biological material in the solution. The experiment was carried out using a CM5 chip that can immobilize ligand very effectively even if low. Before the experiment, the sensor chip surface was activated by preconcentration with 10mM sodium aetate (pH4.5-5.0) and injection of EDC / NHS for optimal Ab fixation. The antibody was diluted to 100 ug / ml in 10 mM sodium acetate (pH 5.0) and injected for 1 minute (5 μl / min). Blocking unreacted activators with 1M ethanolamine (pH 8.5) and immobilization of CM5 chip Ab, the immobilization values are shown in Figure 9 (HilA), Figure 10 (FliC).

< Example  6> Salmonella cell  and HilA , FliC Ag Refractive Index Variation by Concentration of

Cell and Ag concentrations were diluted in PBS beffer to interact with the sensor chip surface for 15 minutes (1 μl / min). The results are shown in FIG. 11 (HilA) and FIG. 12 (FliC). The biosensor chip acted very proportionally with the change of cell concentration and the change of Ag (HilA, FliC) concentration.

< Example  7> AFM ( Atomic force microscopy analysis )

Atomic force microscopy was used to study surface topography of antibody and cell binding. The results are shown in Fig. 13 (two-dimensional image of Hilia), Fig. 14 (three-dimensional image of Hilia), and Fig. 15 (three-dimensional image of FliC). Bacterial cells responded and bound to biosensor chips well in accordance with two-dimensional and three-dimensional images of AFM.

1 is a diagram showing the process of amplifying the Salmonella specific HliA gene of the present invention,

Figure 2 shows a schematic diagram of the process of amplifying the Salmonella specific FilC gene of the present invention,

Figure 3 is a schematic diagram showing the process of overexpressing the HilA protein for detecting Salmonella of the present invention,

4 is a schematic diagram showing the process of overexpressing the FilC protein for detecting Salmonella of the present invention,

Figure 5 shows the schematic diagram showing the process of pure separation and purification of HilA protein for the production of Salmonella antibody,

Figure 6 shows the schematic diagram showing the process of pure separation and purification of FilC protein for the production of Salmonella antibody,

Figure 7 Schematic representation of the procedure for producing Salmonella HilA antibody,

Figure 8 Schematic representation of the procedure for Salmonella FilC antibody production,

9 is a schematic diagram illustrating a process of immobilizing Salmonella HilA polycolnal Antibody prepared on a gold chip,

10 will represented by illustrating a process of fixing a Salmonella FilC polycolnal Antibody production in the gold chip,

11 is Schematic representation of the reaction of flowing cells and HilA Ag to the immobilized Salmonella HilA polycolnal Antibody,

Figure 12 Schematic representation of the reaction by flowing Cell and FilC Ag to the immobilized Salmonella FilC polycolnal Antibody,

FIG. 13 is a schematic of AFM two-dimensional results confirming surface topography of Salmonella HilA antibody and Cell. FIG.

Figure 14 shows the schematic view showing the 3-dimensional AFM results to determine the surface topography of the Salmonella antibody HilA and Cell.

Figure 15 shows the schematic view showing the 2-dimensional AFM results to determine the surface topography of the Salmonella antibody FilC and Cell.

FIG. 16 schematically shows AFM three-dimensional results confirming surface topography of Salmonella FilC antibody and Cell. FIG.

Claims (2)

Production and Application of Prediction 7 Peptide (15 mer; KNEDNIWFKRWKQD-C) Antibody of Salmonella HliA Production and Application of Prediction 4 Peptide (15 mer; GTDQKIDGDLKFDD-C) Antibody of Salmonella FliC

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