Application of toxoplasma gondii elongation factor monoclonal antibody in toxoplasma gondii detection and detection method

Abstract

The embodiment of the invention discloses application of an anti-toxoplasma gondii elongation factor monoclonal antibody in toxoplasma gondii detection and a detection method, and belongs to the technical field of toxoplasma gondii detection, a toxoplasma gondii elongation factor is EF-1alpha, and early diagnosis and discovery of toxoplasma gondii infection are crucial to control of the disease. The monoclonal antibody for resisting the toxoplasma gondii elongation factor EF-1 alpha is an antibody which has great potential to be applied to diagnosis of toxoplasmosis, the expression of the toxoplasma gondii elongation factor 1-alpha is highly conservative, and the monoclonal antibody plays an important role in pathogenesis of parasites and host cell invasion. The monoclonal antibody for resisting the EF-1alpha with the gene number of TGME49286420 is found for the first time in the invention, the expression of the monoclonal antibody in the toxoplasma gondii is very specific, and the toxoplasma gondii can be detected through a Western-blot technology and an indirect immunofluorescence technology.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of Toxoplasma gondii detection, and specifically to an application of a Toxoplasma gondii elongation factor monoclonal antibody in Toxoplasma gondii detection and a detection method. Background Art

[0002] Toxoplasma gondii is a highly harmful obligate intracellular parasite that is distributed worldwide and infects a wide range of hosts, including birds, humans, and mammals. Toxoplasma gondii mainly parasitizes in nucleated cells and flows through the blood to reach various parts of the body, destroying organs such as the brain and heart, causing the host's immunity to decline and even death. The spread of this parasitic disease has not only had a serious impact on the sustainable development of animal husbandry, but also threatens human life and health. Therefore, the prevention and control of toxoplasmosis is very critical, and the detection of Toxoplasma gondii plays an important role in the prevention of Toxoplasma gondii.

[0003] At present, the commonly used detection methods for Toxoplasma gondii mainly include etiological detection, immunological detection and molecular biological detection. Etiological detection has the disadvantages of low sensitivity, long time consumption and large workload. Immunological detection has the characteristics of simple operation and high specificity, but it still has the disadvantage of low sensitivity and cannot detect Toxoplasma gondii infection in time. Molecular detection technology has the advantages of strong specificity and high sensitivity, but it has the disadvantages of long time consumption, complex operation and dependence on large instruments. Therefore, it is urgent to develop a detection method with strong specificity, high sensitivity, convenience and fastness. Summary of the invention

[0004] To this end, the embodiments of the present invention provide an application of a monoclonal antibody of Toxoplasma elongation factor in Toxoplasma detection and a detection method to solve the problems of low sensitivity, complex operation and long time consumption in the prior art due to imperfect Toxoplasma detection technology.

[0005] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0006] According to a first aspect of an embodiment of the present invention, the present invention provides an application of an anti-Toxoplasma elongation factor monoclonal antibody in Toxoplasma detection, wherein the Toxoplasma elongation factor is EF-1α, and the anti-gene number is TGME49_286420.

[0007] According to a second aspect of an embodiment of the present invention, the present invention provides a method for detecting Toxoplasma gondii, which uses the above-mentioned anti-Toxoplasma elongation factor monoclonal antibody for detection.

[0008] Furthermore, the method for detecting Toxoplasma gondii includes Western-blot detection and indirect immunofluorescence experimental identification.

[0009] Furthermore, the Western-blot detection comprises the following steps:

[0010] S1: ME49 and RH strains of Toxoplasma were purified from mice infected with Toxoplasma gondii.

[0011] S2: Prepare protein lysis buffer, take out the purified worms, add 5-10 times the volume of protein lysis buffer to the worms, and mix well in a centrifuge tube; place the centrifuge tube on an ice-water mixture, ultrasonically disrupt the cells until the solution is clear and transparent, and centrifuge for 10-15 minutes;

[0012] S3: Transfer the supernatant obtained by centrifugation to a new pre-cooled centrifuge tube, add 5× Loading Buffer, shake and mix continuously, and heat in a 100°C metal water bath for 10-15 minutes to obtain the insect protein sample;

[0013] S4: Prepare 12% SDS-PAGE protein electrophoresis gel, add the insect body sample obtained in step S3 to the gel well and start electrophoresis, set the voltage to 150V, and set the time to 90min; after the electrophoresis is completed, pry open the glass plate with a plastic plate, and then use another plastic plate to cut off all the concentrated gel and excess separation gel, and transfer the protein in the separation gel to the PVDF membrane by wet transfer;

[0014] S5: Take out the transferred PVDF membrane, immerse it in the prepared skim milk, and block it on a shaker at 4℃ overnight; take out the blocked membrane, cut it according to the required size, add the membrane to an incubation box containing 3mL skim milk and 3uL anti-Toxoplasma elongation factor monoclonal antibody, and incubate it on a shaker in a 37℃ constant temperature incubator for 60min; take out the PVDF membrane and put it in a new clean incubation box, add 3-5mL PBST solution, and wash it on a shaker for 3 times, each time for 10min; add horseradish peroxidase-labeled goat anti-mouse secondary antibody, dilute it with PBST at a ratio of 1:25000; incubate it on a shaker in a 37℃ constant temperature incubator for 60min; take out the incubation box, pour out the secondary antibody, add 3-5mL PBST solution, and wash it on a shaker for 3 times, each time for 10min;

[0015] S6: Prepare super-sensitive luminescent liquid, take out the PVDF membrane, place it on the photosensitive plate, evenly add the super-sensitive luminescent liquid, and observe with a photosensitive instrument.

[0016] Furthermore, in step S2, the protein lysis solution includes 1×PBS, 50mM sodium ascorbate, 1% NP-40, 1% Triton X-100, 1% Tween-20, 5mM EDTA, and 50mM sodium tetraborate; the ultrasonic conditions are: power 10%, ultrasound 5s, interval 10s, a total of 6 times; the centrifugation conditions are: 4°C, 12000rmp.

[0017] Furthermore, in step S4, the transfer conditions of the wet transfer method are 180 mA and 60 min.

[0018] Further, in step S5, the PBST is prepared as follows: take a sterile blue-cap bottle, add 100 mL of 10X PBS buffer, dilute the 10X PBS buffer to 1X concentration with 900 mL of distilled water, stir evenly, add 0.1% Tween-20, and stir to mix evenly.

[0019] Furthermore, the indirect immunofluorescence assay comprises the following steps:

[0020] S1: 1×10 5 The Toxoplasma gondii tachyzoites purified from mice were evenly spread on an adhesive slide and air-dried at room temperature.

[0021] S2: Add 1 mL of 4% paraformaldehyde fixative onto the cell slide and fix for 15 min at room temperature. After removing the fixative, wash with PBS three times.

[0022] S3: Add 1 mL of 0.2% TritonX-100 to the slide, permeabilize for 15 min at room temperature, remove the permeabilization solution, and wash with PBS three times;

[0023] S4: Add 1 mL of 5% skim milk to the slide and block at 37°C for 45 min. After blocking, add anti-Toxoplasma elongation factor monoclonal antibody as primary antibody at a ratio of 1:200 to 1 mL of 5% skim milk, and healthy mouse serum as negative control. Incubate overnight at 4°C, and then wash with PBS 5-6 times.

[0024] S5: Alexa The secondary antibody of 488conjugated goat anti-mouse IgG was diluted in PBS at a ratio of 1:1000 and dropped on the slide, incubated at 37℃ for 45min, and then washed with PBS for 5-6 times;

[0025] S6: After DAPI staining in the dark for 3 minutes, wash with PBS three times, add anti-fluorescence quencher to seal the slide, and observe the results using a confocal fluorescence microscope.

[0026] The embodiments of the present invention have the following advantages:

[0027] Early diagnosis and detection of Toxoplasma infection are crucial for controlling the disease. The anti-Toxoplasma elongation factor EF-1α monoclonal antibody of the present invention is a very potential antibody for the diagnosis of toxoplasmosis. The expression of Toxoplasma elongation factor 1-α is highly conserved, and plays an important role in the pathogenesis of the parasite and the invasion of host cells. The anti-EF-1α monoclonal antibody of gene number TGME49_286420 described in the present invention is discovered for the first time in the present invention, and its expression in Toxoplasma is very specific, and Toxoplasma can be detected by Western-blot technology and indirect immunofluorescence technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0029] Figure 1 The prediction result of the transmembrane region of the Toxoplasma gondii EF-1α protein provided in Example 1 of the present invention;

[0030] Figure 2 The signal peptide prediction result of the Toxoplasma gondii EF-1α protein provided in Example 1 of the present invention;

[0031] Figure 3 Analysis of the hydrophilicity and hydrophobicity of the Toxoplasma gondii EF-1α protein provided in Example 1 of the present invention;

[0032] Figure 4 The specificity and effectiveness verification results of the anti-Toxoplasma EF-1α monoclonal antibody provided in Example 1 of the present invention;

[0033] Figure 5 The Western Blot results of the whole proteins of Toxoplasma gondii RH and ME49 strains in mice provided in Example 2 of the present invention;

[0034] Figure 6 This is the Western Blot result of the whole protein of Toxoplasma gondii ME49 strain purified in vivo and in vitro in mice provided in Example 2 of the present invention.

[0035] Figure 7 This is the indirect immunofluorescence result of Toxoplasma gondii ME49 strain provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0036] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] 1. Bioinformatics analysis of Toxoplasma gondii elongation factor EF-1α

[0039] 1. Use the online prediction software Topcons and SignalP to predict the transmembrane region and signal peptide of EF-1α protein;

[0040] 2. Use the protein sequence physicochemical parameter online prediction software ProtParam to analyze the number of amino acid residues, amino acid composition, relative molecular mass, theoretical isoelectric point, fat coefficient, instability coefficient, extinction coefficient, and protein half-life of EF-1α protein.

[0041] 3. Use the protein hydrophilicity and hydrophobicity online analysis software ProtScale to draw the hydrophilicity and hydrophobicity sequence spectrum of EF-1α protein, observe the folding of the protein, and analyze its hydrophilicity and hydrophobicity.

[0042] Table 1 Basic composition of EF-1α amino acids of the present invention

[0043]

[0044] In the present invention, a highly conserved protein elongation factor (EF)-1α (TGME49_286420) in Toxoplasma gondii was discovered, which has neither a transmembrane region ( Figure 1 ), and no signal peptide ( Figure 2 ), is a highly hydrophilic polymeric ribosomal protein ( Figure 3 , Table 1 ), which are ubiquitous and abundantly expressed in the parasite.

[0045] 2. Specificity and effectiveness verification of anti-Toxoplasma EF1-α monoclonal antibodies

[0046] The anti-Toxoplasma elongation factor EF-1α monoclonal antibody in the present invention is customized by Shanghai Abimat Company. The target of the monoclonal antibody obtained by mass spectrometry and immunoprecipitation experiments is verified to be EF-1α, and the detected molecular weight is consistent with the predicted molecular weight, about 49KDa, and the gene ID is: TGME49_286420.

[0047] Table 2 Basic information of proteins obtained by Co-IP with specific antibodies according to the present invention

[0048]

[0049] The anti-Toxoplasma EF-1α monoclonal antibody prepared using this protein has high specificity.

[0050] Example 2

[0051] This embodiment provides a method for detecting Toxoplasma gondii by Western-blot, comprising the following steps:

[0052] S1: ME49 and RH strains of Toxoplasma gondii were purified from mice infected with Toxoplasma gondii (the ME49 and RH strains of Toxoplasma gondii used were kept by the Zoonotic Diseases Laboratory of the College of Animal Husbandry and Veterinary Medicine of Shenyang Agricultural University);

[0053] S2: Prepare protein lysis buffer (1×PBS, 50mM sodium ascorbate, 1% NP-40, 1% Triton X-100, 1% Tween-20, 5mM EDTA, 50mM sodium tetraborate), take out the purified worms, add 5-10 times the volume of the worms to the protein lysis buffer, and mix in a centrifuge tube; place the centrifuge tube on an ice-water mixture, and ultrasonically disrupt the cells. The ultrasonic conditions are: power 10%, ultrasonic 5s, interval 10s, a total of 6 times; after the solution is clear and transparent, centrifuge at 4°C, 12000rmp for 10-15min;

[0054] S3: Transfer the supernatant obtained by centrifugation to a new pre-cooled centrifuge tube, add 5× Loading Buffer, shake and mix continuously, and heat in a 100°C metal water bath for 10-15 minutes to obtain the insect protein sample;

[0055] S4: Prepare 12% SDS-PAGE protein electrophoresis gel, add the insect body sample obtained in step S3 to the gel well and start electrophoresis, set the voltage to 150V, and set the time to 90min; after the electrophoresis is completed, pry the glass plate open with a plastic plate, and then use another plastic plate to cut off all the concentrated gel and excess separation gel, and transfer the protein in the separation gel to the PVDF membrane by wet transfer method (transfer conditions are 180mA, 60min);

[0056] S5: Take out the transferred PVDF membrane, immerse it in the prepared skim milk, and block it on a shaker at 4°C overnight; take out the blocked membrane, cut it according to the required size, add the membrane to an incubation box containing 3mL skim milk and 3uL anti-Toxoplasma elongation factor monoclonal antibody, and incubate it on a shaker in a 37°C incubator for 60 minutes; take out the PVDF membrane and put it in a new clean incubation box, add 3-5mL PBST solution (the preparation method of PBST is as follows: take a sterile blue-cap bottle, add 100mL of 10X PBS buffer, use 900mL of distilled water to dilute the 10X PBS buffer to 1X concentration, stir evenly, and add 0.1% Tween-20 (1 mL, stir to mix evenly), wash on a shaker for 3 times, 10 min each time; add horseradish peroxidase-labeled goat anti-mouse secondary antibody (Biyuntian, A0216), dilute with PBST at a ratio of 1:25000; incubate on a shaker in a 37°C incubator for 60 min; take out the incubation box, pour out the secondary antibody, add 3-5 mL PBST solution, wash on a shaker for 3 times, 10 min each time;

[0057] S6: Prepare super-sensitive luminescent liquid (Biyuntian, P0018S, prepared according to the Biyuntian ECL super-sensitive luminescent liquid kit method), take out the PVDF membrane, place it on the gelatin plate, evenly add the super-sensitive luminescent liquid, and observe with a gelatin analyzer.

[0058] Depend on Figure 5 , 6 The results show that the antibody has a very high specificity in detecting worms of two different genotypes, RH and ME49 strains, and in two different culture environments, in vivo and in vitro, using Western-blot technology.

[0059] Example 3

[0060] This embodiment provides a method for identifying Toxoplasma gondii by indirect immunofluorescence assay (IFA), comprising the following steps:

[0061] S1: 1×10 5 The Toxoplasma gondii tachyzoites purified from mice were evenly spread on an adhesive slide and air-dried at room temperature.

[0062] S2: Add 1 mL of 4% paraformaldehyde fixative onto the cell slide and fix for 15 min at room temperature. After removing the fixative, wash with PBS three times.

[0063] S3: Add 1 mL of 0.2% TritonX-100 to the slide, permeabilize for 15 min at room temperature, remove the permeabilization solution, and wash with PBS three times;

[0064] S4: Add 1 mL of 5% skim milk to the slide and block at 37°C for 45 min. After blocking, add anti-Toxoplasma elongation factor monoclonal antibody as primary antibody at a ratio of 1:200 to 1 mL of 5% skim milk, and healthy mouse serum as negative control. Incubate overnight at 4°C and then wash with PBS 5 times.

[0065] S5: Alexa 488conjugated goat anti-mouse IgG was used as the secondary antibody and diluted in PBS at a ratio of 1:1000. The antibody was then added to the slides and incubated at 37°C for 45 min, followed by washing with PBS for 5 times.

[0066] S6: After DAPI staining in the dark for 3 minutes, wash with PBS three times, add anti-fluorescence quencher to seal the slide, and observe the results using a confocal fluorescence microscope.

[0067] Depend on Figure 7 The results show that the monoclonal antibody against Toxoplasma gondii elongation factor EF-1α of the present invention is a very potential antibody for diagnosing toxoplasmosis with high specificity.

[0068] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Use of an anti-Toxoplasma elongation factor monoclonal antibody in Toxoplasma gondii detection, characterized in that: The Toxoplasma gondii elongation factor is EF-1α.

2. A method for detecting Toxoplasma gondii, characterized in that: The detection is carried out using the anti-Toxoplasma elongation factor monoclonal antibody described in claim 1.

3. The method according to claim 2, characterized in that The method for detecting Toxoplasma gondii includes Western-blot detection and indirect immunofluorescence experimental identification.

4. The method according to claim 3, characterized in that The Western-blot assay The following steps are involved: S1: ME49 and RH strains of Toxoplasma were purified from mice infected with Toxoplasma gondii. S2: Prepare protein lysis buffer, take out the purified worms, add 5-10 times the volume of protein lysis buffer to the worms, and mix well in a centrifuge tube; place the centrifuge tube on an ice-water mixture, ultrasonically disrupt the cells until the solution is clear and transparent, and centrifuge for 10-15 minutes; S3: Transfer the supernatant obtained by centrifugation to a new pre-cooled centrifuge tube, add 5× Loading Buffer, shake and mix continuously, and heat in a 100°C metal water bath for 10-15 minutes to obtain the insect protein sample; S4: Prepare 12% SDS-PAGE protein electrophoresis gel, add the insect body sample obtained in step S3 to the gel well and start electrophoresis, set the voltage to 150V, and set the time to 90min; after the electrophoresis is completed, pry open the glass plate with a plastic plate, and then use another plastic plate to cut off all the concentrated gel and excess separation gel, and transfer the protein in the separation gel to the PVDF membrane by wet transfer; S5: Take out the transferred PVDF membrane, immerse it in the prepared skim milk, and block it on a shaker at 4℃ overnight; take out the blocked membrane, cut it according to the required size, add the membrane to an incubation box containing 3mL skim milk and 3uL anti-Toxoplasma elongation factor monoclonal antibody, and incubate it on a shaker in a 37℃ constant temperature incubator for 60min; take out the PVDF membrane and put it in a new clean incubation box, add 3-5mL PBST solution, and wash it on a shaker for 3 times, each time for 10min; add horseradish peroxidase-labeled goat anti-mouse secondary antibody, dilute it with PBST at a ratio of 1:25000; incubate it on a shaker in a 37℃ constant temperature incubator for 60min; take out the incubation box, pour out the secondary antibody, add 3-5mL PBST solution, and wash it on a shaker for 3 times, each time for 10min; S6: Prepare super-sensitive luminescent liquid, take out the PVDF membrane, place it on the photosensitive plate, evenly add the super-sensitive luminescent liquid, and observe with a photosensitive instrument.

5. The method according to claim 4, characterized in that In step S2, the protein lysis solution includes 1×PBS, 50mM sodium ascorbate, 1% NP-40, 1% Triton X-100, 1% Tween-20, 5mM EDTA, and 50mM sodium tetraborate; the ultrasonic conditions are: power 10%, ultrasonic 5s, interval 10s, a total of 6 times; the centrifugation conditions are: 4°C, 12000rmp.

6. The method according to claim 4, characterized in that In step S4, the transfer conditions of the wet transfer method are 180 mA and 60 min.

7. The method according to claim 4, characterized in that In step S5, the PBST is prepared as follows: take a sterile blue-cap bottle, add 100 mL of 10X PBS buffer, dilute the 10X PBS buffer to 1X concentration with 900 mL of distilled water, stir evenly, add 0.1% Tween-20, and stir to mix evenly.

8. The method according to claim 3, characterized in that The indirect immunofluorescence assay comprises the following steps: S1: 1×10 5 The Toxoplasma gondii tachyzoites purified from mice were evenly spread on an adhesive slide and allowed to air dry at room temperature. S2: Add 1 mL of 4% paraformaldehyde fixative onto the cell slide and fix for 15 min at room temperature. After removing the fixative, wash with PBS three times. S3: Add 1 mL of 0.2% TritonX-100 to the slide, permeabilize for 15 min at room temperature, remove the permeabilization solution, and wash with PBS three times; S4: Add 1 mL of 5% skim milk to the slide and block at 37°C for 45 min. After blocking, add anti-Toxoplasma elongation factor monoclonal antibody as primary antibody at a ratio of 1:200 to 1 mL of 5% skim milk, and healthy mouse serum as negative control. Incubate overnight at 4°C, and then wash with PBS 5-6 times. S5: Alexa The secondary antibody of 488 conjugated goat anti-mouse IgG was diluted in PBS at a ratio of 1:1000 and dropped on the slide, incubated at 37°C for 45 min, and then washed with PBS 5-6 times; S6: After DAPI staining in the dark for 3 minutes, wash with PBS three times, add anti-fluorescence quencher to seal the slide, and observe the results using a confocal fluorescence microscope.

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