Application of babesia orientalis thioredoxin peroxidase 2 in preparation of babesia orientalis disease detection kit
By preparing a detection kit for Babesia orientalis thioredoxin peroxidase 2 (TPx-2), the problems of complexity and low sensitivity of existing detection methods were solved, and a simple and efficient detection of oriental Babesia disease was achieved.
Patent Information
- Application Number
- CN202511043641.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-10-21
AI Technical Summary
Existing methods for detecting Babesia orientalis have problems such as complex operation, low sensitivity or the need for expensive instruments, which makes it difficult to meet the needs of clinical testing.
A detection kit was prepared using thioredoxin peroxidase 2 (TPx-2) from Babesia orientalis. Its specificity and sensitivity were verified by immunoblotting and indirect immunofluorescence assays, and it was applied to the detection of oriental Babesia infection.
The invention provides a method for detecting oriental babesiosis with simple operation and high sensitivity, good immunogenicity and specificity, and is suitable for clinical detection.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biology, and in particular to application of Babesia orientalis thioredoxin peroxidase 2 in preparing a detection kit for Babesia orientalis disease. Background Art
[0002] Babesia orientalis is a parasitic protozoan that is transmitted by the Rhipicephalus falciformis tick and causes babesiosis in buffaloes. Therefore, buffalo infection with Babesia orientalis is closely linked to the tick's activity season, typically occurring in months like May and June. Babesia orientalis is primarily prevalent in the Yangtze River Basin of my country, where buffalo farming is common. The parasite primarily invades buffaloes' red blood cells, causing symptoms such as fever, anemia, jaundice, and hemoglobinuria. If treatment is not timely, the animals often die, causing significant losses to the buffalo industry.
[0003] Currently, diagnostic methods for Babesia orientalis include microscopic, molecular, and serological testing. Microscopic testing requires extensive clinical experience, as the parasites easily evade immune responses and hide in the host's microvasculature. Furthermore, misdiagnosis can occur when the sample is low in parasite infection or contains mixed infections. Molecular biological testing offers high sensitivity and specificity, but often requires expensive equipment and is cumbersome to perform, making it difficult to use in clinical settings. Serological testing for Babesia orientalis is still in the laboratory development stage and is rarely used clinically. Therefore, there is an urgent need for simple, sensitive methods for detecting Babesia orientalis. Summary of the Invention
[0004] In view of this, the present invention aims to provide a use of Babesia orientalis thioredoxin peroxidase 2 in the preparation of a detection kit for oriental babesiosis. The Babesia orientalis thioredoxin peroxidase 2 provided by the present invention has good immunogenicity, produces antibodies with good specificity and high sensitivity, and is simple to operate, and can be used in a detection kit for oriental babesiosis.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The invention relates to a method for preparing a detection kit for oriental babesiosis using Babesia orientalis thioredoxin peroxidase 2. The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1.
[0007] The present invention also provides a use of Babesia orientalis thioredoxin peroxidase 2 in preparing a specific diagnostic antigen for oriental babesiosis. The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1.
[0008] The present invention also provides a use of Babesia orientalis thioredoxin peroxidase 2 in preparing a specific diagnostic antigen for oriental babesiosis. The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1.
[0009] Beneficial Technical Effects: The present invention provides the use of Babesia orientalis thioredoxin peroxidase 2 in preparing a detection kit for oriental babesiosis. The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 described herein is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1. The Babesia orientalis thioredoxin peroxidase 2 provided herein has good immunogenicity, produces antibodies with good specificity and high sensitivity, is simple to operate, and has been validated by immunoblotting and indirect immunofluorescence assays. The enzyme can be used in detection kits related to oriental babesiosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 This is the electrophoresis diagram of the amplification of the thioredoxin peroxidase gene 2 of Babesia orientalis; where 1: target gene amplified from the total gDNA of Babesia orientalis by cloning primers; 2: target gene amplified from the total cDNA of Babesia orientalis by cloning primers; M: DNA molecular weight standard;
[0011] Figure 2 The SDS-PAGE electrophoresis results of the qualitative analysis of Babesia orientalis thioredoxin peroxidase 2; 1: IPTG-induced pGEX-6P-1-TPx-2 expression product; 2: uninduced pGEX-6P-1-TPx-2; 3: pGEX-6P-1-TPx-2 expression product in the supernatant of IPTG-induced protein; 4: pGEX-6P-1-TPx-2 expression product in the inclusion bodies of IPTG-induced protein; 5: purified pGEX-6P-1-TPx-2 recombinant protein; M: protein molecular weight standard;
[0012] Figure 3The immunogenicity results of Babesia orientalis thioredoxin peroxidase 2 are shown in Figure 1. 1: Reaction of the recombinant protein with positive serum from buffalo infected with Babesia orientalis; 2: Reaction of the recombinant protein with serum from healthy buffalo; M: Protein molecular weight standard.
[0013] Figure 4 Western blot analysis of the native form of Babesia orientalis thioredoxin peroxidase 2. 1: Whole Babesia orientalis antigen reacts with TPx-2 positive polyclonal antiserum; 2: Buffalo erythrocytes react with TPx-2 positive polyclonal antiserum; 3: Whole Babesia orientalis antigen reacts with mouse negative serum; 4: Buffalo erythrocytes react with mouse negative serum.
[0014] Figure 5 Indirect immunofluorescence detection of Babesia orientalis; 5A: Infected erythrocytes incubated with serum containing a polyclonal antibody against BoTPx-2; 5B: Infected erythrocytes incubated with serum from healthy mice; 1: Hoechst staining: Babesia orientalis nuclei appear blue; BoTPx-2 staining with 594 results in BoTPx-2 appearing red; Merge: Superposition of the two reveals the blue Babesia orientalis nuclei within erythrocytes and the presence of TPx-2 in Babesia orientalis; DIC: Bright-field image display; Scale bar, 2 μm.
[0015] Figure 6 This is the map of the pGEX-6P-1-TPx-2 recombinant plasmid constructed in the present invention. DETAILED DESCRIPTION
[0016] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial sources; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.
[0017] Example 1: Purification of Babesia orientalis
[0018] Buffaloes were infected with ticks carrying Babesia orientalis. After infection was confirmed by PCR, smear microscopy, etc., 100 mL of anticoagulated blood (infection rate of approximately 5-10%) was collected from the infected buffaloes and placed in two 50 mL centrifuge tubes. The blood was centrifuged at 3000 rpm for 10 minutes, and the white flocs (leukocytes) between the supernatant and the red blood cells were removed. Three volumes of 1×PBS (3 times the volume of the red blood cells) were slowly added, and the mixture was shaken well. This operation was repeated three times until the supernatant became colorless and transparent. Two volumes of red blood cell lysis buffer were added, and the mixture was shaken well. The mixture was allowed to stand at room temperature for 30 minutes. After repeated pipetting 10 times with a 10 mL syringe, the mixture was centrifuged at 12000 rpm for 10 minutes, and the precipitate was collected. This step was repeated three times until the supernatant became colorless and transparent. The precipitate was dissolved in 1×PBS to obtain a solution of Babesia orientalis parasites.
[0019] Example 2: Extraction of Babesia orientalis gDNA
[0020] The extracted insect body solution was used for gDNA extraction using a blood / cell / tissue genomic DNA extraction kit (DP304, Tiangen Biochemical Technology Co., Ltd.). The extracted gDNA samples were frozen at -20°C for future use.
[0021] Example 3: Cloning and sequence analysis of Babesia orientalis thioredoxin peroxidase gene 2
[0022] 1) Primer design
[0023] Use clone manager software to design upstream and downstream primers:
[0024] F1: 5'-ATGGTGGCTCTTTCCCTACG-3';
[0025] R1: 5'-TTACTCAGTGGGTGGTTTCGGTTTC-3'.
[0026] 2) PCR amplification: Using Babesia orientalis gDNA as a template, PCR amplification was performed using primers F1 and R1 in a 25 μL reaction system. The PCR reaction system was as follows:
[0027]
[0028] PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 55°C for 90 s, 68°C for 1 min, 35 cycles; 72°C for 10 min.
[0029] 3) PCR product identification: After amplification, 10 μL of the PCR product was spotted with 10× nucleic acid loading buffer, and electrophoresed on a 1.0% agarose gel with 1× TAE buffer at 120 V for 30 min to observe the results and obtain the target fragment (see Figure 1 ).
[0030] 4) Cloning, screening and sequencing of target genes
[0031] Using full gold Blunt Cloning Kit (CB101-01, Beijing Quanshijin Biotechnology): Mix 1 μL (50 ng / μL) of pEasy-blunt vector with 3 μL of the gel-recovered target fragment, add 6 μL of Solution I from the kit, mix thoroughly, and ligate overnight at 16°C. Aseptically add 10 μL of the ligation product to E. coli Trans-T1 competent cells. Mix thoroughly by gentle pipetting and place on ice for 30 minutes. Heat shock the cells in a 42°C water bath for 90 seconds, then immediately cool them in an ice bath for 3 minutes. Be careful not to shake. Transfer the bacterial suspension to 500 μL of LB broth preheated to 37°C and gently shake at 150 rpm for 45 minutes at 37°C to restore drug resistance. 40 μL of X-gal (20 mg / mL) and 4 μL of IPTG (200 mg / mL) were added to LB agar plates containing ampicillin (AMP) (100 μg / mL) and spread evenly. After incubation at 37°C for 0.5 h, 150 μL of the bacterial culture was spread onto the plate. The plate was inverted and incubated in a 37°C incubator for 12–16 h. After incubation at 4°C to allow full blue color development, white colonies were selected and inoculated into LB medium containing 150 μg / mL AMP. The culture was shaken vigorously (230 rpm) for 12–16 h before identification. Clones that were positive for PCR amplification were sent to Shanghai Qinke Biotechnology Co., Ltd. for sequencing analysis. A 771-bp gDNA sequence was obtained, indicating a complete open reading frame (ORF). The sequence of the ORF is shown in Sequence Listing Seq_1. Sequence length: 771 bp; sequence characteristics: correct open reading frame (ORF): 771 bp; determining position: start and stop codon positions: ATG, position 1; TAA, position 771.
[0032] 5) The ORF sequence obtained in step 4 above is 771 bp long. The protein encoded by this gene contains 256 amino acids and has a molecular weight of approximately 27.7 kDa. The amino acid sequence was analyzed using BLAST (Basic Local Alignment Search Tool) software from the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov). The nucleotide sequence similarity between different species was compared using the ClustalW method using Megalign software in the DNAstar software package. The results indicate that the full-length ORF coding region sequence of the Babesia orientalis thioredoxin peroxidase gene 2 was obtained.
[0033] Example 4: Construction of prokaryotic expression vector and induced expression in Escherichia coli
[0034] 1) Design of expression primers for Babesia orientalis thioredoxin peroxidase gene 2:
[0035] Homologous primers for expression were designed based on the ORF sequence of the Babesia orientalis thioredoxin peroxidase gene 2:
[0036] F2:
[0037] 5'-TTCTGTTCCAGGGGCCCCTGATGATTGGCGGTGTCACGTC-3';
[0038] R2:
[0039] 5'-GATCGTCAGTCAGTCACGATGTCAAGACTTGGAAAAGGTCTTG GC-3'.
[0040] The homologous primers used to amplify the vector are:
[0041] F3: 5'-CATCGTGACTGACTGACGATC-3';
[0042] R3: 5'-CAGGGGCCCCTGGAACAGAA-3'.
[0043] 2) Construction of recombinant expression plasmid
[0044] TPx-2 was connected to the GST tag vector pGEX-6P-1 using homologous recombination.
[0045] 3) PCR Amplification: PCR amplification was performed using Babesia orientalis gDNA and pGEX-6P-1 plasmid as templates, using primers F2 and R2, and primers F3 and R3, respectively. The reaction system was 50 μL. The PCR reaction system was as follows:
[0046]
[0047] PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 55°C for 90 s, 68°C for 1 min, 35 cycles; 72°C for 10 min.
[0048] 4) PCR product identification: After amplification, the PCR product was spotted with 10× nucleic acid loading buffer and electrophoresed on a 1.0% agarose gel in 1× TAE buffer at 120V for 30 min to observe the results and obtain the target fragment and the linear fragment of the vector.
[0049] 5) Recover the vector and target fragment from gels separately and determine the recovery concentration.
[0050] Take 3.2 μL (32 ng / μL) of pGEX-6P-1 recovered product and mix it with 2.8 μL of TPx-2 recovered by gel, then add 1 μL of Exnase TM II. Mix 2μL of 5×CE buffer and 1μL of sterile water, then ligate at 37°C for 40 minutes. Aseptically add 10μL of the ligation product to E. coli Trans-T1 competent cells. Mix gently by pipetting repeatedly. Place on ice for 30 minutes. Heat shock the culture in a 42°C water bath for 90 seconds, then immediately cool it in an ice bath for 2 minutes. Be careful not to shake it. Transfer the bacterial solution to 500μL of LB medium preheated to 37°C and gently shake at 150 rpm at 37°C for 50 minutes to restore drug resistance. Add 40μL of X-gal (20mg / mL) and 4μL of IPTG (200mg / mL) to an LB agar plate containing ampicillin (AMP) (100μg / mL) and spread evenly. After incubating at 37°C for 0.5 hours, spread 150μL of the bacterial solution onto the plate. Invert the plate and culture in a 37°C constant temperature incubator for 12 to 16 hours. Pick white colonies and inoculate them into LB culture medium containing 150 μg / mL AMP. Shake vigorously (230 rpm) for 12 to 16 hours before identification.
[0051] 6) Construction of pGEX-6P-1-TPx-2 expression vector and identification of positive clones
[0052] The pGEX-6P-1-TPx-2 plasmid (1 μL) was aseptically added to the competent cells of Escherichia coli BL21 (DE3) (100 μL), and the mixture was gently pipetted repeatedly to mix. The mixture was placed in an ice bath for 30 minutes. Heat shock was performed in a 42°C water bath for 90 seconds, and then immediately cooled in an ice bath for 2 minutes. Be careful not to shake. The bacterial solution was transferred to 500 μL of LB culture medium preheated to 37°C, and gently shaken at 150 rpm at 37°C for 45 minutes to restore the bacterial resistance. 150 μL of the bacterial solution was spread on a plate containing ampicillin (AMP + ) (100 μg / mL) on LB agar plates. Invert the plates and incubate at 37°C for 12–16 hours. Select a single colony and inoculate it into LB medium containing 150 μg / mL AMP. Shake vigorously (230 rpm) for 12–16 hours before identification. Simultaneously, transform the empty pGEX-6P-1 vector without any exogenous genes as a negative control.
[0053] Take 1 μL of bacterial solution for PCR, and the reaction system is 25 μL as follows:
[0054]
[0055] PCR reaction conditions: 95°C for 5 min; 94°C for 30 s, 55°C for 90 s, 72°C for 1 min, 35 cycles; 72°C for 10 min.
[0056] The bacterial culture of the clones that were amplified positive was sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing analysis.
[0057] The pGEX-6P-1-TPx-2 recombinant plasmid constructed by the present invention is shown in FIG. Figure 6 shown.
[0058] 7) Prokaryotic expression of pGEX-6P-1-TPx-2 recombinant protein
[0059] The positive pGEX-6P-1-TPx-2 / BL21 (DE3) bacterial solution was added to LB medium containing AMP at a ratio of 1:100 and cultured at 37°C and 200 rpm until OD 600 When the pH is about 0.6, 0.8 mmol / L IPTG is added and induced at 37°C for 4 hours. A large amount of induced expression bacterial solution is collected and centrifuged at 8000 rpm at 4°C for 15 minutes. The supernatant is discarded and the bacterial pellet is resuspended in PBS and crushed three times by pressure. Then, the pellet is centrifuged at 12000 rpm at 4°C for 10 minutes. The pellet is discarded and the supernatant is collected. After microfiltration, the crude protein of recombinant expression is obtained. The results of SDS-PAGE electrophoresis are as follows. Figure 2 shown.
[0060] 8) Purification of recombinant protein
[0061] Purify the recombinant protein using Glutathione Sepharose 4B: Take 1.33 mL of Glutathione Sepharose 4B collagen solution, centrifuge at 1800 rpm for 5 minutes, remove the supernatant, add 10 mL of 1× PBS, shake gently until the Glutathione Sepharose 4B gel is suspended in the solution, centrifuge at 1800 rpm for 5 minutes, and discard the supernatant; add 1 mL of 1× PBS and resuspend to obtain 50% Glutathione Sepharose 4B gel. Induce 200 mL of bacteria, collect the bacteria at 4°C, 12000 rpm, 1 min, then wash the induced bacteria 2 to 3 times with PBS, resuspend the bacteria in 1×PBS (generally 1 / 10 volume), ultrasonically disrupt until clear, centrifuge at 12000 rpm, 20 min to obtain the supernatant, add an appropriate amount of treated 50% Glutathione Sepharose 4B gel to the supernatant (generally 20 mL supernatant corresponds to 1 mL gel), gently shake it on a shaker at room temperature to allow it to adsorb protein for 1 hour; centrifuge at 2000 rpm, 5 min and discard the supernatant; add at least 10 times the volume of PBS, gently shake until the Glutathione Sepharose 4B gel is suspended in the solution, centrifuge at 2000 rpm, 5 min and discard the supernatant; repeat the above steps twice; add 1 mL GST elution Buffer, shake gently for 10 minutes; centrifuge at 2000 rpm for 5 minutes, collect the supernatant; repeat the above steps at least twice; detect protein purity by SDS-PAGE electrophoresis and protein concentration by UV spectrophotometer; store the protein fraction at -80℃. Figure 2 shown.
[0062] Example 5: Identification of recombinant protein immunogenicity and native form
[0063] 1) Identification of immunogenicity
[0064] The purified recombinant protein was first subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane at 50V. After 3 hours, the PVDF membrane was blocked with TBST-5% skim milk powder for 1 hour and washed 3 times with TBST for 5 minutes each. The PVDF membrane was placed in 1:100 diluted buffalo serum positive for Babesia orientalis and healthy buffalo serum, respectively, incubated at room temperature for 1 hour, washed 3 times with TBST for 5 minutes each. The secondary antibody (1:2000 diluted rabbit anti-bovine IgG) was added and incubated at room temperature for 1 hour. The membrane was washed 3 times with TBST for 5 minutes each, and then BCL was added for color development. Western blot results showed that the purified recombinant protein TPx-2 at 40kDa reacted specifically with buffalo serum positive for Babesia orientalis, but did not react with healthy buffalo serum (see Figure 3 ).
[0065] 2) Identification of the natural form of TPx-2
[0066] Whole-parasite antigens of Babesia orientalis were subjected to SDS-PAGE electrophoresis. After transferring the proteins to a PVDF membrane using semi-dry transfer, the membrane was blocked with TBST-5% skim milk powder at 4°C overnight and washed three times with TBST for 5 minutes each. The PVDF membrane was incubated with 1:100 diluted TPx-2 mouse positive serum and negative serum at 37°C for 2 hours and washed three times with TBST for 5 minutes each. Secondary antibody (1:2000 diluted goat anti-mouse IgG) was added and incubated at room temperature for 1 hour. The membrane was washed three times with TBST for 5 minutes each and then developed with BCL. Western blot results showed that Babesia orientalis reacted with TPx-2 positive serum to produce a specific band of approximately 37 kDa, but did not produce a band with negative serum (see Figure 4 ).
[0067] Example 6: Identification of Babesia orientalis using immunofluorescence
[0068] Take 1 μL of worm-stained blood and evenly apply it on a clean glass slide to prepare a blood smear. After drying, fix it with 100% ice methanol at room temperature for 30 minutes, then use 0.5% TritonX-100 to permeabilize for 5 minutes, wash 3 times with PBS, and block it with PBS solution containing 1% (m / v) bovine serum albumin (BSA, purchased from Sigma) at 37°C for 1 hour. Wash 3 times with PBS, 2 minutes each time; add primary antibody (1:100) TPx-2 mouse positive serum and negative serum, incubate at 37°C for 1 hour, wash 3 times with PBS, 2 minutes each time; add secondary antibody (1:1000 diluted 594 fluorescent goat anti-mouse IgG and Hoechst for cell nucleus staining), incubate at room temperature for 1 hour, wash 3 times with PBS, 2 minutes each time, and then use anti-fluorescence quencher to seal the slide. The experimental results were observed under a laser confocal microscope ( Figure 5 ), the nucleus of Babesia orientalis is blue (Hoechst), TPx-2 is red, and the two are superimposed to reveal the blue nucleus of Babesia orientalis in red blood cells and the red TPx-2 protein present in Babesia orientalis (Merge) ( Figure 5 ).
[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. Use of Babesia orientalis thioredoxin peroxidase 2 in preparing a detection kit for oriental Babesia disease, characterized in that: The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1.
2. Use of Babesia orientalis thioredoxin peroxidase 2 in the preparation of a specific diagnostic antigen for Babesia orientalis disease, characterized in that: The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1.
3. Use of Babesia orientalis thioredoxin peroxidase 2 in the preparation of a specific diagnostic antigen for Babesia orientalis disease, characterized in that: The amino acid sequence of the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_2, and the nucleotide sequence of the gene encoding the Babesia orientalis thioredoxin peroxidase 2 is shown in Seq_1.