Application of dunnii dunnii TPx-2 protein in preparation of detection kit for dunnii dunnii disease

Abstract

The invention provides application of Duncan babesia dunnii TPx-2 protein in preparation of a detection kit for Duncan babesia dunnii disease, and belongs to the technical field of molecular biology. According to the application of the TPx-2 protein provided by the invention in preparation of the detection kit for the dunnii dunnii disease, the amino acid sequence of the TPx-2 protein is shown as Seq2. The Duncan-dunnii TPx-2 protein provided by the invention can effectively detect the existence of Duncan-dunnii in mouse blood, and has good immunogenicity, and the generated antibody has good specificity and high sensitivity. Therefore, the dunnii dunnii TPx-2 protein and the antibody generated by the dunnii dunnii TPx-2 protein can be used for detecting the dunnii dunnii disease and can be applied to a detection kit.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of Duncan-Babesia TPx-2 protein in the preparation of a detection kit for Duncan-Babesia disease. Background Technology

[0002] Babesia dulcis causes babesiosis in humans. It is reported to be transmitted primarily through ticks, blood transfusions, and the placenta. The parasite was first detected in the blood of a patient in Washington, D.C., in 1991. With a better understanding of the parasite, its distribution has expanded beyond the Americas to include multiple continents worldwide, including Asia. Infections with Babesia dulcis have been found in several regions and countries, including South Korea, Japan, and Southeast Asia, with the number of cases increasing annually, indicating a global epidemic. Infection with this parasite can cause fever, anemia, jaundice, and hemoglobinuria in humans and rodents, and in severe cases, can even lead to death.

[0003] Currently, there are no specific drugs targeting Babesia dulcis. Because Babesia is morphologically and pathogenically similar to Plasmodium, its treatment primarily follows the treatment regimens for Plasmodium, leading to increasing side effects, including drug resistance. Furthermore, there are no specific diagnostic techniques for Babesia dulcis; clinical diagnosis requires complex and time-consuming methods such as microscopy. Moreover, if the parasite is not in the acute phase of infection, it can easily evade immune responses, hiding in distal venous capillaries, making detection methods like microscopy difficult and prone to false negatives. Therefore, identifying corresponding parasite antigens as diagnostic antigens for application in molecular detection methods has become crucial research. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of Duncan's babesi TPx-2 protein in the preparation of a detection kit for Duncan's babesiosis. This invention obtains the thioredoxin peroxidase gene 2 (TPx-2) through extensive screening. Using bioinformatics methods, multi-faceted analysis of this gene reveals that it is relatively conserved and possesses good antigenicity, showing promise for further research as a candidate diagnostic antigen. Subsequently, this gene is amplified from Duncan's babesi and recombinantly expressed. Immunoblotting and indirect immunofluorescence experiments verify that the antibody produced by the recombinant protein expressed by this gene has good specificity and high sensitivity, and can be used as a detection antigen for Duncan's babesiosis in a detection kit.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The application of Duncan-Babesia TPx-2 protein in the preparation of a detection kit for Duncan-Babesia disease, wherein the amino acid sequence of the TPx-2 protein is shown in Seq_2.

[0007] The present invention also provides the application of Duncan-Babesia TPx-2 protein in the preparation of Duncan-Babesia antibody detection kit, wherein the amino acid sequence of the TPx-2 protein is shown in Seq_2.

[0008] The present invention also provides the application of Duncan-Babesia TPx-2 protein in the preparation of Duncan-Babesia disease-specific diagnostic antigens, the amino acid sequence of which is shown in Seq_2.

[0009] In some embodiments, the method for preparing the Duncan-Babesia TPx-2 protein preferably includes: introducing the encoding gene of the TPx-2 protein into Escherichia coli, constructing a recombinant expression plasmid pGEX-6P-1-TPx-2, transforming it into BL21(DE3) using the competent cell method, screening for highly resistant transformants, and inducing expression to obtain the TPx-2 protein.

[0010] In some embodiments, the screening of highly resistant transformants is preferably performed using ampicillin.

[0011] In some embodiments, the induced expression is preferably induced with 0.8 mmol / L IPTG at 37°C for 4 h.

[0012] In some implementations, the induced expression is preferably followed by separation and purification.

[0013] In some embodiments, the separation and purification steps are preferably as follows: centrifuge the induced bacterial culture at 8000 r / min for 15 min at 4°C, discard the supernatant, add 20 mL PBS to resuspend the bacterial precipitate, and then break it 3 times using a pressure disruptor; centrifuge at 12000 r / min for 10 min at 4°C, collect the supernatant, microfilter it, and then purify it using a GST purification column.

[0014] In some embodiments, the nucleotide sequence of the gene encoding the TPx-2 protein is shown in Seq_1.

[0015] Beneficial Technical Effects: This invention provides the application of Duncan's babesi TPx-2 protein in the preparation of a detection kit for Duncan's babesiosis. The amino acid sequence of the TPx-2 protein is shown in Seq_2. The TPx-2 protein provided by this invention can effectively detect the presence of Duncan's babesi in mouse blood, exhibits good immunogenicity, and produces antibodies with good specificity and high sensitivity. Therefore, both the TPx-2 protein and the antibodies it produces can be used for the detection of Duncan's babesiosis and applied to detection kits. Attached Figure Description

[0016] Figure 1Electrophoresis diagram of the amplification of the Duncan-Babesia thioredoxin peroxidase gene 2; where 1: target gene amplified from total gDNA of Duncan-Babesia using cloning primers; 2: target gene amplified from total cDNA of Duncan-Babesia using cloning primers; M: DNA molecular weight standard;

[0017] Figure 2 SDS-PAGE electrophoresis results for qualitative analysis of thioredoxin peroxidase 2 in Babesia dulcis; among which, Figure 2 In A: 1: pGEX-6P-1-TPx-2 expression product induced by IPTG; 2: uninduced pGEX-6P-1-TPx-2; 3: pGEX-6P-1-TPx-2 expression product in IPTG-induced supernatant; 4: pGEX-6P-1-TPx-2 expression product in IPTG-induced inclusion bodies; Figure 2 B1: Purified pGEX-6P-1-TPx-2 recombinant protein; M: Protein molecular weight standard;

[0018] Figure 3 The results were for the immunogenicity of Duncan's babesi thioredoxin peroxidase 2; among which, Figure 3 A: The recombinant protein reacts with positive serum from mice infected with Duncan's Babesia; Figure 3 B: Reaction of recombinant protein with healthy mouse serum; M: Protein molecular weight standard;

[0019] Figure 4 Western blot identification of the natural form of Duncan-Babesia thioredoxin peroxidase 2. Figure 4 In A, 1: the reaction of Duncan-Babesia whole worm antigen with TPx-2 positive polyclonal antibody serum; 2: the reaction of mouse erythrocytes with TPx-2 positive polyclonal antibody serum. Figure 4 In B, 1: the reaction of Duncan-Babesia whole worm antigen with mouse negative serum; 2: the reaction of mouse erythrocytes with mouse negative serum.

[0020] Figure 5 To detect Duncan's Babesia using indirect immunofluorescence; Hoechst: The nucleus of Duncan's Babesia appears blue; BdTPx2: BdTPx-2 appears red after 594 staining; Merge: The two images are superimposed to show the blue Duncan's Babesia nucleus within erythrocytes and the TPx-2 present in Duncan's Babesia; DIC: Bright field image; Scale bar size is 2 μm;

[0021] Figure 6 The pGEX-6P-1-TPx-2 recombinant plasmid constructed in this invention is shown in the image. Detailed Implementation

[0022] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the materials, reagents, etc., used in the embodiments and experimental examples of the present invention can be obtained commercially; unless otherwise specified, the methods used in the embodiments and experimental examples of the present invention are conventional methods.

[0023] Example 1: Purification of Babesia dunccanii

[0024] First, mice infected with Duncan's babesi (infection rate of approximately 5-10%) were anesthetized, and 100 mL of anticoagulated blood was collected from the orbital cavity into two 50 mL centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min to remove the white flocculent material (white blood cells) between the supernatant and the red blood cells. Three volumes of 1×PBS were slowly added to the red blood cells, and the mixture was shaken well. This operation was repeated three times until the supernatant was 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 min. After repeatedly pipetting the mixture 10 times with a 10 mL syringe, the tubes were centrifuged at 12000 rpm for 10 min to collect the precipitate. This step was repeated three times until the supernatant was colorless and transparent. The precipitate was dissolved in 1×PBS to obtain the Duncan's babesi body solution.

[0025] Example 2: Extraction of gDNA from Babesia dunccanii

[0026] The extracted parasite solution was used for gDNA extraction using a blood / cell / tissue genomic DNA extraction kit (DP304, purchased from Tiangen Biotech Co., Ltd.). The extracted gDNA samples were stored at -20°C for later use.

[0027] Example 3: Cloning and sequence analysis of the Duncan-Babesia thioredoxin peroxidase gene 2

[0028] 1) Primer design

[0029] The upstream and downstream primers were designed using clone manager software: F1: 5'-ATGGCTAGGTTTGCA AAG-3'; R1: 5'-TTAGTTTAATCCTTTTTTAGAGTATTTATT-3'.

[0030] 2) PCR amplification: Using Duncan's Babesia gDNA as a template, PCR amplification was performed using F1 and R1 primers. The reaction volume was 25 μL, and the PCR reaction system was as follows:

[0031]

[0032] PCR reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 90 s, 68℃ for 1 min, 35 cycles; 72℃ for 10 min.

[0033] 3) PCR product identification: After amplification, take 10 μL of the PCR product, load it with 10× nucleic acid loading buffer, electrophoresis on a 1.0% agarose gel, 1× TAE buffer, at 120V for 30 min, and observe the results to obtain the target fragment (see...). Figure 1 ).

[0034] 4) Cloning, screening, and sequencing of the target gene

[0035] Using full gold -Blunt Cloning Kit (CB101-01, purchased from Beijing TransGen Biotech): Mix 1 μL (50 ng / μL) of pEasy-blunt vector with 3 μL of the target fragment recovered from the gel, then add 6 μL of Solution I from the kit. Mix well and incubate overnight at 16°C. Take 10 μL of the ligation product and aseptically add it to E. coli Trans-T1 competent cells. Gently and repeatedly pipette to mix, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, then immediately incubate on ice for 3 min to cool, being careful not to shake. Transfer the bacterial culture to 500 μL of LB medium preheated to 37°C, and gently shake at 150 rpm at 37°C for 45 min to restore bacterial resistance. Add 40 μL of X-gal (20 mg / mL) and 4 μL of IPTG (200 mg / mL) to an LB agar plate containing ampicillin (AMP) (100 μg / mL), spread evenly, and incubate at 37°C for 30 min. Then, spread 150 μL of bacterial culture onto the plate. Invert the plate and incubate at 37°C for 12-16 h. After incubation at 4°C until the blue color is fully developed, pick white colonies and inoculate them into LB medium containing 150 μg / mL AMP. Shake vigorously (230 rpm) for 12-16 h before identification. The bacterial culture of the PCR-positive clone was sent to Shanghai Qinke Biotechnology Co., Ltd. for sequencing analysis, yielding a 792bp gDNA sequence. This gene has a complete open reading frame (ORF), and its ORF sequence is shown in Sequence Listing Seq_1. Sequence length: 792bp, sequence type: gDNA, sequence characteristics: has a correct open reading frame (ORF): 792bp; determining positions: start and stop codon positions: ATG, position 1; TAA, position 792.

[0036] 5) The full-length ORF sequence obtained in step 4 above is 792 bp. The protein encoded by this gene contains 263 amino acids and has a molecular weight of approximately 29 kDa. The amino acid sequence was analyzed using the 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 with the Megalign software in the DNAstar software package. The results showed that the full-length ORF coding region sequence of the Duncan's babesi thioredoxin peroxidase gene 2 was obtained.

[0037] Example 4: Construction of prokaryotic expression vector and induction of expression in Escherichia coli

[0038] 1) Design expression primers for the Duncan-Babesia thioredoxin peroxidase gene 2:

[0039] Homologous primers designed based on the ORF sequence of the Duncan-Babesia thioredoxin peroxidase gene 2 are as follows:

[0040] F2: 5'-TTCTGTTCCAGGGGCCCCTGATGGCTAGGTTTGCAAAG-3';

[0041] R2:

[0042] 5'-GATCGTCAGTCAGTCACGATGTTAGTTTAATCCTTTTTTAGAGTA TTTATT-3'.

[0043] The homologous primers used for amplification vectors are:

[0044] F3: 5'-CATCGTGACTGACTGACGATC-3';

[0045] R3: 5'-CAGGGGCCCCTGGAACAGAA-3'.

[0046] 2) Construction of recombinant expression plasmids

[0047] TPx-2 was linked to the GST tag vector pGEX-6P-1 using homologous recombination.

[0048] 3) PCR amplification: Using Duncan's Babesia gDNA and pGEX-6P-1 plasmid as templates, PCR amplification was performed using primers F2, R2 and F3, R3, respectively. The reaction volume was 50 μL, and the PCR reaction system is as follows:

[0049]

[0050] PCR reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 90 s, 68℃ for 1 min, 35 cycles; 72℃ for 10 min.

[0051] 4) PCR product identification: After amplification, the PCR product was loaded with 10× nucleic acid loading buffer, 1.0% agarose gel, 1× TAE buffer, 120V, and electrophoresis for 30 min to observe the results and obtain the linear fragments of the target fragment and the vector respectively.

[0052] 5) Perform gel recovery of the carrier and target fragment separately, and determine the recovery concentration.

[0053] Take 3.2 μL (32 ng / μL) of pGEX-6P-1 recovered product and mix it with 2.8 μL of TPx-2 recovered from gel electrophoresis, then add 1 μL of LExnase. TM II. Mix 2 μL of 5×CE buffer and 1 μL of sterile water, and ligate at 37°C for 40 min. Take 10 μL of the ligation product and add it aseptically to E. coli Trans-T1 competent cells. Gently and repeatedly pipette to mix, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, then immediately incubate on ice for 2 min to cool, being careful not to shake. Transfer the bacterial culture to 500 μL of LB medium preheated to 37°C, and gently shake at 150 rpm at 37°C for 50 min to restore the bacteria's drug resistance. Add 40 μL of X-gal (20 mg / mL) and 4 μL of IPTG (200 mg / mL) to an LB agar plate containing ampicillin (AMP) (100 μg / mL), spread evenly, and incubate at 37°C for 30 min. Then, spread 150 μL of the bacterial culture onto the plate. Invert the petri dishes and incubate them in a constant temperature incubator at 37℃ for 12-16 hours. Pick white colonies and inoculate them into LB medium containing 150 μg / mL AMP. Shake vigorously (230 rpm) for 12-16 hours before identification.

[0054] 6) Construction of pGEX-6P-1-TPx-2 expression vector and identification of positive clones

[0055] Aseptically add 1 μL of pGEX-6P-1-TPx-2 plasmid to 100 μL of E. coli BL21(DE3) competent cells, gently and repeatedly pipette to mix, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, then immediately incubate on ice for 2 min to cool, being careful not to shake. Transfer the bacterial culture to 500 μL of LB medium preheated to 37°C, and gently shake at 150 rpm at 37°C for 45 min to restore bacterial resistance. Spread 150 μL of the bacterial culture onto a plate containing ampicillin (AMP). + The culture was prepared on LB agar plates containing 100 μg / mL AMP. The plates were inverted and incubated at 37°C for 12–16 hours. Single colonies were then inoculated into LB medium containing 150 μg / mL AMP and shaken vigorously (230 rpm) for 12–16 hours before identification. Simultaneously, the pGEX-6P-1 empty vector without any exogenous gene was transformed as a negative control.

[0056] Take 1 μL of bacterial culture for PCR, and the reaction volume is 25 μL, as follows:

[0057]

[0058] PCR reaction conditions: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 90 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min.

[0059] Clones that amplified positive were sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0060] The pGEX-6P-1-TPx-2 recombinant plasmid constructed in this invention is shown in the following diagram. Figure 6 As shown.

[0061] 7) Prokaryotic expression of pGEX-6P-1-TPx-2 recombinant protein

[0062] The above-mentioned positive pGEX-6P-1-TPx-2 / BL21(DE3) bacterial suspension was added to LB medium containing AMP at a ratio of 1:100 and incubated at 37°C and 200 rpm until OD. 600 The concentration was approximately 0.6. 0.8 mmol / L IPTG was added and the mixture was induced at 37℃ for 4 h. A large amount of the induced bacterial culture was collected, centrifuged at 8000 rpm for 15 min at 4℃, the supernatant was discarded, the bacterial pellet was resuspended in PBS, and the mixture was pressure-disrupted three times. Then, the mixture was centrifuged at 12000 rpm for 10 min at 4℃, the pellet was discarded, the supernatant was collected, and microfiltration yielded the crude recombinant protein. SDS-PAGE electrophoresis was performed to detect the protein. The results are shown below. Figure 2 As shown.

[0063] 8) Purification of recombinant proteins

[0064] Purification of recombinant protein using Glutathione Sepharose 4B: Take 1.33 mL of Glutathione Sepharose 4B collagen solution, centrifuge at 1800 rpm for 5 min to remove the supernatant, add 10 mL of 1×PBS, gently shake until the Glutathione Sepharose 4B gel is suspended in the solution, centrifuge at 1800 rpm for 5 min to remove the supernatant; resuspend in 1 mL of 1×PBS to obtain 50% Glutathione Sepharose 4B gel. Induce 200 mL of bacterial cells at 4°C and 12000 rpm for 1 min to collect the cells. Wash the induced cells 2-3 times with PBS, resuspend the cells in 1×PBS (generally 1 / 10 volume), and sonicate until clear. Centrifuge at 12000 rpm for 20 min and collect 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 on a shaker at room temperature to allow protein adsorption for 1 h. Centrifuge at 2000 rpm for 5 min and discard the supernatant. Add at least 10 times the volume of PBS and gently shake until the Glutathione Sepharose 4B gel is suspended in the solution. Centrifuge at 2000 rpm for 5 min and discard the supernatant. Repeat the above steps twice. Add 1 mL of GST elution. Add buffer and gently shake for 10 min; centrifuge at 2000 rpm for 5 min, collect the supernatant, and repeat the above steps at least twice; assess protein purity by SDS-PAGE electrophoresis and protein concentration by UV spectrophotometry; store the protein aggregator at -80℃. SDS-PAGE electrophoresis results are shown below. Figure 2 As shown.

[0065] Example 5: Identification of Immunogenicity and Natural Form of Recombinant Protein

[0066] 1) Identification of immunogenicity

[0067] The purified recombinant protein was first subjected to SDS-PAGE electrophoresis, 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, followed by TBST washing three times for 5 minutes each time. The PVDF membrane was then placed in 1:500 diluted Duncan's babesi mouse positive serum and healthy mouse serum, respectively, and incubated at room temperature for 1 hour, followed by TBST washing three times for 5 minutes each time. Secondary antibody (1:1000 diluted rabbit anti-mouse IgG) was added, and the membrane was incubated at room temperature for 1 hour, followed by TBST washing three times for 5 minutes each time, and then BCL was added for color development. Western blot results showed that the purified recombinant protein TPx-2 specifically reacted with Duncan's babesi mouse serum at 54 kDa, but did not react with healthy mouse serum (see...). Figure 3 ).

[0068] 2) Identification of the natural form of TPx-2

[0069] Whole-strain antigens of *Babesia dulcis* were subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes using wet transfer, followed by blocking with 5% skim milk dissolved in TBST overnight at 4°C. The membranes were then washed three times with TBST for 5 min each time. The PVDF membranes were incubated separately with 1:500 diluted TPx-2 mouse positive and negative sera at 37°C for 2 h, followed by three TBST washes for 5 min each time. Secondary antibody (1:1000 diluted goat anti-mouse IgG) was added, and the membranes were incubated at room temperature for 1 h, followed by three TBST washes for 5 min each time. BCL was then added for color development. Western blot results showed that *Babesia dulcis* reacted with TPx-2 positive serum, producing a specific band of approximately 29 kDa, while no band was observed with negative serum (see [link to Western blot analysis]). Figure 4 ).

[0070] Example 6: Identification of Duncan's Babesia using immunofluorescence

[0071] Prepare a blood smear by evenly spreading 1 μL of infected blood onto a clean glass slide. After drying, fix with 100% ice-cold methanol at -20℃ for 1 h, then permeate with 0.1% Triton (dissolved in PBS) for 10 min. Wash three times with PBS, then block with PBS solution containing 5% FBS (purchased from Sigma) at 37℃ for 1 h. Wash three times with PBS, 2 min each time. Add primary antibody (1:100) TPx-2 mouse positive and negative sera, incubate at 37℃ for 1 h, wash three times with PBS, 2 min each time. Add secondary antibody (1:1000 diluted 594 fluorescent goat anti-mouse IgG and Hoechst for nuclear staining), incubate at room temperature for 1 h, wash three times with PBS, 2 min each time, and then mount with an anti-fluorescence quencher. Observe the experimental results under a laser confocal microscope. The results are as follows: Figure 5 As shown. By Figure 5 As can be seen, the blue nucleus (Hoechst) of Duncan-Babesia and the red TPx-2 protein are superimposed, revealing the blue Duncan-Babesia inside the red blood cell and the red TPx-2 protein (Merge) present in Duncan-Babesia.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Duncan's babesi TPx-2 protein in the preparation of a detection kit for Duncan's babesiosis, characterized in that, The amino acid sequence of the TPx-2 protein is shown in Seq_2.

2. The application of Duncan's babesi TPx-2 protein in the preparation of Duncan's babesi antibody detection kit, characterized in that, The amino acid sequence of the TPx-2 protein is shown in Seq_2.

3. The application of Duncan's babesi TPx-2 protein in the preparation of Duncan's babesiosis-specific diagnostic antigens, characterized in that, The amino acid sequence of the TPx-2 protein is shown in Seq_2.

4. The application according to any one of claims 1 to 3, characterized in that, The method for preparing Duncan-Babesia TPx-2 protein includes: introducing the encoding gene of TPx-2 protein into Escherichia coli, constructing a recombinant expression plasmid pGEX-6P-1-TPx-2, transforming it into BL21(DE3) using the competent cell method, screening for highly resistant transformants, and inducing expression to obtain TPx-2 protein.

5. The application according to any one of claims 1 to 4, characterized in that, The nucleotide sequence of the gene encoding the Duncan-Babesia TPx-2 protein is shown in Seq_1.

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