Immunogens, polyclonal antibodies, and applications of PRA1 protein with specific histidine methylation modifications

Abstract

This invention discloses an immunogen, polyclonal antibody, and application of a PRA1 protein with specific histidine methylation modification. The immunogen comprises a polypeptide with the amino acid sequence shown in SEQ ID No. 2. Immunizing animals with this immunogen can yield a polyclonal antibody that specifically recognizes the PRA1 protein with specific histidine methylation modification, enabling rapid and simple detection of changes in specific histidine methylation of PRA1 in biological samples. This promotes research on the functional effects of specific histidine methylation on PRA1 proteins, particularly the dynamic changes in specific histidine methylation during Candida albicans infection and anti-Candida albicans processes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immunogen of PRA1 protein with specific histidine methylation modification, a polyclonal antibody that can specifically recognize PRA1 protein with specific histidine methylation modification, and its application. Background Technology

[0002] PRA1 is a zinc-binding protein expressed by the fungus *Candida albicans*, which absorbs zinc ions from the environment for its growth. *Candida albicans* is a common pathogenic fungus in clinical practice, causing both local and systemic life-threatening infections. PRA1 is primarily located on the surface of *Candida albicans*, but it can also be secreted extracellularly. PRA1 expression is pH-regulated; it is typically expressed under neutral pH conditions and generally not expressed below pH 6.0.

[0003] PRA1 is a cell surface protein involved in host-parasite interactions during Candida infection. Along with MP65, it represents a major component of the biofilm matrix. It isolates zinc from host tissues and mediates leukocyte adhesion and migration. As a surface protein, it binds to two human complement regulators, CFH and CFHR1, as well as plasminogen activator PLG, mediating complement evasion and extracellular matrix interactions and / or degradation. As a released protein, it enhances complement control near yeast, creating an additional protective layer that controls host complement attack and helps the fungus escape host surveillance. It binds to host liquid phase C3 and blocks the cleavage of C3 to C3a and C3b, resulting in inhibited complement activation. It also mediates human complement control and complement evasion by binding to another human complement inhibitor, C4BPA, and by binding to host integrins α-M / β-2. It reduces complement-mediated adhesion and uptake of Candida albicans by human macrophages.

[0004] Methylation, as a biochemical strategy to enhance the characteristics of modified residues, is commonly used by cells for recognition and regulation. When discussing protein methylation, lysine or arginine methylation is well-known in the field; however, histidine in proteins can also be methylated. We discovered that histidine at position 292 of the Candida albicans-derived PRA1 (Swiss-Prot: P87202) protein is methylated, and this modification can be mediated by the histidine N1 methyltransferase METTL9. Since METTL9 modifies substrates with a specific motif (XHXH), where the second H is the methylation site, and PRA1 possesses this motif, METTL9 can transfer the methyl group from the methyl donor SAM to the first N atom of histidine at position 292 of the Candida albicans-derived PRA1 protein, thereby achieving methylation of the PRA1 protein.

[0005] However, the specific functions of the PRA1 protein modified by the aforementioned specific histidine methylation have not been studied, and there are no specific antibodies targeting the specific histidine methylation sites. Currently, the only method for identifying histidine methylation on PRA1 is mass spectrometry, but mass spectrometry requires a complex platform, which is generally not available in laboratories. In addition, the entire process of mass spectrometry takes a long time to process the sample, and the results cannot be presented intuitively. Summary of the Invention

[0006] In view of this, the present invention needs to provide an immunogen of PRA1 protein with specific histidine methylation modification. This immunogen can be used to obtain a polyclonal antibody by immunizing animals. This polyclonal antibody can specifically recognize PRA1 protein with specific histidine methylation modification, thereby realizing the identification of specific histidine methylation on PRA1 protein. This can greatly simplify the entire detection process, shorten the detection time, and present the detection results intuitively, thereby promoting the development of research on the functional effects of specific histidine methylation of PRA1 protein and the dynamic changes of specific histidine methylation of PRA1 during Candida albicans infection and anti-Candida albicans processes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The present invention first provides a PRA1 protein immunogen with specific histidine methylation modification, comprising a polypeptide with an amino acid sequence as shown in SEQ ID No. 2.

[0009] In a further embodiment, the PRA1 immunogen modified with specific histidine methylation also includes a carrier protein, which is coupled to the polypeptide.

[0010] Preferably, the carrier protein is selected from hemocyanin.

[0011] The present invention further provides a polyclonal antibody that specifically recognizes PRA1 protein with specific histidine methylation modification, obtained by immunizing animals with the PRA1 protein immunogen with specific histidine methylation modification as described above;

[0012] Preferably, the animal used for immunization is a New Zealand white rabbit.

[0013] This invention further provides a method for preparing a polyclonal antibody that specifically recognizes PRA1 protein modified with specific histidine methylation, comprising the following steps:

[0014] Provides a polypeptide with the amino acid sequence shown in SEQ ID No. 2;

[0015] The polypeptide was coupled to a carrier protein to obtain an immunogen;

[0016] Animals were immunized with the aforementioned immunogen, serum was collected, and polyclonal antibodies were obtained through purification.

[0017] Preferably, the carrier protein is selected from hemocyanin.

[0018] In a further embodiment, the immunized animal is a New Zealand white rabbit, and the specific timing, dosage, and Freund's adjuvant type for each immunization of the New Zealand white rabbit are as follows:

[0019]

[0020] In a further embodiment, the purification is performed using affinity chromatography.

[0021] The present invention further provides the application of the polyclonal antibody as described above or the polyclonal antibody prepared by the method described above in recognizing PRA1 protein with specific histidine methylation modification.

[0022] The present invention further provides a detection reagent for PRA1 protein with specific histidine methylation modification, including the polyclonal antibody as described above or the polyclonal antibody prepared by the preparation method described above.

[0023] The present invention further provides a test strip for detecting PRA1 protein with specific histidine methylation modification, including the detection reagents as described above.

[0024] The present invention further provides a detection kit for PRA1 protein with specific histidine methylation modification, comprising the detection reagents as described above or the test strips as described above.

[0025] The beneficial effects of this invention are as follows:

[0026] Polyclonal antibodies can be prepared using the PRA1 protein immunogen with specific histidine methylation modification provided in this invention. These polyclonal antibodies can highly specifically recognize PRA1 proteins with specific histidine methylation modification and will not recognize unmethylated PRA1 proteins. This enables rapid and simple detection of changes in specific histidine methylation of PRA1 in biological samples, which greatly promotes the study of the functional effects of specific histidine methylation of PRA1 proteins and the dynamic changes in specific histidine methylation of PRA1 during Candida albicans infection and anti-Candida albicans processes. Attached Figure Description

[0027] Figure 1 This is the detection result of the purified polyclonal antibody in Example 4 for the recognition of PRA1 protein modified by histidine methylation at position 292. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] The first aspect of the present invention provides a PRA1 protein immunogen with specific histidine methylation modification, which is used to prepare a polyclonal antibody that can specifically recognize the PRA1 protein with specific histidine methylation modification.

[0031] Specifically, the PRA1 protein immunogen with specific histidine methylation modification includes a polypeptide with an amino acid sequence as shown in SEQ ID No. 2.

[0032] The PRA1 protein mentioned in this article refers to the Candida albicans-derived PRA1 protein (Swiss-Prot: P87202), and the specific histidine mentioned refers to histidine position 292 in the Candida albicans-derived PRA1 protein. The unmethylated peptide of the Candida albicans-derived PRA1 protein containing this histidine position 292 is named polypeptide 1, and its amino acid sequence is: ANSHCHT H ADGEVHC (SEQ ID No. 1), where the underlined part represents histidine at position 292 of the PRA1 protein derived from Candida albicans, and C is added for coupling with the carrier protein. Methylation modification of the N at the π position of a specific histidine in peptide 1 yields the methylated peptide ANSHCHTH(methyl)ADGEVHC, named peptide 2, with the amino acid sequence ANSHCHTXADGEVHC (SEQ ID No. 2), where X in the sequence represents N1-histidine methylation, i.e., methylation modification of the N at the π position of histidine.

[0033] In this embodiment, the methylation modification of histidine at position 292 of the PRA1 protein is all obtained by the histidine N1 methyltransferase METTL9. For details, please refer to the applicant's research paper METTL9 mediated N1-histidine methylation of zinc transporters is required for tumor growth. M Lvet. Protein & Cell. 2021.

[0034] Polyclonal antibodies obtained by immunizing animals with a PRA1 protein immunogen containing a specific histidine methylation-modified polypeptide 2 as shown in SEQ ID No. 2 can specifically recognize PRA1 proteins with specific histidine methylation modification but not those without methylation modification, thus enabling rapid identification of PRA1 proteins with specific histidine methylation modification.

[0035] Furthermore, the PRA1 protein with specific histidine methylation modification described herein also includes a carrier protein, which is coupled to polypeptide 2, thereby facilitating the stimulation of helper T cells and further inducing a B cell immune response. Specifically, the carrier protein can be selected from carrier proteins conventionally used in the art; preferably, the carrier protein is selected from hemocyanin (KLH) or bovine serum albumin (BSA); more preferably, the carrier protein is selected from hemocyanin.

[0036] A second aspect of the present invention provides a polyclonal antibody that specifically recognizes PRA1 protein with specific histidine methylation modification, obtained by immunizing animals with a PRA1 immunogen with specific histidine methylation modification as described above. The immunized animal can be a rabbit or a mouse, preferably a rabbit. In one or more embodiments of the present invention, the immunized animal is a New Zealand white rabbit.

[0037] The third aspect of the present invention provides a method for preparing a polyclonal antibody that specifically recognizes a PRA1 protein modified with a specific histidine methylation, the method comprising steps S101-S105.

[0038] Step S101: Provide a polypeptide with specific histidine methylation modification of PRA1 protein.

[0039] Specifically, polypeptide 2 is artificially synthesized to obtain a polypeptide with specific histidine methylation modification, whose amino acid sequence is ANSHCHT. X ADGEVHC (SEQ ID No. 2).

[0040] Step S102: Couple the peptide modified with specific histidine methylation to the carrier protein to obtain the immunogen.

[0041] Specifically, the polypeptide 2 in step S101 is coupled to the carrier protein using protein conjugation technology. More specifically, the carrier protein is first activated, and then the activated carrier protein is mixed with polypeptide 2 and reacted, followed by overnight dialysis to obtain the immunogen. Preferably, the selection of the carrier protein is similar to that described in the first aspect of the present invention, and will not be elaborated here.

[0042] Step S103: Immunize the animals with the immunogen.

[0043] Specifically, in one embodiment, the animals used for immunization are New Zealand white rabbits. The New Zealand white rabbits are immunized multiple times with the immunogen obtained in step S102. The selected New Zealand white rabbits are young, healthy animals weighing around 2.5 kg, with glossy fur and free movement. Selected animals are pre-fed for about two weeks to eliminate unqualified animals and ensure the smooth progress of subsequent experiments. In one embodiment, the immunogen is injected into the animal after being mixed with an adjuvant, thereby enhancing the body's immune response to the antigen or changing the type of immune response. There are many types of adjuvants. In one embodiment of the present invention, Freund's complete adjuvant and Freund's incomplete adjuvant, which are currently the most commonly used in animal experiments, are preferred. In one embodiment, the volume ratio of adjuvant to immunogen is 1:1. The specific time, dosage, and type of Freund's adjuvant for each immunization are as follows:

[0044]

[0045] The use of multi-site subcutaneous immunization means that the immunization sites include, but are not limited to, the subcutaneous tissue on the back, abdomen, armpits, or limbs.

[0046] Step S104: Collect serum from immunized animals to determine antibody titer.

[0047] Specifically, after immunization, blood is first collected for ELISA testing to determine whether the serum contains the corresponding antibodies and whether the antibody titer meets the standard. The serum collection can be carried out using conventional methods in this field.

[0048] Step S105: After the titer meets the standard, blood is collected for antibody purification.

[0049] Specifically, in one embodiment, the antibody purification in this invention is preferably affinity chromatography purification. First, a peptide containing a specific histidine methylation modification (peptide 2) is used for preliminary purification. Then, the same peptide containing the same unmethylated modification (peptide 1) is used for secondary purification to remove the antibody that recognizes the peptide containing the unmethylated modification. The purified antibody is then identified by ELISA detection.

[0050] In some specific embodiments, the purification steps are as follows: pretreating the affinity chromatography column, then adding serum containing polyclonal antibodies into the pretreated affinity chromatography column for loading, and eluting with elution buffer.

[0051] In some specific implementations, ELISA detection includes the following steps:

[0052] ① Coat an enzyme-linked plate with a polypeptide modified by specific histidine methylation, and wash the plate;

[0053] ② Block the coated ELISA plate, incubate, and discard the blocking solution;

[0054] ③ Add the purified polyclonal antibody, incubate, discard the blocking solution, and wash the plate;

[0055] ④ Add enzyme-labeled secondary antibody, incubate, discard blocking solution, and wash plate;

[0056] ⑤ Add substrate solution to develop color, stop the reaction, and then read the plate.

[0057] The polyclonal antibody sample to be tested is diluted at a certain ratio. In one or more embodiments of the present invention, the dilution ratio is from 1:250 to 1:1024000.

[0058] Experimental results show that the polyclonal antibody prepared by the method described in this invention has an antibody titer of over 1 when diluted at a ratio of 1:250, and an ELISA detection value of around 2.4, which is higher than the positive standard of 1.

[0059] The fourth aspect of this invention provides the application of the polyclonal antibody described in the second aspect of this invention or the polyclonal antibody prepared by the preparation method described in the third aspect of this invention in recognizing PRA1 protein with specific histidine methylation modifications. Specifically, the polyclonal antibody obtained in this invention can be applied to Western blot, or immunofluorescence, immunohistochemistry, flow cytometry, ELISA kits, etc., to detect PRA1 protein with specific histidine methylation modifications, thereby achieving rapid and simple detection of changes in specific histidine methylation of PRA1 in biological samples. This plays a significant role in promoting the study of the functional impact of specific histidine methylated PRA1 protein and the dynamic changes in specific histidine methylation of PRA1 during specific life activities or Candida albicans infection and colonization.

[0060] The fifth aspect of the present invention provides a detection reagent for PRA1 protein with specific histidine methylation modification, including polyclonal antibodies as described in the second aspect of the present invention or polyclonal antibodies prepared by the preparation method described in the third aspect of the present invention.

[0061] The sixth aspect of the present invention provides a test strip for detecting PRA1 protein with specific histidine methylation modification, comprising the detection reagents as described in the fifth aspect of the present invention.

[0062] The seventh aspect of the present invention provides a detection kit for PRA1 protein with specific histidine methylation modification, comprising the detection reagents as described in the fifth aspect of the present invention or the test strips as described in the sixth aspect of the present invention.

[0063] It is understood that specific test kits may also include reagents commonly used in the field, such as diluents, conversion solutions, buffers, washing solutions, elution solutions, and purification solutions, which will not be elaborated here.

[0064] The present invention will be described below through specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present invention in any way. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used can be obtained commercially.

[0065] Example 1: Preparation of polypeptides

[0066] The following peptides were synthesized by Hangzhou Huaan Biotechnology Co., Ltd.:

[0067] The polypeptide with the amino acid sequence shown in SEQ ID No. 1 is named polypeptide 1;

[0068] The polypeptide with the amino acid sequence shown in SEQ ID No. 2 is named polypeptide 2.

[0069] Example 2: Preparation of Candida albicans-derived PRA1 protein immunogen modified by histidine methylation at position 292

[0070] By using protein conjugation technology, peptide 2 from Example 1 was conjugated with hemocyanin (KLH) to obtain the Candida albicans-derived PRA1 immunogen modified with histidine methylation at position 292. The specific steps are as follows:

[0071] 1. Peptide 2 conjugated with KLH

[0072] (1) Dissolve 20 mg KLH in 2 mL of 5 mM EDTA aqueous solution;

[0073] (2) Weigh 8 mg of Sulfo-SMCC and dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well;

[0074] (3) Add the Sulfo-SMCC solution dropwise to the KLH solution while gently shaking, and let it stand at room temperature for 1 hour;

[0075] (4) Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clamp, and dialyze it in 2L of 1×PBS at 4°C with magnetic stirring for 1h.

[0076] (5) Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed KLH into a 15mL imported centrifuge tube, and label the tube with the reagent name, time and concentration, and store in a refrigerator at 4℃;

[0077] (6) Weigh 4 mg of peptide 2, dissolve it in 50 μL LDMSO, add 200 μL of 1×PBS, mix quickly, and then immediately add KLH according to the ratio of peptide:KLH = 1 mg: 680 μg. Incubate overnight at 4°C or react at room temperature for 2 h.

[0078] (7) Place the cross-linked KLH-peptide complex into a dialysis bag, clamp it with a dialysis clamp, and dialyze overnight in 4L of 1×PBS at 4°C with magnetic stirring.

[0079] (8) Take the dialyzed KLH-peptide into a clean 1.5mL centrifuge tube, dispense it according to the immunization dose, and store it in a -20℃ refrigerator.

[0080] 2. Peptide 2 conjugated with carrier protein BSA (peptide 2 is conjugated with BSA for subsequent antibody purification and detection).

[0081] (1) Dissolve 20 mg BSA in 2 mL of 5 mM EDTA aqueous solution;

[0082] (2) Weigh 5 mg of Sulfo-SMCC and dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well;

[0083] (3) Add the Sulfo-SMCC solution dropwise to the BSA solution while gently shaking, and let it stand at room temperature for 1 hour;

[0084] (4) Place the activated BSA solution into a dialysis bag, clamp it with a dialysis clamp, and dialyze it in 2L of 1×PBS at 4°C with magnetic stirring for 1h.

[0085] (5) Replace with fresh 1×PBS and dialyze for 2 hours, repeating once. Place the activated dialyzed BSA into a 15mL imported centrifuge tube, label the tube with the reagent name, time, and concentration, and store at 4℃.

[0086] (6) Weigh 1 mg of polypeptide 2, dissolve it in 50 μL of DMSO, add 150 μL of 1×PBS, mix quickly, then add 100 μL of the above activated BSA solution, and react overnight at 4°C or at room temperature for 2 h.

[0087] (7) Place the cross-linked BSA-Peptide complex into a 1.5 mL centrifuge tube, add 1×PBS to 1 mL, label the item number, concentration and date, and hand it over to the detection group.

[0088] Example 3: Preparation of a polyclonal antibody that specifically recognizes the Candida albicans PRA1 protein modified by histidine methylation at position 292.

[0089] Animal immunization:

[0090] The Candida albicans PRA1 immunogen modified with histidine methylation at position 292 in Example 2 was taken out of the -20°C freezer and thawed at room temperature, avoiding repeated freeze-thaw cycles. The syringe was then labeled. According to the configuration in Table 1, the adjuvant was drawn at a volume ratio of 1:1 between the adjuvant and the Candida albicans PRA1 immunogen modified with histidine methylation at position 292. The adjuvant was fully emulsified. The emulsification standard was that the emulsified immunogen did not disperse when dropped into 37°C water.

[0091] Before the initial immunization, all New Zealand white rabbits were numbered. The initial antigen concentration was 1 mg / mL, with each rabbit receiving 0.5 mL. For the second to fourth immunizations, the antigen concentration was halved. Immunization was performed according to the specific time and dosage described in Table 1. Multiple subcutaneous injections were used for immunization, with 0.2 mL injected at each point. On the 7th day after the third immunization, a small serum sample was collected from the middle ear artery for testing. If the test was satisfactory, a booster immunization was performed 7 days later. Whole blood could be collected 7 days after the booster immunization.

[0092] Table 1. Number, timing, and dosage of immunizations

[0093]

[0094] Antibody titer testing:

[0095] After three immunizations, an ELISA test is performed to determine whether the serum contains the corresponding antibodies and whether the antibody titer meets the target. The specific steps are as follows:

[0096] (1) Plate coating: Dilute the known antigen peptides (peptide 1 and peptide 2, respectively) to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μl to each reaction well of the polystyrene plate, incubate overnight at 4°C, and the next day, discard the solution in the well and wash once with 1xPBST washing buffer at 180 μl per well.

[0097] (2) Blocking: Add 150 μL of 1% BSA (prepared with PBST) to each well for blocking, incubate at 37°C for 1 hour, and then discard the blocking solution;

[0098] (3) Sample addition: Dilute the sample to be tested according to a certain ratio (see Table 2 for dilution ratio), take 50 μL of the diluted antibody into the above-mentioned blocked reaction wells, and set up the negative control wells (PBS) at the same time. Incubate at 37°C for 1 hour, then discard the blocking solution and wash 3 times with 1xPBST washing buffer at 150 μL per well.

[0099] (4) Add enzyme-labeled antibody: Add 50 μL of freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the enzyme-labeled plate and incubate at 37°C for 45 min. Then discard the blocking solution and wash 3 times with 150 μL of 1xPBST buffer per well.

[0100] (5) Add substrate solution for color development and plate reading: Add 50 μl of the temporarily prepared TMB substrate solution to each reaction well, incubate at 37℃ for 5 min, add 50 μL of 1M sulfuric acid to each reaction well to terminate the reaction, place the microplate in a preheated microplate reader (450 nm) for reading, and the results are shown in Table 2.

[0101] Table 2. Results of serum antibody titer detection

[0102]

[0103] Note: In Table 2, 1# and 2# are parallel tests of rabbits numbered RB2666, and 3# and 4# are parallel tests of rabbits numbered RB2665.

[0104] Antibody purification:

[0105] Once the antibody titer meets the standard, immunization is performed 7 days later. Seven days after immunization, whole blood can be collected for antibody purification. The specific steps are as follows:

[0106] (1) The affinity chromatography column was thoroughly washed with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0107] (2) Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0108] (3) Load the filtered serum sample at a flow rate of 40 mL / h and repeat once;

[0109] (4) Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance (T setting) reading to 100.

[0110] (5) Adjust the absorbance of the protein detector (1A range) to 0. At this time, turn on the HD-A computer acquisition device on the computer desktop and adjust the full screen range to 5. Use glycine solution (pH 2.7, 0.2M) to elute the antibody at a rate of 40mL / h. At this time, press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0111] (6) During antibody collection, the pH value of the antibody was adjusted to about 7 with 1M sodium bicarbonate in a timely manner, and the highest peak value of the elution peak was recorded.

[0112] (7) After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tube connected to the collector with pure water.

[0113] (8) Wash the affinity chromatography column with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store it at 4°C.

[0114] (9) The purified antibodies were sent for testing according to different requirements. When purifying methylated antibodies, the serum was first passed through a methylation column, and the eluted antibodies were then passed through a non-methylation column to obtain specific methylated antibodies. The purification of other modified antibodies was similar, and ELISA detection was performed. The results are shown in Table 3.

[0115] Table 3 Results of purified antibody titer assay

[0116]

[0117] Note: In Table 3, 1# and 2# are parallel tests of rabbits numbered RB2666, and 3# and 4# are parallel tests of rabbits numbered RB2665.

[0118] Finished product antibody concentration testing:

[0119] (1) After the amount of purified antibody is sufficient for delivery and the titer of the semi-finished product is qualified, mix all the antibodies and concentrate them using an ultrafiltration concentrator to achieve a certain concentration and volume.

[0120] (2) Place the concentrated antibody in 1L 0.01M PBS (pH7.4) and dialyze at room temperature. Change the medium every 3 hours for a total of 3 times (for overnight dialyze, place in a refrigerator at 2-8℃).

[0121] (4) Take the dialyzed antibody into a clean centrifuge tube, filter the antibody in a clean bench using a 0.22um disposable low adsorption filter, take a small sample for testing, and take another 5ul to test the concentration.

[0122] (5) Concentration was detected using the Protein A280 on the denovix DS-11 micro-volume spectrophotometer.

[0123] After testing, the antibody concentrations of 0.5 mL (RB2666) and 0.15 mL (RB2665) were 0.33 mg / mL (RB2666) and 3.24 mg / mL (RB2665), respectively.

[0124] Example 4: Identification of PRA1 protein derived from Candida albicans with histidine methylation at position 292

[0125] The PRA1 protein derived from Candida albicans was purified in vitro using the GST tag to obtain an unmethylated GST-PRA1 fusion protein;

[0126] Using the histidine N1 methyltransferase METTL9, the GST-PRA1 fusion protein with histidine methylation at position 292 was obtained in vitro, namely the protein GST-PRA1 H292me with methylation at position H292.

[0127] The purified polyclonal antibody prepared in Example 3 was used for Western blotting detection, and the results are as follows: Figure 1 As shown, GST and GST-PRA1 represent the levels of GST and PRA1, respectively; h represents human; hMETTL9 and hMETTL9 mutant represent the protein levels of histidine N1 methyltransferase METTL9 with and without enzyme activity, respectively. H292me represents the methylation level of histidine at position 292 of PRA1 (detected using antibody purified from rabbit RB2665). Figure 1 The detection results show that the polyclonal antibody prepared in Example 3 can recognize PRA1 with histidine methylation at position 292 but cannot recognize PRA1 without methylation.

[0128] Therefore, the immunogen provided in this invention can be used to prepare a highly specific polyclonal antibody that recognizes the Candida albicans PRA1 protein with histidine methylation at position 292. This polyclonal antibody does not recognize unmethylated PRA1 protein and can be widely used in immunohistochemistry, Western blot, immunofluorescence, flow cytometry, and in vitro immunoassays. This enables rapid and simple detection of changes in specific histidine methylation of PRA1 in biological samples, promoting research on the functional effects of specific histidine methylation on PRA1 proteins, particularly the dynamic changes in specific histidine methylation during Candida albicans infection and anti-Candida albicans processes.

[0129] Furthermore, the polyclonal antibody preparation method of this invention is simple to operate and can be used for the large-scale preparation of antibodies that specifically recognize the Candida albicans PRA1 protein modified by histidine methylation at position 292.

[0130] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0131] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A PRA1 protein immunogen with specific histidine methylation modification, characterized in that, The PRA1 protein immunogen is a polypeptide with the amino acid sequence shown in SEQ ID No.

2.

2. The PRA1 protein immunogen with specific histidine methylation modification as described in claim 1, characterized in that, The PRA1 immunogen with specific histidine methylation modification also includes a carrier protein, which is coupled to the polypeptide.

3. The PRA1 protein immunogen with specific histidine methylation modification as described in claim 2, characterized in that, The carrier protein is selected from hemocyanin.

4. A method for preparing a polyclonal antibody that specifically recognizes PRA1 protein modified with a specific histidine methylation, characterized in that, Includes the following steps: Provides a polypeptide with the amino acid sequence shown in SEQ ID No. 2; The polypeptide was coupled to a carrier protein to obtain an immunogen; Animals were immunized with the aforementioned immunogen, serum was collected, and polyclonal antibodies were obtained through purification. The immunized animals are New Zealand white rabbits, and the specific timing, dosage, and Freund's adjuvant type for each immunization of the New Zealand white rabbits are as follows:

5. The preparation method according to claim 4, characterized in that, The carrier protein is selected from hemocyanin.

6. The preparation method according to claim 4, characterized in that, The purification was performed using affinity chromatography.

7. A polyclonal antibody that specifically recognizes PRA1 protein modified with specific histidine methylation, characterized in that, It is prepared by the preparation method described in any one of claims 4-6.

8. The use of the polyclonal antibody as described in claim 7 in the preparation of a product that recognizes a PRA1 protein with specific histidine methylation modification, wherein the product is a detection reagent, a test strip, or a detection kit.

9. A reagent for detecting PRA1 protein with specific histidine methylation modification, characterized in that, Including the polyclonal antibody as described in claim 7.

10. A test strip for detecting PRA1 protein with specific histidine methylation modification, characterized in that, Includes the detection reagent as described in claim 9.

11. A detection kit for PRA1 protein with specific histidine methylation modification, characterized in that, Includes the test reagent as described in claim 9 or the test strip as described in claim 10.

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