CN115991752A - PRA1 protein immunogen modified by specific histidine methylation, polyclonal antibody and application - Google Patents
PRA1 protein immunogen modified by specific histidine methylation, polyclonal antibody and application Download PDFInfo
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Abstract
The invention discloses a PRA1 protein immunogen with specific histidine methylation modification, a polyclonal antibody and application, wherein the PRA1 protein immunogen with specific histidine methylation modification comprises a polypeptide with an amino acid sequence shown as SEQ ID No. 2; the polyclonal antibody specifically recognizing the PRA1 protein with the specific histidine methylation modification can be obtained by immunizing animals with the PRA1 protein immunogen with the specific histidine methylation modification, so that the change of the PRA1 specific histidine methylation in a biological sample can be rapidly and simply detected, and the development of dynamic change of the PRA1 specific histidine methylation in the processes of candida albicans infection and candida albicans resistance can be promoted and studied for the functional influence of the PRA1 protein with the specific histidine methylation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PRA1 protein immunogen with specific histidine methylation modification, a polyclonal antibody capable of specifically recognizing the PRA1 protein with specific histidine methylation modification and application thereof.
Background
The PRA1 protein belongs to zinc ion binding protein, and the fungus belongs to candida albicans and expresses zinc ions in the PRA1 protein uptake environment for self growth. Candida albicans is a clinically common pathogenic fungus that can cause localized and systemic life threatening infections. PRA1 is mainly located on the surface of candida albicans, while it can also be secreted extracellularly. PRA1 expression is regulated by pH, it is typically expressed at neutral pH, and is typically not expressed when pH is below 6.0.
PRA1 is a cell surface protein involved in host-parasite interactions during candida infection. Together with MP65, represent the major component of the biofilm matrix. Isolating zinc from host tissue and mediating leukocyte adhesion and migration. As surface proteins, binding to two human complement modulators CFH and CFHR1 and plasminogen PLG, mediates complement evasion and extracellular matrix interactions and/or degradation. As a released protein, complement control in the vicinity of the yeast is enhanced, thereby creating an additional protective layer that controls host complement attack and aids in the monitoring of fungal escape from the host. Binding to host liquid phase C3 and blocking cleavage of C3 to C3a and C3b results in inhibition of complement activation. Human complement control and complement escape are also mediated by binding to another human complement inhibitor, C4BPA, and by binding to host integrin α -M/β -2. Reduces complement-mediated adhesion, and uptake of candida albicans by human macrophages.
Methylation, a biochemical strategy that increases the characteristics of modified residues, is commonly used by cells for recognition and regulation. Referring to protein methylation, well known in the art are lysine or arginine methylation; indeed, protein histidine can also be methylated. We found that histidine 292 on the protein of Candida albicans source PRA1 (Swiss-Prot: P87202) was identified as having methylation modification and can be mediated by the histidine N1 methylation transferase METTL9, the second H being the site methylated by METTL9 due to the preference of METTL9 modification substrate for a motif XH H; in the PRA1, the motif is that METTL9 can transfer the methyl group on the methyl donor SAM to the first N atom of 292 histidine on candida albicans PRA1 through reaction, so as to achieve the purpose of methylation modification of PRA1 protein.
However, the specific function of PRA1 proteins with specific histidine methylation modifications as described above has not been studied nor has specific antibodies directed against specific histidine methylation modification sites. However, the current means for identifying histidine methylation on PRA1 can only be realized by mass spectrometry, but the mass spectrometry technology requires a complex platform, and a general laboratory does not have such conditions; in addition, the whole flow of the mass spectrum method has long processing time on the sample, and the obtained result cannot be intuitively presented.
Disclosure of Invention
In view of the foregoing, it is desirable to provide a PRA1 protein immunogen modified by specific histidine methylation, which can obtain a polyclonal antibody by immunizing animals, and the polyclonal antibody can specifically recognize the PRA1 protein modified by specific histidine methylation, thereby realizing the identification of the specific histidine methylation on the PRA1 protein, greatly simplifying the whole detection process, shortening the detection time, and intuitively presenting the detection result, so as to promote the research on the functional influence of the PRA1 protein by specific histidine methylation and the development of the dynamic change of the PRA1 specific histidine methylation in the candida albicans infection and candida albicans resisting processes.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention firstly provides a PRA1 protein immunogen modified by specific histidine methylation, which comprises a polypeptide with an amino acid sequence shown as SEQ ID No. 2.
In a further aspect, the specific histidine methylation modified PRA1 immunogen further comprises a carrier protein, said carrier protein being coupled to said polypeptide;
preferably, the carrier protein is selected from hemocyanin.
The invention further provides a polyclonal antibody specifically recognizing the PRA1 protein with specific histidine methylation modification, which is obtained by immunizing animals with the PRA1 protein immunogen with specific histidine methylation modification;
preferably, the animal used for immunization is New Zealand white rabbit.
The invention further provides a preparation method of the polyclonal antibody for specifically recognizing the PRA1 protein with specific histidine methylation modification, which comprises the following steps:
providing a polypeptide with an amino acid sequence shown as SEQ ID No. 2;
coupling the polypeptide with a carrier protein to obtain an immunogen;
immunizing an animal by adopting the immunogen, collecting serum, and purifying to obtain a polyclonal antibody;
preferably, the carrier protein is selected from hemocyanin.
Further, the immunized animal is a New Zealand white rabbit, and the time, the dosage and the Freund adjuvant types of each immunization of the New Zealand white rabbit are specifically as follows:
further, the purification is performed by affinity chromatography.
The invention further provides the use of a polyclonal antibody as described above or a polyclonal antibody prepared by the preparation method, for identifying a specific histidine methylation-modified PRA1 protein.
The invention further provides a detection reagent of PRA1 protein with specific histidine methylation modification, which comprises the polyclonal antibody or the polyclonal antibody prepared by the preparation method.
The invention further provides a detection test paper for PRA1 protein with specific histidine methylation modification, which comprises the detection reagent.
The invention further provides a detection kit of PRA1 protein with specific histidine methylation modification, which comprises the detection reagent or the detection test paper.
The beneficial effects of the invention are as follows:
the polyclonal antibody can be prepared by the PRA1 protein immunogen with specific histidine methylation modification, and can highly specifically identify the PRA1 protein with specific histidine methylation modification and can not identify the PRA1 protein with unmethylation modification, so that the rapid and simple detection of the change of PRA1 specific histidine methylation in a biological sample is realized, and the monoclonal antibody plays a great pushing role in researching the functional influence of the PRA1 protein with specific histidine methylation and the dynamic change of PRA1 specific histidine methylation in the candida albicans resisting process.
Drawings
FIG. 1 shows the results of the detection of PRA1 protein recognition modified by purified polyclonal antibodies with histidine methylation at position 292 in example 4.
Detailed Description
The following detailed description of embodiments of the invention is exemplary and is provided merely to illustrate the invention and is not to be construed as limiting the invention.
Unless defined otherwise, 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 belongs. 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.
In a first aspect, the present invention provides a specific histidine-methylation-modified PRA1 protein immunogen for use in the preparation of polyclonal antibodies specifically recognizing a specific histidine-methylation-modified PRA1 protein.
Specifically, the PRA1 protein immunogen modified by specific histidine methylation comprises a polypeptide with an amino acid sequence shown as SEQ ID No. 2.
The PRA1 protein as described herein refers to the Candida albicans PRA1 protein (Swiss-Prot: P87202), and the specific histidine refers to histidine 292 in the Candida albicans PRA1 protein. The unmethylated peptide fragment of candida albicans source PRA1 protein containing histidine 292 is named as polypeptide 1, and the amino acid sequence is as follows: ANSSCHT (ANCHCHCHCHT)HADGEVHC (SEQ ID No. 1), wherein the underline is the 292 rd histidine in the Candida albicans source PRA1 protein, and C is conventionally added for coupling with carrier protein; methylation modification is carried out on N of a specific histidine pi position in the polypeptide 1, namely a methylation modified peptide segment ANSHCHTH (methyl) ADGEVHC is obtained, the peptide segment is named as a polypeptide 2, and the amino acid sequence is as follows: ANSHCHTXADGEVHC (SEQ ID No. 2), wherein X in the sequence represents N1-histidine methylation, an N-methylation modification of histidine pi.
In this example, the 292 nd histidine methylation modification of PRA1 protein is mediated by histidine N1-methyltransferase METTL9, and the specific method can be seen in the applicant's research paper METTL 9-mediated N1-histidine methylation of zinc transporters is required for tumor growth.M Lv et al protein & cell.2021.
The polyclonal antibody obtained after the animal is immunized by the PRA1 protein immunogen comprising the specific histidine methylation modification of the polypeptide 2 with the amino acid sequence shown in SEQ ID No.2 can specifically identify the PRA1 protein with the specific histidine methylation modification and not identify the PRA1 protein with the unmethylation modification, and can realize the rapid identification of the PRA1 protein with the specific histidine methylation modification.
Further, specific histidine methylation-modified PRA1 proteins herein also include carrier proteins coupled to polypeptide 2, thereby facilitating stimulation of helper T cells, further inducing B cell immune responses. In particular, the carrier protein may be selected from carrier proteins conventionally employed in the art, preferably from hemocyanin (KLH) or Bovine Serum Albumin (BSA), more preferably from hemocyanin.
In a second aspect the invention provides a polyclonal antibody specifically recognizing a specific histidine-methylation-modified PRA1 protein, obtained by immunizing an animal with a specific histidine-methylation-modified PRA1 immunogen as described above, wherein the animal used for immunization may be a rabbit or a mouse, preferably the animal is a rabbit, and in one or more embodiments of the invention the immunized animal used is a new zealand white rabbit.
In a third aspect, the present invention provides a method for preparing a polyclonal antibody specifically recognizing a specific histidine methylation-modified PRA1 protein, the method comprising steps S101 to S105.
Step S101, providing a polypeptide with PRA1 protein specific histidine methylation modification.
Specifically, polypeptide 2 is artificially synthesized to obtain specific histidine methylation modified polypeptide, and the amino acid sequence of the polypeptide is ANSSCHHTXADGEVHC(SEQ ID No.2)。
Step S102, coupling the polypeptide with specific histidine methylation modification with carrier protein to obtain the immunogen.
Specifically, the polypeptide 2 in the step S101 is coupled with carrier protein through a protein coupling technology, more specifically, the carrier protein is activated firstly, then the activated carrier protein and the polypeptide 2 are mixed and reacted, and the mixture is dialyzed overnight to obtain immunogen; preferably, the carrier protein is selected similarly to that described in the first aspect of the invention and will not be described in detail herein.
Step S103, immunizing animals by using the immunogen.
Specifically, in one embodiment, the animals used for immunization are New Zealand white rabbits, the New Zealand white rabbits are immunized for multiple times by using the immunogen obtained in the step S102, the selection of the New Zealand white rabbits requires healthy animals with body weight of about 2.5kg and free movement, the selected animals are kept for about 2 weeks, and thus unqualified animals are eliminated, and the smooth proceeding of the subsequent experiments is ensured; in one embodiment, the immunogen is injected into an animal by mixing the immunogen with an adjuvant to enhance the immune response of the body to the antigen or change the type of immune response, and the adjuvant is of a variety, and in one embodiment of the invention, freund's complete adjuvant and Freund's incomplete adjuvant which are most commonly used in animal tests at present are adopted, and preferably, in one embodiment, the volume ratio of the adjuvant to the immunogen is 1:1, the time, dosage and Freund's adjuvant species of each immunization are specifically:
with multipoint subcutaneous immunization, it is understood that the site of immunization includes, but is not limited to, subcutaneous in the back, subcutaneous in the abdomen, subcutaneous in the armpit, or subcutaneous in the extremities.
Step S104, serum is collected from the immunized animal to determine the antibody titer.
Specifically, after the immunization is finished, blood is firstly collected for carrying out Elisa detection to determine whether the serum contains corresponding antibodies and whether the titers of the antibodies reach the standard, wherein the serum is collected by adopting a conventional method in the field.
And step S105, after the detection titer reaches the standard, taking blood for antibody purification.
Specifically, in one embodiment, the antibody purification in the present invention is preferably affinity chromatography purification, wherein the antibody containing the peptide fragment corresponding to the specific histidine methylation modification (polypeptide 2) is first subjected to primary purification, and then the antibody containing the peptide fragment corresponding to the same peptide fragment containing the unmethylation modification (polypeptide 1) is subjected to secondary purification to remove the identified antibody; the purified antibodies were identified by Elisa assay.
In some embodiments, the purification specific steps are: pretreating the affinity chromatographic column, adding serum containing polyclonal antibody into the pretreated affinity chromatographic column for loading, and eluting with eluent.
In some embodiments, the Elisa assay specifically comprises the steps of:
(1) coating the enzyme-linked plate with specific histidine methylation modified polypeptide, and washing the plate;
(2) closing the coated enzyme-linked plate, incubating, and discarding the sealing liquid;
(3) adding the polyclonal antibody obtained by purification, incubating, discarding the sealing solution, and washing the plate;
(4) adding enzyme-labeled secondary antibodies, incubating, discarding sealing liquid, and washing the plate;
(5) adding substrate liquid for color development, and reading the plate after stopping the reaction.
The polyclonal antibody sample to be tested is diluted in a ratio, and in one or more embodiments of the invention, diluted in a ratio of 1:250 to 1:1024000.
Experimental results show that the polyclonal antibody prepared by the preparation method disclosed by the invention has the following structure that: when diluted with 250, the antibody titer was more than 1, and the Elisa detection value was about 2.4, which was higher than the positive standard of 1.
The fourth aspect of the present invention provides an application of the polyclonal antibody according to the second aspect of the present invention or the polyclonal antibody prepared by the preparation method according to the third aspect of the present invention in identifying PRA1 protein modified by specific histidine methylation, in particular, the polyclonal antibody obtained in the present invention may be applied to Westernblot, or immunofluorescence, immunohistochemistry, flow detection, ELISA kit, etc. for detecting PRA1 protein modified by specific histidine methylation, thereby realizing rapid and simple detection of changes of PRA1 specific histidine methylation in biological samples, and playing a great role in researching functional effects of PRA1 protein methylated by specific histidine and dynamic changes of PRA1 specific histidine methylation in specific vital activity or candida albicans infection field planting process.
In a fifth aspect, the invention provides a reagent for detecting a specific histidine methylation-modified PRA1 protein, comprising a polyclonal antibody according to the second aspect of the invention or a polyclonal antibody produced by the production method according to the third aspect of the invention.
In a sixth aspect, the invention provides a test strip for detecting a specific histidine methylation-modified PRA1 protein, comprising a detection reagent according to the fifth aspect of the invention.
In a seventh aspect, the invention provides a kit for detecting a specific histidine methylation-modified PRA1 protein, comprising a detection reagent according to the fifth aspect of the invention or comprising a detection strip according to the sixth aspect of the invention.
It will be appreciated that reagents such as diluents, converting solutions, buffers, washes, eluents and purification solutions, which are conventional in the art, may also be included in the particular assay kit and will not be described in detail herein.
The present invention will be illustrated by the following examples, which are given for illustrative purposes only and are not intended to limit the scope of the present invention in any way, and unless otherwise specified, the conditions or procedures not specifically described are conventional and the reagents and materials employed are commercially available.
Example 1 preparation of Polypeptides
The following polypeptides were synthesized by the assigned Hangzhou Hua An Biotechnology Co., ltd:
a polypeptide with an amino acid sequence shown as SEQ ID No.1, which is named as polypeptide 1;
the polypeptide with the amino acid sequence shown as SEQ ID No.2 is named as polypeptide 2.
EXAMPLE 2 preparation of histidine-methylation-modified Candida albicans-derived PRA1 protein immunogen at position 292
Coupling the polypeptide 2 of example 1 with hemocyanin (KLH) by protein coupling technology to obtain the 292 nd histidine methylation modified candida albicans source PRA1 immunogen, which comprises the following specific steps:
1. polypeptide 2 conjugated KLH
(1) 20mg of KLH was dissolved in 2mL of 5mM aqueous EDTA;
(2) 8mg of Sulfo-SMCC is weighed and completely dissolved in 50 mu L of DMSO, then 150 mu L of 1XPBS is added and uniformly mixed;
(3) Dropwise adding the Sulfo-SMCC solution into the KLH solution, gently shaking while adding, and standing at room temperature for 1h;
(4) Placing the activated KLH solution into a dialysis bag, clamping the dialysis bag, and dialyzing the dialysis bag for 1h in 2L of 1XPBS under magnetic stirring of a refrigerator at 4 ℃;
(5) The new 1XPBS was exchanged for dialysis for 2h and repeated once; placing activated and dialyzed KLH into a 15mL inlet centrifuge tube, marking the names, time and concentration of the reagents on the tube, and preserving the tube in a refrigerator at 4 ℃;
(6) 4mg of polypeptide 2 was weighed, dissolved in 50. Mu.L of LDMSO, and 200. Mu.L of 1XPBS was added to the solution, followed by rapid mixing according to the polypeptide: klh=1 mg: immediately adding KLH in 680 mug proportion, and reacting for 2 hours at 4 ℃ overnight or at room temperature;
(7) Placing the crosslinked KLH-peptide crosslinked compound into a dialysis bag, clamping the crosslinked KLH-peptide crosslinked compound by a dialysis clamp, and dialyzing the crosslinked KLH-peptide crosslinked compound in 4L of 1XPBS under magnetic stirring at a temperature of 4 ℃ for overnight;
(8) And taking out the dialyzed KLH-peptide into a clean 1.5mL centrifuge tube, subpackaging according to the immune dose, and preserving in a refrigerator at-20 ℃.
2. Polypeptide 2 coupled to carrier protein BSA (polypeptide 2 coupled to BSA for use in post purification antibodies and antibody detection)
(1) 20mg of BSA was dissolved in 2mL of 5mM EDTA aqueous solution;
(2) Weighing 5mg of Sulfo-SMCC, completely dissolving in 50 mu L of DMSO, adding 150 mu L of 1XPBS, and uniformly mixing;
(3) Dropwise adding the Sulfo-SMCC solution into the BSA solution, gently shaking while adding, and standing at room temperature for 1h;
(4) Placing the activated BSA solution into a dialysis bag, clamping the BSA solution by a dialysis clamp, and dialyzing the BSA solution for 1h in 2L of 1XPBS under magnetic stirring of a refrigerator at 4 ℃;
(5) The dialysis was repeated once by replacing fresh 1XPBS for 2 h. Placing activated and dialyzed BSA into a 15mL inlet centrifuge tube, marking the names, time and concentration of the reagents on the tube, and preserving the BSA in a refrigerator at 4 ℃;
(6) 1mg of polypeptide 2 is weighed, dissolved in 50 mu L of DMSO, added with 150 mu L of 1XPBS, quickly mixed, then added with 100 mu L of the activated BSA solution, and reacted for 2 hours at 4 ℃ in a refrigerator overnight or at room temperature;
(7) The crosslinked BSA-Peptide complex is added into a 1.5mL centrifuge tube, 1XPBS is added to 1mL, the project number, the concentration and the date are marked, and the BSA-Peptide complex is crossed with a detection group.
EXAMPLE 3 preparation of polyclonal antibodies specifically recognizing the 292 th histidine methylation-modified Candida albicans-derived PRA1 protein
Animal immunization:
taking the 292 nd histidine methylation modified candida albicans source PRA1 immunogen in the embodiment 2 out of a refrigerator at the temperature of minus 20 ℃, dissolving at normal temperature, avoiding repeated freeze thawing, and marking a syringe; according to the configuration in table 1, the volume ratio of adjuvant to the candida albicans source PRA1 immunogen modified by histidine methylation at position 292 is 1:1 extracting an adjuvant, fully emulsifying, wherein the emulsification standard is as follows: the emulsified immunogen is dripped into 37 ℃ water and is not dispersed as being qualified.
Before primary immunization, numbering all New Zealand white rabbits, wherein the concentration of primary antigen is 1mg/mL, the concentration of rabbits is 0.5 mL/rabbit, the amount of secondary-quaternary antigen is halved, immunization is carried out according to specific time and dosage shown in table 1, and multipoint subcutaneous injection is adopted during immunization, wherein each point is 0.2mL; and 3, detecting small sample serum of middle ear artery at 7 days after three-immunity of the rabbit, wherein the detection is qualified, and taking whole blood after 7 days after the three-immunity of the rabbit and the three-immunity of the rabbit.
TABLE 1 immunization times, times and doses
Antibody titer detection:
after three immunizations, the Elisa test is carried out on whether the serum contains the corresponding antibody and whether the titer of the antibody reaches the standard, and the specific steps are as follows:
(1) And (3) wrapping the plate: with coating buffer (coating buffer: na 2 CO 3 And NaHCO 3 Buffer) the known antigen polypeptides (polypeptide 1 and polypeptide 2, respectively) were diluted to 1. Mu.g/mL, 50. Mu.l were added to the reaction wells of each polystyrene plate, overnight at 4 ℃, the next day the in-well solution was discarded, and 1XPBST wash buffer was used to wash 1 time at 180. Mu.l per well;
(2) Closing: blocking with 150 μl of 1% BSA (prepared by PBST) per well, incubating at 37deg.C for 1 hr, and discarding blocking solution;
(3) Sample adding: diluting the sample to be detected according to a certain proportion (the dilution proportion is shown in Table 2), taking 50 mu L of diluted antibody into the closed reaction hole, setting a negative control hole (PBS), incubating for 1 hour at 37 ℃, discarding the sealing liquid, and washing with 150 mu L of 1xPBST washing buffer solution for 3 times;
(4) Adding enzyme-labeled antibody: fresh diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) was added to the wells of the enzyme-labeled plate at 50. Mu.L/well, incubated at 37℃for 45min, after which the blocking solution was discarded and washed 3 times with 150. Mu.L of 1xPBST buffer per well;
(5) Adding substrate liquid for color development, and reading a plate: 50 μl of TMB substrate solution prepared temporarily was added to each reaction well, the reaction was stopped by adding 50 μl of 1M sulfuric acid to each reaction well, and the ELISA plate was read in a preheated ELISA (450 nm), the results are shown in Table 2.
TABLE 2 serum antibody titre detection results
Note that: in Table 2, 1# and 2# are rabbit parallel test number RB2666, and 3# and 4# are rabbit parallel test number RB 2665.
Antibody purification:
after the titer of the antibody meets the standard, the antibody is added for 7 days, and whole blood can be collected after the addition of the antibody for 7 days, and the antibody is purified, specifically comprising the following steps:
(1) The affinity column was washed thoroughly with 20mL of pure water and 1XPBS (pH 7.4) in this order, at a flow rate of 70 mL/h;
(2) Taking 10mL of serum to be purified in a 50mL centrifuge tube, and carrying out suction filtration by using a microporous filter membrane with the aperture of 0.45 mu m and the diameter of 25 mm;
(3) Loading the filtered serum sample at a flow rate of 40mL/h, and repeating the steps once;
(4) Washing the column with 20mL 1XPBS (pH 7.4) at a flow rate of 70mL/h, connecting the protein detector after 10min, and adjusting the light transmittance (T grade) of the instrument to be 100 in the washing process;
(5) Adjusting the absorbance (1A grade) indication of the protein detector to 0, opening an HD-A computer collector on a computer desktop, adjusting the full screen range to 5, eluting the antibody at a speed of 40mL/h by using glycine solution (pH 2.7,0.2M), pressing a green elution record button to start eluting, and collecting the antibody when the indication of the instrument starts to rise;
(6) In the process of collecting the antibody, adjusting the pH value of the antibody to about 7 in time by using 1M sodium bicarbonate, and recording the highest peak value of the elution peak;
(7) After the antibody is collected, the pH value is regulated to about 7, the volume of the eluted antibody is recorded, and then the rubber tube connected with the collector is washed by purified water;
(8) Sequentially cleaning the affinity chromatography column with 20mL of 1XPBS and pure water at a speed of 70mL/h, adding 20% ethanol, sealing, and storing in a refrigerator at 4 ℃;
(9) The purified antibodies were submitted to different tests according to different requirements, when the methylated antibodies were purified, serum was passed through a methylation column, the eluted antibodies were passed through a non-methylation column to obtain specific methylated antibodies, other modified types of antibodies were purified similarly, and Elisa was performed, with the results shown in Table 3.
TABLE 3 purification of antibody titre detection results
Note that: in Table 3, 1# and 2# are rabbit parallel test number RB2666, and 3# and 4# are rabbit parallel test number RB 2665.
And (3) detecting the concentration of the finished antibody:
(1) After the amount of the antibody which can be delivered is purified and the detection titer of the semi-finished product is qualified, mixing all the antibodies, concentrating by using an ultrafiltration concentration tube to reach a certain concentration and volume;
(2) Placing the concentrated antibody in 1L 0.01M PBS (pH 7.4), dialyzing at room temperature, changing liquid once every 3h, and changing liquid for 3 times (overnight dialyzing in refrigerator at 2-8deg.C);
(4) Taking out the dialyzed antibody to a clean centrifuge tube, filtering the antibody in an ultra-clean workbench by using a disposable low-adsorption filter head with the diameter of 0.22um, taking a small sample for inspection, and taking 5ul of detection concentration;
(5) The concentration was measured on an ultra-micro spectrophotometer (densovix DS-11) Protein A280 application.
After detection, the volumes of the antibodies were 0.5mL (RB 2666) and 0.15mL (RB 2665), respectively, and the antibody concentrations were 0.33mg/mL (RB 2666) and 3.24mg/mL (RB 2665), respectively.
EXAMPLE 4 identification of histidine-methylation-modified Candida albicans-derived PRA1 protein at position 292
Purifying candida albicans source PRA1 protein in vitro by adopting GST tag to obtain unmethylated GST-PRA1 fusion protein;
the GST-PRA1 fusion protein with the methylation modification of the 292 rd histidine is obtained by utilizing the histidine N1 site methylation transferase METTL9 through in vitro reaction, namely, the protein GST-PRA 1H 292me with the methylation modification of the H292 site;
western blotting was performed using the purified polyclonal antibody prepared in example 3, and the detection results are shown in FIG. 1, wherein GST and GST-PRA1 represent the levels of GST and PRA1, respectively, h represents human, and hMETTL9 variant represent the levels of protein having enzyme activity and enzyme activity deficiency of histidine N1 methyltransferase METTL9, respectively. H292me represents the level of histidine methylation at position 292 of PRA1 (detected by antibodies purified from rabbit RB 2665). From the results of the assay in FIG. 1, it can be seen that the polyclonal antibody prepared in example 3 can recognize PRA1 with methylation modification of histidine 292 but cannot recognize PRA1 with non-methylation modification.
Therefore, the immunogen provided by the invention can be used for preparing the polyclonal antibody capable of highly specifically recognizing the PRA1 protein of the candida albicans source modified by the 292 th histidine methylation, and the polyclonal antibody can not recognize the PRA1 protein modified by unmethylation, can be widely used for immunohistochemistry, westernblot, immune silver light, flow detection, in-vitro immunoassay and the like, realizes rapid and simple detection of the change of the PRA1 specific histidine methylation in a biological sample, and promotes research on the functional influence of the PRA1 protein of the specific histidine methylation so as to promote the development of the PRA1 specific histidine methylation dynamic change in the candida albicans infection and candida albicans resisting process.
The preparation method of the polyclonal antibody is simple to operate, and can be used for preparing a large amount of antibodies for specifically recognizing 292 nd histidine methylation modified candida albicans source PRA1 protein.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A PRA1 protein immunogen with specific histidine methylation modification, which is characterized by comprising a polypeptide with an amino acid sequence shown as SEQ ID No. 2.
2. The specific histidine-methylation-modified PRA1 protein immunogen of claim 1, further comprising a carrier protein coupled to said polypeptide;
preferably, the carrier protein is selected from hemocyanin.
3. A polyclonal antibody specifically recognizing a specific histidine-methylation-modified PRA1 protein, characterized in that it is obtained by immunizing an animal with the specific histidine-methylation-modified PRA1 protein immunogen according to claim 1 or 2;
preferably, the animal used for immunization is New Zealand white rabbit.
4. A method for preparing a polyclonal antibody specifically recognizing a PRA1 protein with specific histidine methylation modification, comprising the steps of:
providing a polypeptide with an amino acid sequence shown as SEQ ID No. 2;
coupling the polypeptide with a carrier protein to obtain an immunogen;
immunizing an animal by adopting the immunogen, collecting serum, and purifying to obtain a polyclonal antibody;
preferably, the carrier protein is selected from hemocyanin.
5. The method according to claim 4, wherein the immunized animal is New Zealand white rabbit, and the time, dosage and Freund's adjuvant type of each immunization of the New Zealand white rabbit are specifically:
6. the method of claim 4, wherein the purifying is performed by affinity chromatography.
7. Use of the polyclonal antibody of claim 3 or the polyclonal antibody produced by the method of any one of claims 4-6 for the recognition of a specific histidine-methylation-modified PRA1 protein.
8. A reagent for detecting a PRA1 protein modified by specific histidine methylation, comprising the polyclonal antibody according to claim 3 or the polyclonal antibody produced by the production method according to any one of claims 4 to 6.
9. A test strip for detecting a PRA1 protein having a specific histidine methylation modification, comprising the detection reagent of claim 8.
10. A kit for detecting a PRA1 protein with specific histidine methylation modification, comprising the detection reagent according to claim 8 or the detection strip according to claim 9.
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| CN (1) | CN115991752A (en) |
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2022
- 2022-12-07 CN CN202211565029.5A patent/CN115991752A/en active Pending
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