CN110079507B - Monoclonal antibody of anti-flounder mucosal immunoglobulin IgT and application thereof - Google Patents
Monoclonal antibody of anti-flounder mucosal immunoglobulin IgT and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody of a specific anti-flounder mucous membrane immunoglobulin IgT, which is named as: the hybridoma cell JF-IgT-1D8 with the preservation number as follows: CCTCC NO, C201971, the preservation unit is: china center for type culture Collection, Address: the hybridoma is secreted by Wuhan university in Wuhan city, Hubei province, with the preservation date of 2019, 04 and 29 days. The immunoblotting result shows that the monoclonal antibody of the invention can specifically identify the natural IgT protein of the paralichthys olivaceus with the molecular weight of about 66kDa in the serum and the mucus of the paralichthys olivaceus, and does not generate cross reaction with the IgM protein. The invention prepares the specific anti-flounder IgT monoclonal antibody for detecting the IgT molecule and the flounder IgT+B lymphocytes provide an important tool.
Description
Technical Field
The invention relates to a preparation method of a monoclonal antibody, in particular to a monoclonal antibody of anti-flounder (Paralichthys olivaceus) mucosal immunoglobulin IgT and a preparation method thereof, belonging to the technical field of fish molecular immunology.
Background
Immunoglobulins (igs) are important functional molecules in adaptive immunity of vertebrates and can specifically recognize and neutralize invading antigens. Mammals have five types of immunoglobulins, IgG, IgD, IgA, IgM, and IgE, with IgA being the major component of the body's mucosal defense system. Teleost immunoglobulin research has started late compared to mammals and has long been recognized as having only two classes of immunoglobulins, IgM and IgD, where IgM is considered the only immunoglobulin capable of reacting to pathogens in the system and mucosa. With the development of molecular biology research, a third class of immunoglobulin was found in teleosts in 2005, named IgT in rainbow trout and IgZ in zebrafish. It is reported that homologous IgT/IgZ is present in almost all fish studied, such as carp, rainbow trout, grass carp, Atlantic salmon, and weever, among others. In recent years, a number of studies have analyzed the characteristics of IgT/IgZ from the molecular level, which was found to be an immunoglobulin ubiquitous in mucosal immune responses. However, due to the lack of IgT/IgZ antibodies, there are few studies on the function of IgT/IgZ at the protein and cellular levels.
In recent years, antibodies against IgT have been developed in some teleost fish species, such as zebrafish, rainbow trout and carp, and IgT/IgZ was found to play an important role in mucosal immunity at the protein and cellular level, functioning similarly to mammalian IgA, while revealing new knowledge of different B-cell subsets of lymphoid organs. The paralichthys olivaceus is an important marine culture economic fish in China, however, due to the lack of the paralichthys olivaceus IgT antibody, the functional research of the paralichthys olivaceus IgT on the protein and cell level and the complete description of the paralichthys olivaceus B cell subset are lacked, and the functional difference research of IgM and IgT in mucosal immunity can not be carried out, so that the preparation of the paralichthys olivaceus IgT antibody is necessary. At present, researches find that IgM and IgT play a role in system and mucosal immunity simultaneously, and the bastard halibut IgT and IgM amino acid sequences have certain similarity, so that in order to avoid the cross reaction of the prepared bastard halibut IgT antibody and IgM, an IgT antigen peptide segment with low similarity to IgM is obtained by screening, and further the IgT antibody with strong specificity and non-cross with IgM is the primary key problem to be solved in the preparation process of the bastard halibut IgT antibody. Only by solving the problem, IgM and IgT can be strictly distinguished, so that the functions and differences of the IgM and the IgT in systemic and mucosal immune responses are researched, and a powerful tool is provided for deeply researching the adaptive immune response mechanism of fishes.
Disclosure of Invention
The invention aims to provide an anti-flounder IgT monoclonal antibody and a preparation method thereof, which are used for detecting flounder IgT protein molecules and IgT+B cells provide an important tool for further understanding the role of flounder IgT in systemic and mucosal immune response.
The invention firstly provides a hybridoma cell which is prepared by taking flounder IgT polypeptide with an amino acid sequence of HLLPGEDIEKKQDS (SEQ ID NO:1) as an antigen to immunize a Balb/c mouse;
the flounder IgT polypeptide is coupled with a hemocyanin (KLH) carrier and then is used as an antigen to immunize a Balb/c mouse;
the invention provides a hybridoma cell JF-IgT-1D8 strain with highest monoclonal antibody expression efficiency, wherein the preservation number is as follows: CCTCC NO of C201971, the preservation unit is: china center for type culture Collection, Address: the preservation date of Wuhan university in Wuhan city of Hubei province is as follows: 29/04/2019.
The hybridoma provided by the invention is used for preparing a monoclonal antibody of specific anti-flounder mucosa immunoglobulin IgT;
the invention also provides a monoclonal antibody of anti-flounder mucous membrane immunoglobulin IgT, which is secreted by the hybridoma cell, and the antibody can selectively identify the IgT protein and does not identify the flounder IgM protein.
The provided monoclonal antibody of the flounder IgT is applied to the preparation of a reagent for detecting the content change of the flounder IgT in each immune tissue or a reagent for researching the immune response of a system and a mucous membrane.
The invention uses bioinformatics software to analyze and predict the B cell epitope of the flounder IgT and selects the peptide segment with the dominant B cell epitope. Meanwhile, the software is used for analyzing and comparing the amino acid sequences of the IgT and IgM of the paralichthys olivaceus, and selecting the sequence with lower similarity, thereby effectively avoiding the paralichthys olivaceusIgT antibodies cross-react with IgM. Constructing a flounder IgT tertiary structure model, and selecting a peptide segment positioned on the surface of an IgT protein molecule. Finally, the selected IgT polypeptide-KLH compound is used as an antigen to immunize a mouse, hybridoma cells are prepared by adopting a cell fusion method, and a specific anti-flounder IgT monoclonal antibody is screened out by an immunological detection screening method. The preparation technical route is novel, strict and reasonable in design. The specific anti-flounder IgT monoclonal antibody prepared by the invention is used for detecting flounder IgT protein molecules and IgT+B lymphocytes provide an important tool.
Drawings
FIG. 1: analyzing a secondary structure of the flounder IgT molecule;
FIG. 2: DNAStar software analyzes the epitope parameter analysis chart of the predicted flounder IgT molecule;
wherein a is hydrophilic, b is flexible, c is alpha helix, d is beta turn, e is antigenic, and f is surface accessibility.
FIG. 3: IEDB software predicts the B cell linear epitope prediction map of the analyzed flounder IgT;
FIG. 4: comparing and analyzing the results of the flounder IgT and IgM amino acid sequences by using BioEdit software;
FIG. 5: tertiary structure model of flounder IgT protein molecule.445HLLPGEDIEKKQDS458Is a finally selected peptide fragment;
FIG. 6: analyzing a specific binding map of the monoclonal antibody and the flounder IgT polypeptide which is not coupled with the KLH by an ELISA method;
wherein 1 represents the OD value of the binding reaction of the monoclonal antibody and the flounder IgT polypeptide which is not coupled with the KLH; 2 represents the OD value of the reaction of myeloma cell supernatant and flounder IgT polypeptide which is not coupled with KLH, namely negative control; 3 represents a blank control, i.e. a flounder IgT polypeptide group not coated with non-coupled KLH;
FIG. 7: analyzing a specific binding reaction diagram of the IgT monoclonal antibody and the surface of the peripheral blood leukocytes of the paralichthys olivaceus by an indirect immunofluorescence method;
wherein A1 shows that the monoclonal antibody observed under 100 times objective lens has specific binding reaction with peripheral blood leukocyte surface, and has strong fluorescence signal, A2 is DAPI-stained nucleus, and A3 is the superposition of A1 and A2; b1 is a negative control obtained by incubating peripheral blood leukocytes of paralichthys olivaceus with myeloma cell supernatant, B2 is a DAPI-lined nucleus, and B3 is an overlay of B1 and B2.
FIG. 8: analyzing a result graph of whether the IgT monoclonal antibody and the paralichthys olivaceus IgM protein are subjected to cross reaction by an immunoblotting method;
wherein A is SDS-PAGE picture, M is molecular weight standard protein; 1: purified paralichthys olivaceus IgM protein;
b is an immunoblotting result graph, and M is a molecular weight standard protein; the reaction result of IgM mab with lane 1 of FIG. A; 2: a reaction result diagram of the flounder IgT monoclonal antibody and the lane 1 of the diagram A;
FIG. 9: analyzing a specific binding reaction graph of the monoclonal antibody, the paralichthys olivaceus serum and skin mucus by an immunoblotting method;
wherein A is SDS-PAGE picture, M is molecular weight standard protein; 1: bastard halibut skin mucus; 2: paralichthys olivaceus serum.
B is an immunoblotting result graph, and M is a molecular weight standard protein; 1. 3, reaction results of flounder IgT monoclonal antibody, myeloma cells and lane 1 of the figure A are shown. 2. 4, the results of the reaction of the flounder IgT monoclonal antibody and myeloma cells are shown in lane 2 of the graph A;
FIG. 10: analyzing a specific binding reaction chart of the monoclonal antibody and the paralichthys olivaceus head kidney, gill, intestinal tract, spleen and peripheral blood leukocyte by flow cytometry;
wherein A1, B1, C1, D1 and E1 respectively represent scatter diagrams of head kidney, branchia, intestinal tract, spleen and peripheral blood leukocyte; a2, B2, C2, D2 and E2 respectively represent the IgT of paralichthys olivaceus in head kidney, branchia, intestinal tract, spleen and peripheral blood+Flow histogram of B cell fraction.
Detailed Description
The invention uses bioinformatics software (DNAStar, IEDB software and the like) to analyze and predict the B cell epitope of the flounder IgT and selects the peptide segment with the dominant B cell epitope. In order to avoid cross reaction between the prepared flounder IgT antibody and IgM, the BioEdit software is used for comparing the flounder IgT and IgM amino acid sequences and selecting a peptide segment with lower similarity with IgM. Meanwhile, a flounder IgT tertiary structure model is constructed, and a peptide segment positioned on the surface of an IgT protein molecule is selected.
Final slave symbolThe final selection of a plurality of peptide segments which meet the requirement obtains a peptide segment (with strong antigenicity, lower similarity with paralichthys olivaceus IgM and positioned on the surface of an IgT protein molecule)445HLLPGEDIEKKQDS458) The antigenic peptide fragment used in the present invention. One part of the peptide segment is coupled with a KLH carrier, and the polypeptide is synthesized by a synthesis technology, and the other part of the peptide segment is not coupled with the carrier. The preparation method comprises the steps of immunizing a Balb/c mouse by polypeptide coupled with a carrier, preparing hybridoma cells by cell fusion, and screening the hybridoma cells secreting a specific anti-flounder IgT monoclonal antibody by an indirect enzyme-linked immunosorbent assay, an immunofluorescence assay and an immunoblotting assay. To avoid screening for antibodies against KLH, IgT polypeptides unconjugated to the KLH vector were used in the screening process to screen for antibodies. Then, the hybridoma cell strain obtained by the method is subjected to amplification culture by a conventional method.
Collecting cell culture supernatant, and purifying to obtain the specific anti-flounder IgT monoclonal antibody. The result of an indirect enzyme-linked immunoreaction experiment of the prepared monoclonal antibody for resisting the flounder IgT shows that the monoclonal antibody can be specifically combined with the IgT polypeptide which is not coupled with the KLH. The monoclonal antibody is specifically combined with the surface of part of white blood cells of the paralichthys olivaceus to present a fluorescence positive signal, and a green fluorescence signal can be observed to be scattered and distributed on the surface of the cell membrane of part of the white blood cells under a 100-time oil lens. The monoclonal antibody can specifically recognize the natural flounder IgT protein with the molecular weight of about 66kDa in the serum and the skin mucus of the flounder, does not recognize the IgM protein of the flounder, and does not cross with the IgM antibody.
The invention is further illustrated by the following specific examples in connection with the accompanying drawings.
Example 1: development of flounder immunoglobulin IgT monoclonal antibody
Preparation of antigenic peptides
(1) The SOPMA Server is used for predicting the secondary structure of the flounder IgT, and researches show that the epitope is mostly positioned at the beta-turn and the random coil position in the secondary structure. The result shows that the flexible structure in the flounder IgT gene accounts for 58.34 percent of the total number of amino acids, wherein the random coil accounts for 52.72 percent, and the beta-turn is 5.62 percent. The alpha-helix and the extended chain, i.e.the beta-sheet, account for 5.98% and 35.69%, respectively, and the distribution of the various structures in the flounder IgT amino acid sequence is shown in FIG. 1.
(2) DNASAR software is adopted to analyze flounder IgT epitope parameters, which mainly comprise Hydrophilicity (hydropathicity Plot-Kyte-Doolittle), flexibility (Flexible Regions-Karplus-Schulz), antigenicity (Antigenic Index-Jameson-Wolf) and Surface accessibility (Surface Probability Plot-Emini). By synthesizing various parameters, amino acid segments with hydrophilicity index more than or equal to 0, surface accessibility index more than or equal to 1 and antigenicity index more than or equal to 0 are taken as potential epitopes in the beta-turn and irregular coil regions. The possible existing positions of the flounder IgT molecular epitope can be seen from figure 2.
(3) And (3) adopting IEDB online prediction software and combining DNAStar software analysis results to further predict the flounder IgT epitope, and selecting a region with higher comprehensive score as a candidate B cell linear epitope. The 12 possible B cell linear epitopes were predicted to be predominantly located in the 24-37, 72-84, 129-144, 178-202, 318-333, 338-347, 375-396, 418-425, 435-441, 450-459, 482-506, 528-544 amino acid segments (FIG. 3).
(4) The BioEdit software compares the Paralichthys olivaceus IgT and IgM amino acid sequences and selects sequences with low similarity (FIG. 4).
(5) And synthesizing the prediction data to finally determine the candidate antigenic peptide. First, epitopes located in transmembrane and intracellular regions are excluded, leaving antigenic peptides located in extracellular segments and Ig-like domains. Second, antigenic peptides are located in random coil and β -turn regions in the secondary structure. Then, the parameters of the antigen epitope and the predicted potential antigen epitope are analyzed, the specific position of the antigen peptide is determined, and meanwhile, the difficulty degree of artificially synthesizing the polypeptide is referred. And finally, selecting a sequence with lower similarity between the flounder IgT and the IgM to avoid crossing. Constructing a tertiary structure model of the flounder IgT protein molecule, and finally selecting the flounder IgT antigen peptide445HLLPGEDIEKKQDS458Located on the protein surface (FIG. 5). The selected flounder IgT polypeptide is synthesized by a company and coupled with a hemocyanin carrier, and the purity is verified by mass spectrometry.
Second, immunization
A lefteye flounder IgT polypeptide-KLH compound is used as an antigen to immunize a mouse. The dose of each immunization is 1mg, the immunization is carried out for 4 times in total, the first 2 times are intraperitoneal injections, the immunization interval is 2 weeks, the last 2 times are tail vein injections, and the immunization interval is 1 week:
(1) basic immunization: mixing IgT polypeptide-KLH compound and Freund's complete adjuvant in the same amount (V/V) to obtain antigen;
(2) and (3) boosting immunity: mixing IgT polypeptide-KLH compound and Freund's incomplete adjuvant in the same amount (V/V) to obtain antigen;
(3) secondary boosting immunization: IgT polypeptide-KLH complex as antigen;
(4) three days prior to fusion, amplified immunization: IgT polypeptide-KLH complexes as antigens.
Cell fusion
(1) After anesthetizing a mouse by using ether, aseptically taking out spleen and thymus, respectively sieving the spleen and the thymus by a 100-mesh sieve, and blowing and beating by using RPMI-1640 solution to form single-cell suspension. The treatment of the experimental animals meets the requirements of animal welfare.
(2) Respectively centrifuging spleen cell suspension and thymocyte suspension at 1000rpm for 3min, discarding supernatant, resuspending spleen cell precipitate with RPMI-1640 solution, and resuspending thymocyte precipitate with RPMI-1640 containing 1% HAT (containing 10% fetal calf serum) selective cell culture solution.
(3) Take 3X 107Centrifuging P3-X63-Ag8U1 myeloma cells in logarithmic growth phase at 1000rpm for 3min, removing supernatant, and then resuspending with RPMI-1640 solution;
(4) uniformly mixing the spleen cell suspension and the tumor cell suspension, centrifuging at 1000rpm for 3min, completely absorbing supernatant, and flicking the bottom of a centrifugal tube to fully and uniformly mix two cell precipitates into paste; sucking polyethylene glycol solution lml preheated to 37 ℃ by a suction pipe, dripping into a centrifugal pipe, and finishing addition within 1 min; then standing in water bath at 37 ℃ for 5 min;
(5) continuously dripping 15ml of RPMI-1640 solution which is preheated to 37 ℃ to dilute the PEG to lose the effect;
(6) supplementing RPMI-1640 solution to 40ml, centrifuging at 1000rpm for 3min, and removing supernatant;
(7) resuspending the precipitated cells in 3ml of 37 ℃ RPMI-1640 (containing 10% fetal calf serum) cell culture medium, and freezing to store 2 ml;
(8) adding the rest 1ml of cell suspension into the thymocyte suspension prepared in the step (2), uniformly mixing, and then dropwise adding into a 96-hole culture plate;
(9) the plates were placed at 37 ℃ in CO2Culturing in 4.5% incubator, observing cell growth with inverted microscope, and taking hybridoma cell culture supernatant for detection after about two weeks.
Fourth, screening and cloning
(1) Screening: after fusion, detection is started when the hybridoma cell population grows to the bottom area 1/3 of the 96-well culture plate, and positive hybridoma cells are screened by adopting an indirect enzyme-linked immunosorbent assay technology.
Coating antigen: diluting IgT polypeptide without coupling KLH to the concentration of 50 mu g/ml by using carbonate coating solution (pH 9.6), adding the diluted IgT polypeptide into a 96-well enzyme label plate (100 mu l/well), and coating the IgT polypeptide at 4 ℃ overnight;
② sucking out the coating liquid, washing with Phosphate Buffer Solution (PBST) containing 0.05 percent of Tween-20, 5min for each time, and washing three times;
③ adding 200 mul of 3 percent bovine serum albumin (PBS) into each hole, and sealing for 1h at 37 ℃;
washing three times by the same method;
taking hybridoma cell culture supernatant as a first antibody, adding 50 mul of the first antibody into an enzyme label plate per hole, and incubating for 1h at 37 ℃;
sixthly, washing three times by the same method;
seventhly, adding alkaline phosphatase-labeled goat anti-mouse Ig (diluted 1: 4000) serving as a second antibody into an enzyme label plate according to 50 mu l of each hole, and incubating for 1h at 37 ℃;
eighthly, washing for three times by the same method as the second method; then adding 100 mul of 4-nitrophenol phosphate (pNPP) application liquid into each hole, reacting for 5-20 min in a dark place, adding 50 mul of 2M NaOH into each hole, stabilizing for 3-5 min, and determining the OD value by using a working wavelength of 405 nm.
Taking myeloma cell supernatant as a negative control, measuring the light absorption value of each hole at the wavelength of 405nm, and calculating the ratio (P/N) of the light absorption value of each experimental hole to the light absorption value of the negative control, wherein the hole is positive when the P/N is more than or equal to 2.1.
(2) Cloning: the detected positive hybridoma cells are cloned by adopting a limiting dilution method, and the steps are as follows:
firstly, anaesthetizing a mouse by using ether, taking out the thymus aseptically, grinding the thymus on a 100-mesh screen, and blowing and beating the thymus by using RPMI-1640 solution to form single-cell suspension;
② centrifuging the thymocyte suspension for 3min at 1000rpm, discarding supernatant, and resuspending the thymocyte sediment by 10ml RPMI-1640 (containing 10% fetal calf serum) cell culture solution;
thirdly, counting the cells in the positive cell hole to be cloned by using a blood cell counting plate, then diluting the cells by using culture solution in a 10-fold gradient manner, taking out 100 hybridoma cells, and putting the hybridoma cells into thymocyte suspension;
blowing and beating the cell suspension uniformly by using a dropper, and dripping the cell suspension into a culture plate with 96 holes, wherein each hole contains 100 mu l of hybridoma cells on average;
fifth, CO is added2Culturing in an incubator;
sixthly, detecting culture supernatant of the hybridoma cells in each hole by an indirect enzyme-linked immunosorbent assay after two weeks, and cloning the hybridoma cells of the obtained positive cloning holes once again according to the method so as to ensure that a monoclonal antibody is formed.
Fifthly, freezing and storing
Collecting hybridoma cells with vigorous growth and good morphology, making into cell suspension, centrifuging for 5min at 200g, removing supernatant, adding frozen stock solution (9 parts RPMI-1640 culture medium +1 part dimethyl sulfoxide) to obtain final cell density of 5 × 106And (2) putting 1ml of cell suspension into a 2ml freezing tube, screwing a screw cover, putting the freezing tube into a small box filled with cotton, putting the small box in an ultra-low temperature refrigerator at minus 80 ℃ for overnight (8-12 hours), and immersing the small box in liquid nitrogen for long-term storage. The hybridoma cell obtained by the invention has vigorous growth and good shape.
Observed under an inverted microscope, the hybridoma with good growth state has vigorous cell division, full appearance, round shape, strong refractivity, uniform cell size and good adherence; the hybridoma cells have the capacity of unlimited division and proliferation; the hybridoma cells with good growth vigor are cultured for 2-3 days conventionally, the culture medium is changed from peach red to yellow, and the culture medium contains a large amount of specific anti-flounder IgT monoclonal antibodies secreted by the hybridoma cells.
Finally, screening out a hybridoma cell with good growth state and high antibody secretion efficiency. The name is as follows: the hybridoma cell JF-IgT-1D8 is preserved in China center for type culture Collection of Wuhan, Wuhan university in China, with the preservation number: CCTCC NO of C201971, and the preservation date is as follows: 29/04/2019.
Example 2: the indirect enzyme-linked immunoassay method for monoclonal antibody of the invention
(1) Coating antigen: diluting IgT polypeptide without coupling KLH to the concentration of 50 mu g/ml by using carbonate coating solution (pH 9.6), adding the diluted IgT polypeptide into a 96-well enzyme label plate (100 mu l/well), and coating the IgT polypeptide at 4 ℃ overnight;
(2) sucking out the coating solution, washing with PBST for 5min each time for three times;
(3) adding 200 μ l of 3% bovine serum albumin (PBS) to each well, and sealing at 37 deg.C for 1 h;
(4) washing for three times by the same method as the method (2);
(5) adding the screened and cloned hybridoma cell culture supernatant serving as a first antibody into an enzyme label plate according to 50 mu l of each hole, and incubating for 1h at 37 ℃ in an incubator;
(6) washing for three times by the same method as the (2);
(7) adding alkaline phosphatase-labeled goat anti-mouse Ig (diluted 1: 4000) serving as a second antibody into an enzyme label plate according to 50 mu l of each hole, and incubating for 1h at 37 ℃ in an incubator;
(8) washing for three times by the same method as the (2); then adding 100 mul of pNPP application liquid into each hole, reacting for 5-20 min in a dark place, adding 50 mul of 2M NaOH into each hole, stabilizing for 3-5 min, and determining the OD value by using a working wavelength of 405 nm.
Myeloma cell supernatant was used as a negative control, coated PBS was used as a blank control, the absorbance of each well at a wavelength of 405nm was measured, and the ratio of the absorbance of the positive well to that of the control well (P/N) was calculated, and the wells were positive when P/N was 2.1 or more.
As a result: the light absorption value of the positive hole is 0.4073; the negative control was 0.0931 and the blank was 0.0911. The results confirmed that the monoclonal antibody of the present invention specifically binds to the flounder IgT polypeptide (FIG. 6).
The result of the prepared anti-flounder IgT monoclonal antibody is shown by indirect enzyme-linked immunoreaction experimentThe monoclonal antibody can be specifically combined with IgT polypeptide which is not coupled with KLH. The immunoblotting result shows that the monoclonal antibody of the invention can specifically identify the IgT protein with the molecular weight of about 66kDa in the serum and the mucus of the paralichthys olivaceus, and does not identify the IgM protein of the paralichthys olivaceus. The immunofluorescence detection result shows that the monoclonal antibody is specifically combined with the surface of peripheral blood leukocytes of the paralichthys olivaceus part, and a fluorescence positive signal is presented. Flow cytometry analysis results show that IgT exists in peripheral blood, spleen, head kidney, gill and intestinal tract of paralichthys olivaceus+B lymphocytes, with the highest content in the spleen.
Example 3: the indirect immunofluorescence method identification of the monoclonal antibody
(1) Resuspending white blood cells of peripheral blood of Percoll in 0.01M PBS, and adjusting the concentration to 1 × 107One per ml.
(2) The blood cell suspension was dropped on a clean glass slide, 10. mu.l each, settled in a wet box at room temperature for 1 hour, taken out, fixed in acetone for 20 minutes, taken out, and air-dried.
(3) Hybridoma supernatant (hybridoma culture fluid) was used as the primary antibody, and myeloma cell supernatant was used as the negative control, and the antibody was applied to a blood cell sample of a slide glass, and incubated in a wet box at 37 ℃ for 45 minutes.
(4) The slides were removed and washed three times with 0.01M PBS for 5 minutes each.
(5) A fluorescein isothiocyanate-labeled goat anti-mouse antibody was used as the second antibody, which was added to the blood cell sample and incubated in a wet box at 37 ℃ for 45 minutes.
(6) And taking out the glass slide, soaking and washing the glass slide, and sealing the glass slide by glycerol.
(7) And (4) observing under a fluorescence microscope.
As a result: the monoclonal antibody of the invention is specifically combined with the surface of part of leukocytes in the peripheral blood of paralichthys olivaceus to present a fluorescence positive signal, and a green fluorescence signal can be observed to be scattered and distributed on the surface of a cell membrane under a 100-fold oil lens, which is consistent with the fact that IgT protein is distributed on the surface of B lymphocytes, while a negative control does not observe a fluorescence signal (figure 7).
Example 4 transfer immunoblotting identification of the monoclonal antibody of the present invention
(1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis:
adding purified paralichthys olivaceus IgM protein into a sample buffer solution containing sodium dodecyl sulfate in equal proportion, and boiling for 3-5 min in boiling water;
adding the processed samples into sample loading holes, adding 10 mu l of samples into each hole, using low current (30-40mA) at the beginning under the condition of constant current, increasing the current (50-70mA) after the samples are partially concentrated into a line in the concentrated gel, carrying out electrophoresis until the bromophenol blue indicator reaches the bottom edge, stopping the electrophoresis, and taking out the gel; the gel portion was used for membrane transfer and the portion was stained in Coomassie brilliant blue stain for 1 h.
③ cut a piece of nitrocellulose membrane (pore diameter 0.22 μm) with the same size as the electrophoresis gel, wet with an electrotransfer buffer (electrotransfer buffer: 25mmol/L Tris-Base, 192mmol/L glycine, 20% methanol, pH 8.3), and place on the gel after electrophoresis. A corner was cut on the nitrocellulose membrane to mark the beginning of the sample sequence. Supporting with wet filter paper, and attaching a second piece of wet filter paper to the other side of the gel sheet; forming a sandwich with the glue block, the nitrocellulose membrane and the filter paper according to the placing sequence;
putting the gel sandwich into an electrophoresis tank containing an electrotransfer buffer solution, and enabling the nitrocellulose membrane to face the anode; electrophoresis constant voltage is 30V, and electrification is carried out for 1.5 hours;
fifthly, taking out the nitrocellulose membrane after the transfer is finished.
(2) Immunoblotting:
washing the cellulose nitrate membrane with PBS for 10 minutes, and then sealing the cellulose nitrate membrane in 3 percent bovine serum albumin solution (PBS) for 1 hour at 37 ℃;
washing with PBST for 3 times, 5 minutes each time;
thirdly, respectively placing the nitrocellulose membrane in culture supernatants of the paralichthys olivaceus IgM monoclonal antibody (1:1000) and the hybridoma cells, and slowly shaking for 1 hour at 37 ℃; washing for three times by the same method;
adding the nitrocellulose membrane into an alkaline phosphatase-labeled goat anti-mouse Ig antibody (diluted at a ratio of 1: 4000), and slowly shaking for 1 hour at 37 ℃; washing for three times by the same method;
fifthly, putting the nitrocellulose membrane into an alkaline phosphatase coloring solution (NBT-BCIP coloring solution) for coloring until the color is clear; the reaction was terminated by washing with deionized water. The nitrocellulose membrane was sandwiched between filter papers and dried. Storing in dark place.
Sixthly, simultaneously boiling the gel in the Coomassie brilliant blue until the bands are clear, and enabling the positions of the purple brown bands on the nitrocellulose membrane to correspond to the gel.
As a result: the paralichthys olivaceus IgM monoclonal antibody can react with the purified paralichthys olivaceus IgM protein at about 74kDa, showing a purple brown band, while the paralichthys olivaceus IgT monoclonal antibody does not react with the purified IgM (fig. 8).
Example 5: the monoclonal antibody of the invention is identified by immunoblotting
(1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis:
adding the bastard halibut skin mucus and serum into a sample buffer solution containing sodium dodecyl sulfate in equal proportion, and boiling in boiling water for 3-5 min;
the procedure was the same as in example 4.
(2) Immunoblotting:
washing the cellulose nitrate membrane with PBS for 10 minutes, and then sealing the cellulose nitrate membrane in 3 percent bovine serum albumin solution (PBS) for 1 hour at 37 ℃;
washing with PBST for 3 times, 5 minutes each time;
thirdly, placing the nitrocellulose membrane in culture supernatant of the hybridoma cells, and slowly shaking for 1 hour at 37 ℃; incubating a nitrocellulose membrane with myeloma cell supernatant as a negative control;
washing three times by the same method;
fifthly, adding the nitrocellulose membrane into an alkaline phosphatase-labeled goat anti-mouse Ig antibody (diluted 1: 4000), and slowly shaking for 1 hour at 37 ℃;
sixthly, washing three times by the same method;
seventhly, putting the cellulose nitrate membrane into an alkaline phosphatase coloring liquid (NBT-BCIP coloring liquid) for coloring until the color is clear;
and washing with deionized water to terminate the reaction. The nitrocellulose membrane was sandwiched between filter papers and dried. Storing in dark place.
Ninthly, simultaneously boiling the gel placed in Coomassie brilliant blue in boiling water until the bands are clear, enabling the position of the purple-brown band on the nitrocellulose membrane to correspond to the gel, carefully cutting off the corresponding band and sending the band to a mass spectrum.
As a result: the flounder IgT monoclonal antibody can react with the skin mucus and serum of the flounder at about 66kDa, and no band is displayed in negative control (figure 9). And cutting the gel on the gel corresponding to the 66kDa and sending the cut gel to a mass spectrum, wherein the mass spectrum result proves that the protein identified by the monoclonal antibody is the flounder IgT protein.
Example 6: the monoclonal antibody of the invention is used for detecting IgT in head kidney, gill, intestinal tract, spleen and peripheral blood leukocyte of paralichthys olivaceus+Use of B cell number ratio
(1) Selecting healthy paralichthys olivaceus temporarily cultured for one week, wherein the body length of the healthy paralichthys olivaceus is 15cm, anesthetizing by using ether, extracting peripheral blood leukocytes of the paralichthys olivaceus from the tail vein, and simultaneously taking spleen, head kidney, gill and intestinal tract tissues of the paralichthys olivaceus, and obtaining leukocytes of the peripheral blood, spleen, head kidney, gill and intestinal tract of the paralichthys olivaceus by using discontinuous density gradient centrifugation of percoll. Diluting the extracted white blood cells of each tissue of Paralichthys olivaceus with sterile PBS buffer solution to obtain a cell density of 1 × 106cells/ml cell suspension.
(2) Two 500. mu.l leukocyte suspensions are prepared from each tissue sample, one is a detection sample, the other is a control sample, 500. mu.l flounder IgT monoclonal antibody is added into the detection sample, 500. mu.l myeloma cell supernatant is added into the same tissue control group, and the mixture is incubated for 1h at 37 ℃.
(3) Cells were harvested by centrifugation at 680 Xg at 4 ℃ and washed three times for 5min with sterile PBS.
(4) The centrifuged cell pellet was washed in resuspension (3) with 1ml of FITC-labeled goat anti-mouse Ig mab and incubated at 37 ℃ for 1 h.
(5) Cells were harvested by centrifugation at 680 Xg at 4 ℃ and washed three times for 5 minutes in sterile PBS resuspension.
(6) After completion of the centrifugation wash, the cells were resuspended in 500. mu.l sterile PBS for flow cytometry analysis.
As a result: the flow analysis of negative control samples of each tissue has no positive reaction cell group, and the flow analysis of the white blood cells of each tissue of the paralichthys olivaceus incubated with the monoclonal antibody has double peaks, which shows that the monoclonal antibody of the invention can be specifically combined with the white blood cells of part of the paralichthys olivaceus. The detection result shows that the IgT in the peripheral blood leucocyte, spleen, head kidney, intestinal tract and gill of the paralichthys olivaceus+The B cell number ratios were 2.75%, 6.25%, 4.51%, 1.65% and 1.98%, respectively (fig. 10).
Those skilled in the art will appreciate that modifications, additions and substitutions are possible, without departing from the scope of the invention as disclosed in the accompanying claims.
Claims (4)
1. A hybridoma cell JF-IgT-1D8, wherein the hybridoma cell is prepared by taking flounder IgT polypeptide with an amino acid sequence of HLLPGEDIEKKQDS as antigen to immunize Balb/c mice; the preservation number of the hybridoma cell is CCTCC NO: C201971.
2. The use of the hybridoma of claim 1 for preparing a monoclonal antibody against flounder mucosal immunoglobulin IgT.
3. A monoclonal antibody against Paralichthys olivaceus mucosal immunoglobulin IgT, which is secreted by the hybridoma of claim 1, and which specifically recognizes Paralichthys olivaceus IgT protein and does not recognize Paralichthys olivaceus IgM protein.
4. The monoclonal antibody of claim 3, in the preparation of a reagent for detecting the content change of flounder IgT in each immune tissue or the application of flounder IgT in a reagent for researching the immune response of a system and a mucous membrane.
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