CN114456235A - Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody and preparation method and application thereof - Google Patents
Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody, and a preparation method and application thereof, and belongs to the technical field of fish molecular immunology. The polyclonal antibody of the paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha comprises the amino acid sequence: APRVIASTRSPSLT the CD8 alpha epitope peptide-KLH complex of paralichthys olivaceus is used to immunize a New Zealand white rabbit, and the serum of the new Zealand white rabbit is purified. Indirect immunofluorescence and immunoblotting experiments show that the rabbit polyclonal antibody can react with HEK293 cells and paralichthys olivaceus head kidney lymphocytes of transfected paralichthys olivaceus CD8 alpha eukaryotic plasmids. Flow cytometry detection of CD8 alpha in paralichthys olivaceus peripheral blood lymphocytes+The number ratio of T lymphocytes is 5.6 +/-0.8%, and the ratio of T lymphocytes to spleen lymphocytesCD8α+The proportion of the number of T lymphocytes is 14.8 +/-1.6%, and the CD8 alpha in the head and kidney lymphocytes+The proportion of the number of T lymphocytes was 19.5. + -. 0.9%. These results indicate that the polyclonal antibody can be used as a reagent for identifying T cell subsets of Paralichthys olivaceus or as a reagent for studying CD8 of Paralichthys olivaceus+An agent for T lymphocyte immune response.
Description
Technical Field
The invention belongs to the technical field of fish molecular immunology, and particularly relates to a Paralichthys olivaceus (Schmidt: (Schmidt))Paralichthys olivaceus) T lymphocyte surface marker molecule CD8 alpha antibody and its preparation method and application.
Background
T lymphocytes are the main immune cells of the body and are involved in the humoral and cellular immune responses of the body. Among them, CD8+ T lymphocytes, one of the major functional subsets of T cells, exert multiple immune functions during the course of immunization, such as in the context of the relevant immune defenses against pathogenic invasion, CD8+T lymphocytes play a crucial role. CD8+After recognizing endogenous antigen peptide-MHC class I molecule complexes on the surface of APC, TCR on the surface of T cell is activated into cytotoxic T cell, and the cytotoxic T cell is directly contacted with target cell invaded by pathogen to generate killing factors such as granzyme and perforin or directly kill the target cell through death receptor approach, thereby eliminating the pathogen. In mammals, CD8+ The characteristics and immune function of the T lymphocyte subsets are well researched, and a new situation is created for developing disease prevention and treatment, evaluating the immune state of an organism and the like.
The CD8 molecule is a membrane-bound extracellular receptor consisting of either α α homodimers or α β heterodimers. CD8 a and CD8 β consist of an IgV-like extracellular domain, a transmembrane domain and a short cytoplasmic tail, the α and β peptides being linked by disulfide bonds. Heterodimers of CD8 are found primarily in mature cytotoxic T cells and thymocytes, while homodimers are expressed in NK cells, Dendritic Cells (DCs), and γ δ T cells. CD8 as an identification CD8+The two CD8 chains of the marker molecules of the T lymphocyte subpopulation are cloned in a plurality of fishes, such as rainbow trout, crucian, puffer, sea bass, Atlantic salmon, paralichthys olivaceus and the like. In earlier researches of the inventor, the recombinant protein of the paralichthys olivaceus CD8 beta has been recombined and expressed, and a polyclonal antibody of the anti-paralichthys olivaceus CD8 beta recombinant protein is developed and used for identifying the CD8 beta of the paralichthys olivaceus+T lymphocyte subpopulations (Xing et al, 2017, mol. Immunol.). Because of the particularity of the paralichthys olivaceus CD8 alpha protein, the paralichthys olivaceus CD8 alpha recombinant protein cannot be prepared, and the related paralichthys olivaceus CD8 alpha and CWhether the D8 beta peptide chain is co-expressed on the surface of a cell membrane and whether a paralichthys olivaceus CD8 alpha homodimer exists is not reported scientifically. Korean scholars use polypeptide to prepare monoclonal antibody for recognizing CD8 alpha molecule of Paralichthys olivaceus and identify CD8 alpha of Paralichthys olivaceus+ T cells, found CD8 alpha+ T cells are involved in antiviral responses (Jung et al, 2021, int. j. mol. Sci.).
Left-eyed flounder (1)Paralichthys olivaceus) Is an important economic fish for mariculture in northern China, and the frequent culture diseases in recent years are important factors which hinder the development of the culture industry. Many studies and practices indicate that vaccines are an effective measure for controlling aquatic diseases. The role of vaccines depends on the activation of adaptive immune mechanisms in the body, during which T cells play an important role. Therefore, develop the CD8 alpha of Paralichthys olivaceus+Antibodies specific for marker molecules of T lymphocytes, useful for the identification of CD8 alpha at the cellular level+ T lymphocyte subset for detecting CD8 alpha of paralichthys olivaceus+The number of T lymphocyte subpopulations was varied, and the level of cellular immune response was assessed.
Disclosure of Invention
The first purpose of the invention is to provide a polyclonal antibody of a surface marker molecule CD8 alpha of a T lymphocyte of paralichthys olivaceus, which can specifically identify CD8 alpha of the paralichthys olivaceus+ T lymphocyte subset for researching flounder CD8 alpha+ The function of T lymphocyte subpopulations provides an important immunological tool.
The second purpose of the invention is to provide a preparation method of the flounder T lymphocyte surface marker molecule CD8 alpha polyclonal antibody.
The third purpose of the invention is to provide a method for preparing a reagent for identifying the T cell subset of the paralichthys olivaceus or preparing a paralichthys olivaceus CD8 polyclonal antibody by using the paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha polyclonal antibody+ Application of T lymphocyte immune response research reagent.
The purpose of the invention is realized by the following technical scheme:
an epitope peptide of a paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha is characterized in that the amino acid sequence of the epitope peptide is as follows:134APRVIASTRSPSLT147。
a polyclonal antibody of a surface marker molecule CD8 alpha of a paralichthys olivaceus T lymphocyte is characterized in that the antibody has an amino acid sequence134APRVIASTRSPSLT147The flounder CD8 alpha epitope peptide-KLH compound is used for immunizing a New Zealand white rabbit and purifying serum of the new Zealand white rabbit.
The flounder CD8 alpha epitope peptide (of the invention)134APRVIASTRSPSLT147) KLH complexes were obtained by epitope screening by analysis of the amino acid sequence CD8 alpha.
The anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody provided by the invention can react with HEK293 cells transfected with paralichthys olivaceus CD8 alpha eukaryotic plasmids through indirect immunofluorescence experiments, and a two-color fluorescence signal appears, which indicates that the antibody can react with natural CD8 alpha molecules of paralichthys olivaceus.
The anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody provided by the invention can react with paralichthys olivaceus head and kidney lymphocytes through indirect immunofluorescence experiments, and red fluorescence signals are distributed on the surface of cell membranes, which is consistent with the fact that CD8 alpha protein is mainly distributed on the surface of T lymphocytes.
The anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody provided by the invention is shown by immunoblotting reaction to be capable of reacting with HEK293 cells transfected with paralichthys olivaceus CD8 alpha eukaryotic plasmids, and a specific strip is shown at 51kDa and is consistent with the molecular weight of paralichthys olivaceus CD8 alpha protein and tag protein. The rabbit polyclonal antibody of the invention reacts with the paralichthys olivaceus head kidney lymphocytes, shows a specific strip at 26kDa, and has the same molecular weight with the molecular weight of the natural CD8 alpha protein of the paralichthys olivaceus.
The anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody provided by the invention is used for detecting peripheral blood, spleen and head and kidney lymphocytes through flow cytometry, the antibody is found to be specifically combined with partial lymphocytes of paralichthys olivaceus, and the detection result shows that CD8 alpha in the peripheral blood lymphocytes of the paralichthys olivaceus+The number ratio of T lymphocytes is 5.6 +/-0.8%, and the CD8 alpha in spleen lymphocytes+The number ratio of T lymphocytes is 14.8 +/-1.6%, and the CD8 alpha in the head and kidney lymphocytes+The proportion of the number of T lymphocytes was 19.5. + -. 0.9%.
The invention provides a toothPreparation of flounder T cell subset identification reagent from polyclonal antibody of flounder T lymphocyte surface marker molecule CD8 alpha, or preparation of flounder CD8+ Application of T lymphocyte immune response research reagent.
The invention has the advantages that: the invention obtains the paralichthys olivaceus CD8 alpha molecular epitope polypeptide with good antigenicity by screening, and prepares the highly specific anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody, so as to identify CD8 alpha from the cell level+ T lymphocyte subset, detecting paralichthys olivaceus CD8 alpha+The quantitative change of T lymphocyte subpopulation and the evaluation of cellular immune response level provide important tools.
Drawings
FIG. 1 is an analysis chart of the epitope of the CD8 alpha molecule of Paralichthys olivaceus.
In the figure, the dotted rectangle is the 134-147 amino acid sequence with higher screening score, the X-axis is the amino acid position, and the Y-axis is the epitope score.
FIG. 2 is an immunofluorescence chart of hFO transfected HEK293 cells with GFP-tagged Paralichthys olivaceus CD8 alpha eukaryotic plasmid.
In the figure, A is GFP green fluorescent protein, B is a differential interference pattern, and C is a superimposed graph of A and B.
FIG. 3 is an indirect immunofluorescence of HEK293 cells against Paralichthys olivaceus CD8 alpha rabbit polyclonal antibody and transfected Paralichthys olivaceus CD8 alpha eukaryotic plasmid.
In the figure, A is DAPI lining staining cell nucleus, B is GFP green fluorescent protein, C is a red positive fluorescence signal displayed by incubating anti-CD 8 alpha rabbit polyclonal antibody, and D is an overlay image of A, B and C; e is DAPI lining stained cell nucleus, F is GFP green fluorescent protein, G is incubation rabbit negative serum as control, H is E, and the superposition graph of F and G.
FIG. 4 is an indirect immunofluorescence of anti-Paralichthys olivaceus CD8 alpha rabbit polyclonal antibody reacting with Paralichthys olivaceus head kidney leukocytes.
In the figure, A is a red positive fluorescence signal displayed by incubating anti-CD 8 alpha rabbit polyclonal antibody, B is DAPI lining nucleus, and C is an overlay image of A and B; d is incubation rabbit negative serum as control, E is DAPI-stained nuclei, and F is the overlay of D and E.
FIG. 5 is an immunoblot of the reaction of anti-Paralichthys olivaceus CD8 alpha rabbit polyclonal antibody with HEK293 cells transfected with Paralichthys olivaceus CD8 alpha eukaryotic plasmid and Paralichthys olivaceus head kidney leukocytes.
In the figure, M is Marker, lane 1 is the immunoblotting result of the reaction of anti-paralichthys olivaceus CD8 alpha rabbit polyclonal antibody and HEK293 cells transfected with paralichthys olivaceus CD8 alpha eukaryotic plasmid, lane 2 is a negative control, lane 3 is the immunoblotting result of the reaction of anti-paralichthys olivaceus CD8 alpha rabbit polyclonal antibody and paralichthys olivaceus head kidney leukocytes, and lane 4 is a negative control.
FIG. 6 shows the detection of CD8 alpha in peripheral blood, spleen and head kidney lymphocytes by anti-Paralichthys olivaceus CD8 alpha rabbit polyclonal antibody+ Flow diagram of T lymphocytes.
Detailed Description
In order to explain the technical scheme of the invention more clearly, the invention is further explained by the specific embodiment in combination with the attached drawings.
Example 1: screening and synthesis of paralichthys olivaceus CD8 alpha molecular epitope peptide
According to the paralichthys olivaceus CD8 alpha gene sequence published by NCBI, after being translated into an amino acid sequence, IEDB online prediction software (http:// tools. immuneepitope. org/bcll /) is used for analyzing the epitope of the paralichthys olivaceus CD8 alpha molecule (figure 1), and finally, polypeptide chains are selected as the polypeptide chains134APRVIASTRSPSLT147Polypeptide is synthesized and coupled with hemocyanin (KLH) by Nanjing Kingsrey Bio Inc., and the purity is verified to be more than 95% by mass spectrum.
Example 2: construction of prokaryotic and eukaryotic plasmid for expressing paralichthys olivaceus CD8 alpha molecule
(1) According to the paralichthys olivaceus CD8 alpha gene sequence published by NCBI, a paralichthys olivaceus CD8 alpha gene is synthesized and connected to pTag-GFP plasmid.
(2) And transfecting the successfully connected pTag-GFP-CD8 alpha plasmid into HEK293 cells, and observing green fluorescence to judge the expression effect of the paralichthys olivaceus CD8 alpha molecule.
As a result: the eukaryotic plasmid of the paralichthys olivaceus CD8 alpha molecule is successfully constructed, and after the eukaryotic plasmid is transfected into cells, green fluorescent protein (see figure 2) is displayed, which indicates that the paralichthys olivaceus CD8 alpha molecule is successfully expressed in HEK293 cells.
Example 3: preparation of anti-paralichthys olivaceus CD8 alpha molecular rabbit polyclonal antibody
(1) The synthesized flounder CD8 alpha polypeptide-KLH compound is used as an antigen to immunize a New Zealand white rabbit, the immunization dose is 0.6mg each time, the immunization is carried out for 4 times in total, the interval of the former 2 times of immunization is 2 weeks, and the interval of the latter 2 times of immunization is 1 week.
(2) Basic immunity: the CD8 alpha polypeptide-KLH complex was mixed with equal amounts (V/V) of Freund's complete adjuvant as antigen and injected subcutaneously at 6 points in the back.
(3) And (3) boosting immunity: the CD8 alpha polypeptide-KLH complex was mixed with equal amounts (V/V) of Freund's incomplete adjuvant as an antigen and injected subcutaneously at 6 points in the back.
(4) Secondary boosting immunization: CD8 alpha polypeptide-KLH complex (600 μ l) was injected intravenously at the ear margin as antigen.
(5) And (3) boosting again: CD8 alpha polypeptide-KLH complex (600 μ l) was injected intravenously at the ear margin as antigen.
(6) After the last immunization, five days are separated for heart blood sampling. The collected blood was allowed to stand at room temperature for 2 hours at an incline and overnight in a refrigerator at 4 ℃. The next day, the centrifuge tube was centrifuged at 8000G and 4 ℃ for 10min, the supernatant was carefully extracted and purified by protein G affinity column chromatography, and the purified product was rabbit anti-Paralichthys olivaceus CD8 alpha molecular polyclonal antibody.
As a result: after four times of immunization, the purified rabbit serum is the rabbit anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody.
Example 4: specificity of identifying paralichthys olivaceus CD8 alpha molecular rabbit polyclonal antibody by indirect immunofluorescence method
(1) Selecting healthy Paralichthys olivaceus (weight 0.7 + -0.2 kg) temporarily cultured for one week, anesthetizing by MS-222, taking head and kidney, lightly grinding into single cell suspension, centrifuging for 5min at 100 Xg to remove erythrocytes, and performing Percoll discontinuous density gradient (1.02/1.07 g/cm)3) And (4) centrifuging to obtain the lymphocytes of the head and kidney of the paralichthys olivaceus. Collecting HEK293 cells transfected with Paralichthys olivaceus CD8 alpha eukaryotic plasmid, and diluting the extracted Paralichthys olivaceus head and kidney lymphocytes and HEK293 cells with sterile PBS buffer solution to obtain cells with density of 1 × 106cells/ml cell suspension.
(2) And dropwise adding the lymphocyte and HEK293 cell suspension on a clean glass slide, wherein each drop is 10 mu l, taking out the drop after settling for 1 hour in a wet box at room temperature, putting the drop into acetone for fixing for 20 minutes, taking out and air-drying.
(3) An anti-paralichthys olivaceus CD8 alpha molecular rabbit polyclonal antibody is taken as a primary antibody (diluted 1: 1000), and the primary antibody is dripped on a cell sample of a glass slide and incubated for 1.5h in a 37 ℃ wet box.
(4) Slides were removed, washed three times for 5 minutes in PBST, and a Cy 3-labeled goat anti-rabbit Ig antibody was used as the secondary antibody (1: 1000 dilution), added dropwise to the cell samples, and incubated for 45 minutes in a 37 ℃ wet box.
(5) The slides were removed and washed three times in PBST, 5 minutes each, and after approximately 10min of DAPI lining of the nuclei, glycerol mounting, observation under a fluorescent microscope and photographing.
As a result: the rabbit polyclonal antibody of the invention is specifically combined with HEK293 cells with GFP labels, presents red fluorescent signals, and the green and red fluorescent signals are overlapped, which shows that the prepared rabbit polyclonal antibody can be specifically combined with HEK293 cells expressing paralichthys olivaceus CD8 alpha protein, namely can specifically react with natural CD8 alpha protein of paralichthys olivaceus (see figure 3). The rabbit polyclonal antibody of the invention is specifically combined with the surface of the partial lymphocytes of the paralichthys olivaceus, presents a fluorescence positive signal, can see the distribution of a red fluorescence signal on the surface of a cell membrane under 100 times of oil lens, which is consistent with the fact that CD8 alpha protein is distributed on the surface of T lymphocytes, and no fluorescence signal is observed in a negative control (see figure 4).
Example 5: specificity of identifying paralichthys olivaceus CD8 alpha molecular rabbit polyclonal antibody by immunoblotting method
(1) Collecting HEK293 cells transfected with Paralichthys olivaceus CD8 alpha eukaryotic plasmids, extracting Paralichthys olivaceus head and kidney lymphocytes for gel electrophoresis, and transferring proteins on the gel to a nitrocellulose membrane.
(2) The nitrocellulose membrane was blocked in 3% bovine serum albumin solution (PBS) for 1h at 37 ℃.
(3) PBST rinse the cellulose nitrate membrane 3 times, each time 5 minutes, will be cellulose nitrate membrane placed in rabbit anti-Paralichthys olivaceus CD8 alpha molecular polyclonal antibody (10 mL PBS with 10 u l antibody), 37 degrees C slow shaking 1.5h, with rabbit negative serum incubation cellulose nitrate membrane as negative control.
(4) The nitrocellulose membrane was rinsed 3 times by PBST for 5 minutes each, added to horseradish peroxidase-labeled goat anti-rabbit Ig antibody (1:4000 dilution), and shaken slowly for 1 hour at 37 ℃.
(5) PBST rinsing the cellulose nitrate membrane for 3 times, each time for 5 minutes, putting the cellulose nitrate membrane into a coloring liquid for color development, and photographing by using a chemiluminescence apparatus to record the result.
As a result: the rabbit polyclonal antibody reacts with HEK293 cells transfected with Paralichthys olivaceus CD8 alpha eukaryotic plasmids, shows a specific band at 51kDa, and has the same molecular weight as the Paralichthys olivaceus CD8 alpha protein plus a tag protein. The rabbit polyclonal antibody of the invention reacts with the paralichthys olivaceus head kidney lymphocytes, shows a specific band at 26kDa, and has the same molecular weight with the molecular weight of the natural CD8 alpha protein of the paralichthys olivaceus (see figure 5).
Example 6: detection of flounder CD8 alpha by flow cytometry+Proportion of T lymphocytes
(1) Selecting healthy Paralichthys olivaceus (with the weight of 0.7 +/-0.2 kg) temporarily cultured for one week, anesthetizing by MS-222, extracting peripheral blood of the Paralichthys olivaceus from the tail vein, and simultaneously taking spleen and head kidney. Using a Percoll discontinuous density gradient (1.02/1.07 g/cm)3) And (4) centrifuging to obtain the lymphocytes of the peripheral blood, the spleen and the head kidney of the paralichthys olivaceus. Diluting the extracted lymph cells of each tissue of Paralichthys olivaceus with sterile PBS buffer solution to obtain cells with density of 1 × 107cells/ml cell suspension.
(2) Two 500. mu.l lymphocyte suspensions were taken from each tissue sample, one was a test sample and the other was a control sample, and 1. mu.l rabbit anti-paralichthys olivaceus CD 8. alpha. polyclonal antibody was added to the test sample, and 1. mu.l rabbit negative serum was added to the control group, and incubation was carried out at 37 ℃ for 1.5 hours.
(3) Cells were harvested by centrifugation at 680g at 4 ℃ and washed three times for 5 minutes in sterile PBS resuspension.
(4) Mu.l FITC-labeled goat anti-rabbit Ig monoclonal antibody was added to the resuspended cells and incubated at 37 ℃ for 1 h.
(5) Cells were harvested by centrifugation at 680g 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 histogram of each tissue negative control sample in flow analysis shows a single peak without a positive reaction cell population, while the histogram of each tissue lymphocyte in flow analysis of the paralichthys olivaceus incubated with the rabbit polyclonal antibody in the invention shows a double peak, which indicates that the monoclonal antibody in the invention can be specifically combined with partial lymphocytes of the paralichthys olivaceus, and the detection result shows that the CD8 alpha in the peripheral blood lymphocytes of the paralichthys olivaceus+The number ratio of T lymphocytes is 5.6 +/-0.8%, and the CD8 alpha in spleen lymphocytes+The proportion of the number of T lymphocytes is 14.8 +/-1.6%, and the CD8 alpha in the head and kidney lymphocytes+The ratio of the number of T lymphocytes was 19.5. + -. 0.9% (see FIG. 6).
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. An epitope peptide of a paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha is characterized in that the amino acid sequence of the epitope peptide is as follows: APRVIASTRSPSLT are provided.
2. A polyclonal antibody of a surface marker molecule CD8 alpha of a paralichthys olivaceus T lymphocyte is characterized in that the polyclonal antibody is prepared by the following amino acid sequences: APRVIASTRSPSLT the CD8 alpha epitope peptide-KLH complex of paralichthys olivaceus is used to immunize a New Zealand white rabbit, and the serum of the new Zealand white rabbit is purified.
3. The use of the polyclonal antibody of the paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha according to claim 2 in the preparation of a reagent for identifying T cell subsets of paralichthys olivaceus.
4. The use of the polyclonal antibody of the surface marker molecule CD8 alpha of the T lymphocyte of Paralichthys olivaceus as claimed in claim 2 in the preparation of CD8+ T lymphocyte immune response research reagentThe use of (1).
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| JAE WOOK JUNG: "Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder ( Paralichthys olivaceus)", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| RENG QIU: "Characterization of TCR+ and CD8+ head kidney leucocytes in Japanese flounder (Paralichthys olivaceus) with antisera against TCRα and CD8α", 《JOURNAL OF FISH BIOLOGY 》 * |
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