CN114456235B - Paralichthys olivaceus T lymphocyte surface marker molecule CD8 alpha antibody, and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a flounder T lymphocyte surface marker molecule CD8 alpha antibody, a preparation method and application thereof, belonging to the technical field of fish molecular immunology. The polyclonal antibody of the flounder T lymphocyte surface marker molecule CD8 alpha comprises the following amino acid sequences: APRVIASTRSPSLT the CD8 alpha epitope peptide-KLH complex of the paralichthys olivaceus is used for immunizing New Zealand white rabbits, and the serum is purified. Indirect immunofluorescence and immunoblotting experiments show that the rabbit polyclonal antibody disclosed by the invention can react with HEK293 cells transfected with flounder CD8 alpha eukaryotic plasmids and flounder head and kidney lymphocytes. Flow cytometry detection of CD8 alpha in peripheral blood lymphocytes of paralichthys olivaceus + The ratio of the number of T lymphocytes is 5.6+/-0.8 percent, and CD8 alpha in spleen lymphocytes + The ratio of the number of T lymphocytes is 14.8+/-1.6 percent, and CD8 alpha in the head and kidney lymphocytes + The ratio 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 subset of paralichthys olivaceus or for researching CD8 of paralichthys olivaceus + Reagents for T lymphocyte immune responses.
Description
Technical Field
The invention belongs to the technical field of fish molecular immunology, and in particular relates to a flounder #Paralichthys olivaceus) T lymphocyte surface marker molecule CD8 alpha antibody and its preparation process and application.
Background
T lymphocytes are the primary immune cells of the body and are involved in both the humoral and cellular immune responses of the body. Wherein, CD8 + T lymphocytes, which are one of the main functional sub-populations of T cells, exert a variety of immune functions during the immune process, such as CD8, in the associated immune defenses against pathogen invasion and the like + T lymphocytes play a vital role. CD8 + After the TCR on the surface of the T cell recognizes the endogenous antigen peptide-MHC class I molecular complex on the surface of the APC, the T cell is activated into cytotoxic T cells, and the cytotoxic T cells are directly contacted with target cells invaded by pathogens to generate killing factors such as granzyme, perforin and the like or directly kill the target cells through death receptor paths, so that the pathogens are cleared. In mammals, CD8 + The characteristics and immune function of the T lymphocyte subpopulation are thoroughly 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 an alpha homodimer or an alpha beta heterodimer. CD8 a and CD8 β consist of an IgV-like extracellular domain, a transmembrane domain and a short cytoplasmic tail, with the α and β peptides linked by disulfide bonds. The heterodimers of CD8 are found predominantly in mature cytotoxic T cells and thymocytes, while the homodimers are expressed in NK cells, dendritic Cells (DCs) and γδ T cells. CD8 as identification CD8 + Marker molecules of T lymphocyte subpopulations, two CD8 chains have been cloned in a number of fish species, such as rainbow trout, crucian, globefish, jewfish, atlantic salmon, and paralichthys olivaceus. In the previous research of the inventor, the flounder CD8 beta recombinant protein has been expressed recombinantly, and a polyclonal antibody against the flounder CD8 beta recombinant protein has been developed for identifying the flounder CD8 beta + T lymphocyte subpopulations (Xing et al, 2017, mol. Immunol.). Because of the specificity of the flounder CD8 alpha protein, the flounder CD8 alpha recombinant protein cannot be prepared, and scientific reports about whether the flounder CD8 alpha and CD8 beta peptide chains are co-expressed on the surface of a cell membrane or whether the flounder CD8 alpha homodimer exists are lacking. Monoclonal antibody for identifying CD8 alpha molecule of paralichthys olivaceus prepared by Korean scholars by using polypeptide and identifying 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.).
Paralichthys olivaceus (L.) kuntzeParalichthys olivaceus) Is important in the north of China, and frequently-occurring aquaculture diseases in recent years are important factors for preventing the development of the aquaculture industry. Many studies and practices have shown that vaccines are an effective means of controlling aquatic diseases. The action of the vaccine depends on the activation of the adaptive immune mechanism of the organism, in which process T cells play an important role. Thus, the development of the Paralichthys olivaceus CD8 alpha + Specific antibodies to marker molecules of T lymphocytes, useful for identifying CD8 alpha at the cellular level + T lymphocyte subpopulation for detecting flounder CD8 alpha + The number of T lymphocyte subpopulations varied and cellular immune response levels were assessed.
Disclosure of Invention
The first object of the invention is to provide a flounder T lymphocyte surface marker molecule CD8 alpha polyclonal antibody which can specifically identify the flounder CD8 alpha + T lymphocyte subpopulation for the study of Paralichthys olivaceus CD8 alpha + The function of T lymphocyte subsets provides an important immunological tool.
The second object of the invention is to provide a preparation method of the flounder T lymphocyte surface marker molecule CD8 alpha polyclonal antibody.
The third object of the invention is to provide a reagent for preparing and identifying the flounder T cell subset by using the flounder T lymphocyte surface marker molecule CD8 alpha polyclonal antibody, or preparing the flounder CD8 + Use of T lymphocyte immune response research reagents.
The invention aims at realizing the following technical scheme:
an epitope peptide of a flounder T lymphocyte surface marker molecule CD8 alpha, which is characterized in that the amino acid sequence of the epitope peptide is as follows: 134 APRVIASTRSPSLT 147 。
a polyclonal antibody of CD8 alpha as the marker molecule of T lymphocyte of paralichthys olivaceus features that its amino acid sequence is 134 APRVIASTRSPSLT 147 CD8 alpha antigen table of paralichthys olivaceusThe polypeptide-KLH complex is obtained by immunizing New Zealand white rabbits and purifying serum.
The invention relates to a flounder CD8 alpha epitope peptide 134 APRVIASTRSPSLT 147 ) The KLH complex is obtained by epitope screening by analysis of the CD8 alpha amino acid sequence.
The indirect immunofluorescence experiment shows that the polyclonal antibody against the paralichthys olivaceus CD8 alpha molecule provided by the invention can react with HEK293 cells transfected with eukaryotic plasmids of the paralichthys olivaceus CD8 alpha, and a bicolor fluorescence signal appears, which indicates that the antibody can react with natural CD8 alpha molecules of the paralichthys olivaceus.
The indirect immunofluorescence experiment shows that the anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody provided by the invention can react with the paralichthys olivaceus head and kidney lymphocytes, and red fluorescence signals are distributed on the surface of cell membranes, which is consistent with the fact that CD8 alpha proteins are mainly distributed on the surface of T lymphocytes.
The anti-paralichthys olivaceus CD8 alpha molecular polyclonal antibody provided by the invention can react with HEK293 cells transfected with the eukaryotic plasmids of paralichthys olivaceus CD8 alpha through immunoblotting reaction, shows a specific band at 51kDa, and is consistent with the molecular weight of the protein of the paralichthys olivaceus CD8 alpha plus a tag protein. The rabbit polyclonal antibody reacts with the flounder head and kidney lymphocytes, shows a specific band at 26kDa, and has the same molecular weight as the natural CD8 alpha protein of the flounder.
The anti-paralichthys olivaceus CD8 alpha molecule polyclonal antibody provided by the invention detects peripheral blood, spleen and head kidney lymphocytes through flow cytometry, and finds that the antibody can be specifically combined with part of the paralichthys olivaceus lymphocytes, and the detection result shows that CD8 alpha in the peripheral blood lymphocytes of the paralichthys olivaceus + The ratio of the number of T lymphocytes is 5.6+/-0.8 percent, and CD8 alpha in spleen lymphocytes + The ratio of the number of T lymphocytes is 14.8+/-1.6 percent, and CD8 alpha in the head and kidney lymphocytes + The ratio of the number of T lymphocytes was 19.5.+ -. 0.9%.
The polyclonal antibody of the flounder T lymphocyte surface marker molecule CD8 alpha provided by the invention is used for preparing and identifying the reagent of the flounder T cell subgroup or preparing the flounder CD8 + Use of T lymphocyte immune response research reagents.
The advantages of the inventionThe point is that: the invention obtains the flounder CD8 alpha molecular epitope polypeptide with good antigenicity through screening, and prepares the highly specific anti-flounder CD8 alpha molecular polyclonal antibody by using the same, in order to identify CD8 alpha from cell level + T lymphocyte subpopulation for detecting flounder CD8 alpha + The number of T lymphocyte subpopulations varies, providing an important tool for assessing cellular immune response levels.
Drawings
FIG. 1 is an analysis chart of the epitope of the CD8 alpha molecule of the paralichthys olivaceus.
In the figure, the dashed rectangle is the score of the epitope with the X-axis being the amino acid position and the Y-axis being the amino acid position of the 134-147 amino acid sequence with higher scores.
FIG. 2 is an immunofluorescence of the eukaryotic plasmid transfected HEK293 cells with the GFP tagged Paralichthys olivaceus CD 8. Alpha. As a marker.
In the figure, A is GFP green fluorescent protein, B is differential interference diagram, and C is superposition diagram of A and B.
FIG. 3 is an indirect immunofluorescence of anti-Paralichthys olivaceus CD8 alpha rabbit polyclonal antibody and HEK293 cells transfected with Paralichthys olivaceus CD8 alpha eukaryotic plasmid.
In the figure, A is DAPI lining cell nucleus, B is GFP green fluorescent protein, C is red positive fluorescent signal displayed by incubating anti-CD 8 alpha rabbit polyclonal antibody, and D is superposition diagram of A, B and C; e is DAPI-stained nuclei, F is GFP green fluorescent protein, G is incubation rabbit negative serum as a control, and H is a superposition of E, F and G.
FIG. 4 is an indirect immunofluorescence of the anti-Paralichthys CD 8. Alpha. Rabbit polyclonal antibody reacted with Paralichthys olivaceus head kidney leucocytes.
In the figure, A is a red positive fluorescent signal displayed by incubating anti-CD 8 alpha rabbit polyclonal antibody, B is DAPI-lined cell nucleus, and C is a superposition diagram of A and B; d is incubation rabbit negative serum as control, E is DAPI-lined nuclei, and F is a superposition 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 leucocytes.
In the figure, M is Marker, lane 1 is immunoblotting result of reaction of anti-flounder CD8 alpha rabbit polyclonal antibody and HEK293 cells transfected with flounder CD8 alpha eukaryotic plasmid, lane 2 is negative control, lane 3 is immunoblotting result of reaction of anti-flounder CD8 alpha rabbit polyclonal antibody and flounder head kidney leucocyte, and lane 4 is negative control.
FIG. 6 shows the detection of CD8 alpha in peripheral blood, spleen and head and kidney lymphocytes by anti-Paralichthys olivaceus CD8 alpha rabbit polyclonal antibody + Flow chart of T lymphocytes.
Detailed Description
In order to more clearly explain the technical solution of the invention, the invention is further described below by means of specific embodiments in conjunction with the accompanying drawings.
Example 1: screening and synthesis of flounder CD8 alpha molecular antigen epitope peptide
According to the NCBI published CD8 alpha gene sequence, after translation into amino acid sequence, the on-line prediction software of IEDB (http:// tools. Immuneepsilon. Org/bcell /) is used for analyzing the CD8 alpha molecular epitope of the Paralichthys olivaceus (figure 1), and parameters such as hydrophilicity, flexibility, antigenicity, surface accessibility and the like are synthesized, and finally the polypeptide chain is selected as 134 APRVIASTRSPSLT 147 Polypeptide is synthesized and hemocyanin (KLH) is coupled in Nanjing Jinsri biological limited company, and the purity is up to more than 95 percent through mass spectrum verification.
Example 2: construction of prokaryotic and eukaryotic plasmids for expressing flounder CD8 alpha molecules
(1) Based on NCBI published sequence of the flounder CD 8. Alpha. Gene, the flounder CD 8. Alpha. Gene was synthesized and ligated to pTag-GFP plasmid.
(2) And (3) transfecting the successfully connected pTag-GFP-CD8 alpha plasmid into HEK293 cells, and observing green fluorescence to judge the expression effect of the flounder CD8 alpha molecule.
Results: eukaryotic plasmids successfully constructed from the flounder CD8 alpha molecules, and after transfection of cells, showed green fluorescent proteins (see FIG. 2), indicating that the flounder CD8 alpha molecules were successfully expressed in HEK293 cells.
Example 3: preparation of anti-paralichthys olivaceus CD8 alpha molecule rabbit polyclonal antibody
(1) The synthesized paralichthys olivaceus CD8 alpha polypeptide-KLH complex is used as an antigen to immunize New Zealand white rabbits, the immunization dose is 0.6mg each time, the immunization is carried out for 4 times, the immunization interval of the first 2 times is 2 weeks, and the immunization interval of the last 2 times is 1 week.
(2) Basic immunization: the CD8 alpha polypeptide-KLH complex was mixed with Freund's complete adjuvant in equal amounts (V/V) as antigen and injected subcutaneously at 6 points on the back.
(3) Boosting: the CD8 alpha polypeptide-KLH complex was mixed with Freund's incomplete adjuvant in equal amounts (V/V) as antigen and injected subcutaneously at 6 points on the back.
(4) Secondary boost: CD8 alpha polypeptide-KLH complex (600 μl) was used as antigen for otic margin intravenous injection.
(5) Boosting again: CD8 alpha polypeptide-KLH complex (600 μl) was used as antigen for otic margin intravenous injection.
(6) Five days after the last immunization, heart blood collection was performed. The collected blood was allowed to stand obliquely at room temperature for 2 hours overnight in a refrigerator at 4 ℃. The next day, the centrifuge tube was centrifuged at 4℃for 10min under 8000G conditions, the supernatant was carefully aspirated, and the supernatant was purified by protein G affinity chromatography, and the purified product was rabbit anti-Paralichthys CD 8. Alpha. Molecule polyclonal antibody.
Results: after four times of immunization, the purified rabbit serum is the rabbit anti-paralichthys olivaceus CD8 alpha molecule polyclonal antibody.
Example 4: indirect immunofluorescence method for identifying specificity of flounder CD8 alpha molecule rabbit polyclonal antibody
(1) Selecting healthy Paralichthys olivaceus (weight 0.7+ -0.2 kg) temporarily raised for one week, taking head and kidney after anesthesia by MS-222, gently grinding into single cell suspension, centrifuging at 100×g for 5min to remove red blood cells, and performing discontinuous density gradient (1.02/1.07 g/cm) 3 ) Centrifuging to obtain lymphocytes of the head and kidney of the paralichthys olivaceus. Simultaneously collecting HEK293 cells transfected with the paralichthys olivaceus CD8 alpha eukaryotic plasmid, diluting the extracted paralichthys olivaceus head kidney lymphocytes and HEK293 cells with sterile PBS buffer solution to obtain the cell density of 1 multiplied by 10 6 cell suspension of cells/ml.
(2) Lymphocyte and HEK293 cell suspensions were added dropwise to a clean glass slide, 10 μl of each drop, and after 1 hour of sedimentation in a wet box at room temperature, taken out and put into acetone for fixation for 20 minutes, taken out and air-dried.
(3) Anti-paralichthys olivaceus CD8 alpha molecule rabbit polyclonal antibody is taken as a first antibody (diluted 1:1000), and is dripped on a cell sample of a glass slide, and incubated for 1.5h in a 37 ℃ wet box.
(4) The slides were removed, washed three times with PBST for 5 minutes each, with Cy 3-labeled goat anti-rabbit Ig antibody as secondary antibody (1:1000 dilution), added dropwise to the cell samples, and incubated in a 37℃wet box for 45 minutes.
(5) The slides were removed, washed three times with PBST for 5 minutes each, and after staining nuclei with DAPI for about 10 minutes, the slides were glycerol blocked, observed under a fluorescence microscope and photographed.
Results: the rabbit polyclonal antibody provided by the invention is specifically combined with HEK293 cells with GFP labels, presents red fluorescent signals, and overlaps green and red fluorescent signals, which indicates that the prepared rabbit polyclonal antibody can be specifically combined with HEK293 cells expressing flounder CD8 alpha protein, namely, can specifically react with flounder natural CD8 alpha protein (see figure 3). The rabbit polyclonal antibody of the invention specifically binds with the lymphocyte surface of a part of paralichthys olivaceus, presents fluorescence positive signals, and can see the distribution of red fluorescence signals on the cell membrane surface under a 100-time oil mirror, which is consistent with the fact that CD8 alpha protein is distributed on the surface of T lymphocytes, and no fluorescence signals are observed in negative control (see figure 4).
Example 5: identification of specificity of flounder CD8 alpha molecule rabbit polyclonal antibody by immunoblotting
(1) HEK293 cells transfected with the flounder CD8 alpha eukaryotic plasmid are collected, meanwhile, the flounder head and kidney lymphocytes are extracted for gel electrophoresis, and then, the protein on the gel is transferred to a nitrocellulose membrane.
(2) Nitrocellulose membranes were blocked in 3% bovine serum albumin solution (PBS formulation) for 1h at 37 ℃.
(3) The nitrocellulose membrane was rinsed 3 times with PBST for 5 minutes each, placed in polyclonal antibody against the flounder CD 8. Alpha. Molecule (10. Mu.l antibody added to 10mL PBS), slowly shaken at 37℃for 1.5 hours, and incubated with rabbit negative serum as a negative control.
(4) The PBST was rinsed 3 times for 5 minutes each, and the nitrocellulose was added to horseradish peroxidase-labeled goat anti-rabbit Ig antibody (1:4000 dilution) and slowly shaken at 37℃for 1 hour.
(5) The nitrocellulose membrane was rinsed 3 times for 5 minutes each time by PBST, and the nitrocellulose membrane was put into a color developing solution to develop color, and the result was recorded by photographing with a chemiluminescent apparatus.
Results: the rabbit polyclonal antibody reacts with HEK293 cells transfected with the flounder CD8 alpha eukaryotic plasmid, shows a specific band at 51kDa, and is consistent with the molecular weight of the flounder CD8 alpha protein plus a tag protein. The rabbit polyclonal antibody of the invention reacts with the flounder head and kidney lymphocytes, shows a specific band at 26kDa, and has the same molecular weight as the natural CD8 alpha protein of the flounder (see figure 5).
Example 6: flow cytometry detection of flounder CD8 alpha + Proportion of T lymphocytes
(1) Healthy paralichthys olivaceus (weight 0.7+/-0.2 kg) temporarily raised for one week is selected, and after anesthesia by MS-222, peripheral blood of the paralichthys olivaceus is extracted from tail veins, and spleen and head and kidney are taken. Using a Percoll discontinuous density gradient (1.02/1.07 g/cm) 3 ) Centrifuging to obtain lymphocyte of the peripheral blood, spleen and head kidney of the paralichthys olivaceus. Diluting the extracted lymphocyte of each tissue of Paralichthys olivaceus with sterile PBS buffer to obtain cell density of 1×10 7 cell suspension of cells/ml.
(2) Two 500. Mu.l lymphocyte suspensions were taken from each tissue sample, one as a test sample and one as a control sample, 1. Mu.l polyclonal antibody against the flounder CD 8. Alpha. Molecule was added to the test sample, 1. Mu.l rabbit negative serum was added to the control group, and incubated at 37℃for 1.5h.
(3) Cells were collected by centrifugation at 680g at 4℃and washed three times for 5 minutes with sterile PBS.
(4) Mu.l of FITC-labeled goat anti-rabbit Ig mab was added to the resuspended cells and incubated at 37℃for 1h.
(5) Cells were collected by centrifugation at 680g at 4℃and washed three times for 5 minutes with sterile PBS.
(6) After the centrifugation wash was completed, the cells were resuspended in 500 μl sterile PBS for flow cytometry analysis.
Results: the histogram of the flow analysis of each tissue negative control sample shows a single peak, has no positive reaction cell population, and shows a double peak with the flow analysis histogram of each tissue lymphocyte of the paralichthys olivaceus incubated with the rabbit polyclonal antibody, which shows that the monoclonal antibody can be specifically combined with part of lymphocyte of the paralichthys olivaceus, and the detection result shows that CD8 alpha in the peripheral blood lymphocyte of the paralichthys olivaceus + The ratio of the number of T lymphocytes is 5.6+/-0.8 percent, and CD8 alpha in spleen lymphocytes + The ratio of the number of T lymphocytes is 14.8+/-1.6 percent, and 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 (2)
1. An epitope peptide of a flounder T lymphocyte surface marker molecule CD8 alpha, which is characterized in that the amino acid sequence of the epitope peptide is as follows: APRVIASTRSPSLT.
2. The use of the epitope peptide according to claim 1 for preparing polyclonal antibody of flounder T lymphocyte surface marker molecule CD8 alpha.
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