Sheep scavenger receptor A antigen polypeptide as well as polyclonal antibody, preparation method and application thereof

Abstract

The invention belongs to the technical field of immunology, and particularly relates to a sheep scavenger receptor A antigen polypeptide and a polyclonal antibody, a preparation method and application of the sheep scavenger receptor A antigen polypeptide, the amino acid sequence of the sheep scavenger receptor A antigen polypeptide is KSLDEKQVHLEQELKG, the rabbit-derived SRA polyclonal antibody is prepared from the antigen polypeptide, the rabbit-derived SRA polyclonal antibody has good reactivity and specificity, and the rabbit-derived SRA polyclonal antibody can be used for preparing the rabbit-derived SRA polyclonal antibody. The kit can be used for detecting the sheep SRA protein.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to sheep scavenger receptor A antigen polypeptide and its polyclonal antibody, preparation method and application. Background Art

[0002] Scavenger receptor A (SRA), also known as macrophage scavenger receptor 1 (MSR1) or CD204, belongs to a class of pattern recognition receptors that are mainly expressed in macrophages. Scavenger receptor A is a member of the scavenger receptor family. SRA exists in three types: type I, type II, and type III, which are derived from alternative splicing of the same gene. Type I and type II isoforms are functional receptors that can mediate the endocytosis of low-density lipoproteins, while type III isoforms are not embedded in the membrane and have no uptake function. However, when they are co-expressed, type III isoforms can inhibit the functions of type I and type II isoforms, suggesting a regulatory mechanism for scavenger receptor activity in macrophages.

[0003] The macrophage scavenger receptor was first discovered in 1979. Subsequently, the scavenger receptor was classified according to its structure, and together with several other scavenger receptors that appeared later, it formed the scavenger receptor family. The scavenger receptor family includes 12 different subclasses (AL). SRA was first identified, and a coiled coil region, a collagen-like region and a C-terminal globular domain form a trimer structure. The collagen region is the main site for ligand binding. The structures of each member of SRA are relatively similar, generally consisting of an N-terminal cytoplasmic region, a transmembrane helix and a C-terminal extracellular part. The main difference between the three different isomers of SRA protein is that type I molecules have a cysteine-rich α-helical coiled domain, while type II lacks a cysteine-rich C-terminal region. Although the structures of these two isomers are slightly different, their functions are basically the same, while type III is trapped in the endoplasmic reticulum and has no function of mediating phagocytosis.

[0004] Currently, the SRA genes of humans, pigs, etc. have been cloned and identified, but there are fewer studies on SRA in sheep, and there is a lack of antigenic peptides for preparing antibodies. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides sheep scavenger receptor A antigen polypeptide and its polyclonal antibody, preparation method and application.

[0006] A sheep scavenger receptor A antigen polypeptide, the amino acid sequence of the antigen polypeptide is KSLDEKQVHLEQELKG.

[0007] A method for preparing polyclonal antibodies against scavenger receptor A comprises the following steps: immunizing an animal with a conjugate of hemocyanin and the antigen polypeptide according to claim 1 as an immunogen, and obtaining polyclonal antibodies against scavenger receptor A from the animal.

[0008] Preferably, the antigen polypeptide is linked to cysteine ​​and then coupled to hemocyanin to obtain a complete antigen;

[0009] emulsifying the complete antigen, and immunizing rabbits with the emulsified complete antigen;

[0010] The serum of the immunized rabbits was collected to obtain the scavenger receptor A polyclonal antibody.

[0011] Preferably, the method of immunizing rabbits is to immunize once every two weeks after the first immunization, for a total of 7 immunizations.

[0012] Preferably, the first immunization dose is 450 μg to 500 μg per mouse, and the subsequent immunization doses are 200 μg to 300 μg per mouse.

[0013] Preferably, the blood of rabbits containing the scavenger receptor A polyclonal antibody is collected, left to stand at 4° C. for 12 h to 24 h, and then separated to obtain serum, thereby obtaining the scavenger receptor A polyclonal antibody.

[0014] Preferably, the coupled scavenger receptor A antigen polypeptide is dissolved in 1×PBS, mixed with Freund's complete adjuvant, and emulsified on ice using an emulsifier.

[0015] A sheep scavenger receptor A polyclonal antibody is prepared by the preparation method.

[0016] The invention discloses an application of the sheep scavenger receptor A polyclonal antibody in preparing a reagent for detecting sheep scavenger receptor A protein.

[0017] SRA is a type II transmembrane glycoprotein that forms a homotrimer on the cell surface. Cell membrane surface proteins play an important role in cell signal transduction, substance transport, and cell-to-cell interactions. SRA has a very short cytoplasmic tail and no identifiable signaling domain, so it is believed to work by forming a multi-molecular signaling complex. However, it cannot be ruled out that SRA directly transduces signals through its cytoplasmic tail. Human SRA has a 50-amino acid cytoplasmic tail, and mice have 55 amino acids. In silico analysis of the SRA protein sequence showed that there is a conserved serine residue in humans and mice. According to UniProt, this residue can be phosphorylated, suggesting a downstream phosphorylation cascade.

[0018] In order to detect the expression of sheep SRA, the present invention predicts the antigenic epitope of SRA through multiple software, and avoids the appearance of phosphorylation sites, glycosylation sites, etc. on the antigenic epitope. The antigenic polypeptide cannot be too long, generally 12AA to 15AA. If it is too long, the cost will increase, and if it is too short, the antibody specificity will be affected. The dominant antigenic epitope is screened out and coupled with hemocyanin to immunize New Zealand white rabbits and Kunming mice to prepare polyclonal antibodies against SRA. The use of synthetic peptides to immunize animals to produce antibodies improves the specificity of antibodies against the target antigen. The combination of antigenic polypeptides and carrier proteins is one of the keys to the successful preparation of antibodies. The antigenic polypeptide is coupled with hemocyanin (KLH) to enhance its immunogenicity, and KLH is far away from mammalian proteins in phylogeny, which reduces the generation of false positives in the preparation of antibodies by immunizing mammals. It is worth noting that the animals should be fed normally for 1 week before immunization, and their mental and dietary status should be observed to ensure that the animals are healthy before immunization. The effectiveness of immunization and animal welfare are the keys to obtaining antiserum.

[0019] In order to detect the expression of sheep scavenger receptor A protein, SRA antigen peptides were prepared by chemical synthesis and immunized New Zealand white rabbits and Kunming mice to prepare polyclonal antibodies. The titer was detected by indirect ELISA, and the specificity was detected by IFA and Western blot.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention successfully prepared polyclonal antibodies of rabbit SRA and mouse SRA. The results of indirect ELISA showed that both mouse SRA and rabbit SRA had good reactivity, and the rabbit SRA antibody titer exceeded 10 5 , the polyclonal antibody can also be used for IFA specificity verification, and fluorescent positive cells can be observed in cells overexpressing SRA. Therefore, the preparation of antibodies for antigenic peptides should be mainly based on immunizing rabbits, and the antibody prepared by immunizing mice has a slightly weaker reactivity. The preparation of polyclonal antibodies against SRA can not only be used to detect the expression of SRA, but is also very important for the functional study of SRA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the comparison and analysis of SRA antigen peptide sequences;

[0023] Figure 2 It is the conservation analysis of SRA antigen peptide sequence;

[0024] Figure 3 IFA analysis of rabbit-derived SRA polyclonal antibody;

[0025] Figure 4 It was shown that the rabbit-derived SRA antibody specifically recognized the overexpressed SRA protein;

[0026] Figure 5 The results show that the expression of SRA protein in sheep alveolar macrophages was detected using rabbit antibodies. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0028] The present invention uses two healthy 6-month-old male New Zealand white rabbits (weight 3 kg) and 6 6-8-week-old female Kunming mice.

[0029] DH5α competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; eukaryotic expression vector pcDNA3.1(+) was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; CHO cells were provided by the clinical veterinary laboratory repository of Shihezi University. The sources of the main reagents are shown in Table 1.

[0030] Table 1 Sources of main reagents

[0031] Reagent name Part Number Manufacturer Reagentname CatalogNumber Productioncompany DAPI stain C0065 Beijing Solable Technology Co., Ltd. Opti-MEM medium 11058021 Invitrogen F12K medium 21127022 Gibco USA Freund's complete adjuvant F5881 Sigma Freund's incomplete adjuvant F5506 Sigma HRP-goat anti-rabbit IgG E030120-01 Earthox USA HRP-goat anti-rabbit fluorescent antibody SA2113 Wuhan Costan Biotechnology Co., Ltd. 1× PBS P1020 Beijing Solable Technology Co., Ltd.

[0032] Example 1

[0033] Prediction of Sheep SRA Antigen Epitopes

[0034] NovoFocusTM antigen prediction software was used to predict the antigen of sheep SRA ORF. The website of NovoFocusTM antigen prediction software is https: / / www.novopro.cn / tools / peptide-antigen-desi gn.html. The final selected antigen peptide sequence is: KSLDEKQVHLEQELKG. When used, the antigen peptide is connected to a cysteine ​​for coupling with hemocyanin, that is, KSLDEKQVHLEQELKG-cys. SRA consists of 290 amino acid sequences, and the selected antigen peptide is located at the amino acid sequence of positions 138-145. It was sent to Shanghai Newpro Biotechnology Co., Ltd. for antigen peptide synthesis, coupled with hemocyanin (KLH) using the Fmoc solid phase method, and purified and identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS).

[0035] result

[0036] The online software SVMTriP was used to predict the antigenic epitopes of sheep SRA (Table 2). The dominant antigenic determinants are between 138 and 240. Immunizing animals with antigens prepared in the SRA (138-145) region may produce antibodies with higher specificity.

[0037] Table 2 Prediction of SRA antigen peptides

[0038] sequence Score Sequence region Sequence number KSLDEKQVHLEQELKG 23 138-145 SEQ ID NO.1 SLDEKQVHLEQELKGE 22.6 227-242 SEQ ID NO.2 MKSLDEKQVHLEQELK 21.6 225-240 SEQ ID NO.3

[0039] The conservation analysis of sheep SRA antigen peptides was carried out by comparing with the sequences of cattle, pigs, goats, humans, etc. The results are as follows Figure 1 and Figure 2 As shown, it was found that the sheep SRA antigen polypeptide was completely consistent with that of goats, and the homology with cattle, pigs, and humans was 87.5%, 75%, and 62.5%, respectively; the SRA antigen epitope was relatively conservative, and the SRA antibody prepared with this antigen could be used for goat SRA protein detection, and it is speculated that it can be used for cattle SRA protein detection.

[0040] Example 2

[0041] Preparation of SRA polyclonal antibody, the specific steps are:

[0042] Before the first immunization, blood was collected from the ear vein of rabbits and from the tails of mice, and serum was used as a negative control for indirect ELISA.

[0043] The coupled SRA antigen polypeptide lyophilized powder was dissolved in sterile 1×PBS, mixed with Freund's complete adjuvant in a 1:1 ratio, emulsified on ice with an emulsifier, and injected subcutaneously at multiple points into rabbits and Kunming mice for the first immunization. The amount of antigen polypeptide injected into rabbits was 500 μg / mouse, and the amount of antigen polypeptide injected into Kunming mice was 100 μg / mouse.

[0044] After that, boost immunization every 14 days: dissolve the coupled SRA antigen polypeptide in 1×PBS, mix it with Freund's incomplete adjuvant in a 1:1 ratio, emulsify it with an emulsifier on ice, and inject it subcutaneously into rabbits and Kunming mice for immunization. The amount of antigen polypeptide injected into rabbits is 250μg / mouse, and the amount of antigen polypeptide injected into Kunming mice is 50μg / mouse. Blood was collected from rabbits and mice on the seventh day after the 7 immunizations. Rabbits were blooded by heart, and mice were blooded by removing eyeballs. The collected whole blood was first placed at 37°C for 30 minutes, and then placed at 4°C overnight to precipitate serum. The horizontal speed was centrifuged at 3000r for 10 minutes, and the serum was collected and stored in 100μL / tube at -80°C to obtain rabbit SRA polyclonal antibodies and mouse SRA polyclonal antibodies, respectively.

[0045] Example 3

[0046] SRA polyclonal antibody titer analysis

[0047] Detect antibody titer using indirect ELISA:

[0048] (1) Dilute the antigen peptide with coating solution, add 100 μL to each well (antigen peptide 1 μg / well), apply sealing film, and place at 4°C overnight;

[0049] (2) Pour off the coating solution, block with 5% BSA at room temperature for 2 h (100 μL / well), pour off the blocking solution, wash with 300 μL PBST per well, wash three times, and spin dry;

[0050] (3) Dilute the serum to be tested with PBS according to the proportion (100 μL / well), react at 37°C for 30 min, wash with PBST three times, and spin dry;

[0051] (4) Dilute the secondary antibodies: HRP-goat anti-rabbit (1:10000) and HRP-goat anti-mouse (1:5000), 100 μL per well, incubate at 37°C for 25 min, wash three times with PBST at the end of the incubation, and spin dry;

[0052] (5) Add TMB colorimetric solution and gently shake to mix. Incubate at 37°C in the dark for 10 min (turn blue). Add 2N concentrated sulfuric acid stop solution in the same proportion (turn yellow). Measure OD with an enzyme reader. 450 value.

[0053] (6) Determine the antibody titer: If the OD of the well to be tested is 450 >0.1, and positive serum OD 450 / Negative serum OD 450 If >2.1, it is judged as positive, and the highest dilution multiple of the positive serum is the serum antibody titer.

[0054] The results of indirect ELISA showed that the antibody titer of mouse SRA was 10 2 As shown in Table 3, the antibody titer of rabbit SRA exceeds 10 5 , as shown in Table 4, indicating that mouse and rabbit SRA polyclonal antibodies are reactive with the antigenic polypeptide.

[0055] Table 3 Mouse SRA antibody titer detection

[0056]

[0057] Table 4 Rabbit SRA antibody titer detection

[0058]

[0059] Example 4

[0060] Indirect Immunofluorescence (IFA) Analysis:

[0061] (1) Cell culture slides: When CHO cells reach the fourth generation, the cells are digested with trypsin, added to a 12-well plate, inoculated onto the slides, and cultured in a cell culture incubator at 37°C and 5% CO2.

[0062] (2) Transfection: After 24 hours of cell attachment, transfect CHO cells with pcDNA3.1-MSR1 plasmid, and transfect empty vector as a control, with 3 replicates per group. Dilute 3 μg pcDNA3.1-MSR1 with 320 μL Opti-MEM medium and mix well. Dilute 9 μL liposome nucleic acid transfection reagent with 320 μL Opti-MEM medium, incubate at room temperature for 5 minutes, mix the diluted plasmid DNA with liposome nucleic acid transfection reagent, shake gently to mix, incubate at room temperature for 20 minutes, add 200 μL of the premixed solution to each well of the cells, and culture in a cell culture incubator at 37°C and 5% CO2. After 5 hours of transfection, replace with F12K medium containing 1% fetal bovine serum. After 24 hours of transfection, discard the medium and wash with PBS 3 times, 5 minutes each time.

[0063] (3) Fixation: Fix with 4% paraformaldehyde at room temperature for 15 min, and wash with PBS three times, 5 min each time.

[0064] (4) Permeabilization: 1 mL of 0.2% Triton x-100 per well, permeabilize for 10 min at room temperature, and wash three times with PBS, each time for 5 min.

[0065] (5) Blocking: Block with 2% BSA at room temperature for 30 min; wash with PBS three times, 5 min each time.

[0066] (6) Antibody incubation: Rabbit SRA polyclonal antibody was diluted with PBS (1:100) and incubated at room temperature for 1 h. Goat anti-rabbit fluorescent secondary antibody was diluted with PBS (1:400) and incubated at room temperature in the dark for 50 min. Wash with PBS three times, 5 min each time.

[0067] (7) Staining and sealing: Add DAPI ready-to-use staining solution and stain for 10 min at room temperature in the dark. After sealing with sealing agent, observe and photograph under a fluorescence microscope.

[0068] The specificity of rabbit SRA polyclonal antibody was tested by IFA. The results showed that compared with the empty vector transfected cell samples, only the SRA overexpressing cells had fluorescent positive cells, such as Figure 3 , rabbit SRA polyclonal antibody can specifically detect cells overexpressing SRA.

[0069] Western blot analysis

[0070] 293t cells were transfected with pCDNA3.1-MSR1 plasmid for 24 hours, and protein expression was detected by western blot.

[0071] (1) Sample preparation: The collected cell samples were added to RIPA cell lysis buffer containing 1% protease inhibitors, 10 μL per well of a 96-well plate cell and 150 μL per 25 mg of tissue. The tissue was minced and added to the lysis buffer and homogenized using a tissue homogenizer. The tissue was lysed on ice for 20 min, and oscillated every 5 min to ensure full lysis. The sample was centrifuged at 12,000 r / min and 4°C for 10 min. The supernatant was collected and added to 5× SDS PAGE Loading Buffer, and boiled in boiling water for 10 min.

[0072] (2) Protein gel preparation: Assemble a 1.0 mm gel glass plate, fill it with distilled water to check for leaks, if not, drain the water, and prepare stacking gel and separation gel according to the instructions of the One step PAGE gel rapid preparation kit (10%). Stacking gel preparation: 0.5 mL Stacker A, 0.5 mL Stacker B, APS 10 μL; separation gel preparation: 2.5 mL Stacker A, 2.5 mL Stacker B, APS 40 μL.

[0073] (3) Electrophoresis: Assemble the gel and electrophoresis tank, add SDS-PAGE electrophoresis buffer to cover the electrode wires, pull the comb vertically upward, spot the sample, and add 10 μL of protein marker as a molecular size indicator. Connect the power supply to 80 V constant voltage until the marker band begins to separate. Adjust the voltage to 120 V constant voltage and continue electrophoresis for about 70 minutes. Stop the electrophoresis when the marker runs away completely at 50 kDa to 70 kDa.

[0074] (4) Protein immunoblotting: Measure the length and width of the gel, cut an NC membrane with the same length and width as the gel and 6 filter papers slightly larger than the length and width of the gel, and soak them in 1× transfer solution. The assembly order is: positive electrode-sponge-3 layers of filter paper-gel-membrane-3 layers of filter paper-sponge-negative electrode. Remove the bubbles. After assembly, close the transfer clamp and place it in the full wet transfer tank. Add enough transfer solution and transfer ice pack, cover the lid, connect the power supply, and use 300mA constant current transfer for 70 minutes to detect the size of SRA protein. After the transfer is completed, remove the membrane.

[0075] (5) Blocking: Prepare a blocking solution containing 5% skimmed milk powder in 1× TBST. Completely immerse the transferred membrane in the blocking solution and block at low speed for 2 h at room temperature. Carefully pour off the blocking solution and wash the membrane four times with 1× TBST at high speed for 5 min each time.

[0076] (6) Antibody incubation: Primary antibody incubation: Rabbit SRA polyclonal antibody diluted 1:1000 in 1×TBST, incubated overnight at 4°C, and the membrane was washed four times with 1×TBST at high speed, each time for 5 min. Secondary antibody incubation: HRP-goat anti-rabbit diluted 1:10000, incubated at room temperature at low speed for 50 min, and the membrane was washed four times with 1×TBST at high speed, each time for 5 min.

[0077] (7) Exposure: In a dark room, prepare the luminescent solution using the ECL colorimetric luminescence kit, mix solution A and solution B in a 1:1 ratio, add the mixture onto the NC film and color it for 5 min, then expose it using a multifunctional imaging system.

[0078] The results showed that the prepared rabbit anti-SRA antibody could specifically recognize SRA protein, such as Figure 4 shown.

[0079] Using the sheep alveolar macrophage protein sample, the rabbit SRA antibody successfully detected the SRA protein, which was about 55Kda in size. Figure 5 shown.

[0080] The above results show that the rabbit SRA polyclonal antibody has good reactivity and specificity. These research results lay the foundation for future research on the function of sheep SRA.

[0081] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0082] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0083] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A sheep scavenger receptor A antigen polypeptide, characterized in that: The amino acid sequence of the antigen polypeptide is KSLDEKQVHLEQELKG.

2. A method for preparing a scavenger receptor A polyclonal antibody, characterized in that: The following steps are involved: An animal is immunized with a conjugate of hemocyanin and the antigen polypeptide according to claim 1 as an immunogen, and a scavenger receptor A polyclonal antibody is obtained from the animal.

3. The preparation method according to claim 2, characterized in that: The antigen polypeptide is connected to cysteine ​​and then coupled to hemocyanin to obtain a complete antigen; emulsifying the complete antigen, and immunizing rabbits with the emulsified complete antigen; The serum of the immunized rabbits was collected to obtain the scavenger receptor A polyclonal antibody.

4. The preparation method according to claim 3, characterized in that: The method of immunizing white rabbits is to immunize once every two weeks after the first immunization, for 7 times.

5. The preparation method according to claim 4, characterized in that: The first immunization dose was 450 μg to 500 μg per mouse, and the subsequent immunization doses were 200 μg to 300 μg per mouse.

6. The preparation method according to claim 3, characterized in that: The blood of rabbits containing the scavenger receptor A polyclonal antibody is collected, and the blood is allowed to stand at 4° C. for 12 to 24 hours, and the serum is separated to obtain the scavenger receptor A polyclonal antibody.

7. The preparation method according to claim 3, characterized in that: Dissolve the coupled scavenger receptor A antigen polypeptide in 1×PBS, mix with Freund's complete adjuvant, and emulsify it on ice using an emulsifier.

8. A sheep scavenger receptor A polyclonal antibody, characterized in that It is prepared by the preparation method according to any one of claims 2 to 7.

9. Use of the sheep scavenger receptor A polyclonal antibody according to claim 8 in the preparation of a reagent for detecting sheep scavenger receptor A protein.

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