Sheep scavenger receptor a antigen polypeptide and polyclonal antibody, preparation method and application thereof

Abstract

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

Description

TECHNICAL FIELD

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

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

[0003] Macrophage scavenger receptor was first discovered in 1979, and then classified according to the structure of scavenger receptor, and together with other several scavenger receptors that appeared later, it formed the scavenger receptor family. The scavenger receptor family includes 12 different subtypes (A-L). SRA was first identified, and a coiled coil region, a collagen-like region and a C-terminal globular domain form a trimeric structure. The collagen region is the main site for binding to ligands. The structures of each member of SRA are relatively close, and generally consist of an N-terminal cytoplasmic region, a transmembrane helix and a C-terminal extracellular portion. The three different isoforms of SRA protein mainly differ in that the I type molecule has a cysteine-rich alpha-helical coiled domain, and the II type lacks a cysteine-rich C-terminal region. Although the structures of these two isoforms are slightly different, their functions are basically the same, while the III type is trapped in the endoplasmic reticulum and has no phagocytosis function.

[0004] At present, the SRA genes of humans, pigs and the like have been cloned and identified, but the study of SRA in sheep is less, and there is a lack of antigen polypeptide for preparing antibodies. SUMMARY

[0005] To solve the above technical problems, the present application provides a sheep scavenger receptor A antigen polypeptide and a polyclonal antibody thereof, a preparation method and application.

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

[0007] The application discloses a preparation method of scavenger receptor A polyclonal antibody, and comprises the following steps: immunizing animals with a conjugate of hemocyanin and the antigen polypeptide, and obtaining the scavenger receptor A polyclonal antibody from the animals.

[0008] Preferably, the antigen polypeptide is coupled with hemocyanin after being connected with cysteine to obtain a complete antigen.

[0009] The complete antigen is emulsified, and a big white rabbit is immunized with the emulsified complete antigen.

[0010] The serum of the immunized big white rabbit is collected to obtain the scavenger receptor A polyclonal antibody.

[0011] Preferably, the method for immunizing the big white rabbit is to immunize the big white rabbit once every two weeks for 7 times after the first immunization.

[0012] Preferably, the first immunization amount is 450 μg / one to 500 μg / one, and the subsequent immunization amount is 200 μg / one to 300 μg / one.

[0013] Preferably, the blood of the big white rabbit containing the scavenger receptor A polyclonal antibody is collected, and the serum is separated by standing at 4 ℃ for 12 h to 24 h to obtain the scavenger receptor A polyclonal antibody.

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

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

[0016] The sheep scavenger receptor A polyclonal antibody is applied to the preparation of 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, and cell membrane surface proteins play an important role in cell signal transduction, material transport and cell-cell interaction. SRA has a very short cytoplasmic tail and no recognizable signal domain, and is therefore considered to play a role by forming a multi-molecular signal complex. However, it cannot be ruled out that SRA directly transduces signals through its cytoplasmic tail. Human SRA has a cytoplasmic tail of 50 amino acids, and mouse SRA has a cytoplasmic tail of 55 amino acids. In silico analysis of the SRA protein sequence shows that there is a conserved serine residue in humans and mice, which can be phosphorylated according to UniProt, which implies a downstream phosphorylation cascade.

[0018] In order to detect the expression of sheep SRA, the application predicts the antigen epitope of SRA through multiple software, and avoids the presence of phosphorylation sites, glycosylation sites and the like on the antigen epitope, the antigen polypeptide cannot be too long, generally 12 AA~15 AA, and the cost is increased if it is too long, and the antibody specificity is affected if it is too short; the advantage antigen epitope is screened out, coupled with hemocyanin, and New Zealand rabbits and Kunming mice are immunized to prepare polyclonal antibodies against SRA. The use of synthetic peptides to immunize animals to produce antibodies improves the specificity of the antibodies against the target antigen. The combination of antigen polypeptide and carrier protein is one of the keys to the success of antibody preparation, and the coupling of antigen polypeptide and hemocyanin (KLH) enhances its immunogenicity, and KLH is far away from the protein of mammals in phylogenetics, which reduces the generation of false positives in the preparation of antibodies by immunizing mammals. It is worth noting that the animals should be normally fed for one week before immunization, and the mental and dietary states of the animals are observed to ensure the health of the animals before immunization, and the effectiveness of immunization and animal welfare are the keys to obtain antiserum.

[0019] In order to detect the expression of sheep SRA, the application predicts the antigen epitope of SRA through multiple software, and avoids the presence of phosphorylation sites, glycosylation sites and the like on the antigen epitope, the antigen polypeptide cannot be too long, generally 12 AA~15 AA, and the cost is increased if it is too long, and the antibody specificity is affected if it is too short; the advantage antigen epitope is screened out, coupled with hemocyanin, and New Zealand rabbits and Kunming mice are immunized to prepare polyclonal antibodies against SRA. The use of synthetic peptides to immunize animals to produce antibodies improves the specificity of the antibodies against the target antigen. The combination of antigen polypeptide and carrier protein is one of the keys to the success of antibody preparation, and the coupling of antigen polypeptide and hemocyanin (KLH) enhances its immunogenicity, and KLH is far away from the protein of mammals in phylogenetics, which reduces the generation of false positives in the preparation of antibodies by immunizing mammals. It is worth noting that the animals should be normally fed for one week before immunization, and the mental and dietary states of the animals are observed to ensure the health of the animals before immunization, and the effectiveness of immunization and animal welfare are the keys to obtain antiserum.

[0020] Compared with the prior art, the application has the beneficial effects that:

[0021] The application successfully prepares the polyclonal antibodies of rabbit SRA and mouse SRA, the indirect ELISA result shows that the rabbit SRA and the mouse SRA both have good reactivity, the antibody titer of the rabbit SRA is more than 10 5 The polyclonal antibodies can also be applied to IFA specificity verification, and the fluorescent positive cells can be observed in the SRA overexpressed cells. Therefore, the antigen polypeptide for preparing antibodies should be mainly immunized with rabbits, and the reactivity of the antibodies prepared by immunizing mice is slightly weak. The preparation of the SRA polyclonal antibodies can not only be applied to the detection of the expression of SRA, but also is very important for the functional research of SRA. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 SRA antigen polypeptide sequence alignment analysis;

[0023] Figure 2 SRA antigen polypeptide sequence conservation analysis;

[0024] Figure 3 IFA analysis of rabbit SRA polyclonal antibodies;

[0025] Figure 4 The rabbit SRA antibody specifically recognizes the overexpressed SRA protein;

[0026] Figure 5 Results of detecting expression of SRA protein in sheep lung alveolar macrophages using rabbit-derived antibodies are shown. DETAILED DESCRIPTION

[0027] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative work are within the scope of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0028] Two healthy 6-month-old male New Zealand white rabbits (weight 3 kg) and six 6-8 week-old female Kunming mice were used in the present application.

[0029] DH5α competent cells were purchased from Tiangen Biosciences (Beijing) Co., Ltd.; eukaryotic expression vector pcDNA3.1 (+) was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; CHO cells were provided by the Clinical Veterinary Laboratory Repository of Shihezi University. The main reagent sources are shown in Table 1.

[0030] Table 1. Main reagent sources

[0031]

[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, and the website of NovoFocusTM antigen prediction software is https: / / www.novopro.cn / tools / peptide-antigen-design.html. The final selected antigen polypeptide sequence is KSLDEKQVHLEQELKG. When used, the antigen polypeptide is connected with a cysteine to facilitate coupling with hemocyanin, i.e. KSLDEKQVHLEQELKG-cys. SRA is composed of 290 amino acid sequences, and the selected antigen polypeptide is located in the amino acid sequence at positions 138-145. The antigen polypeptide was synthesized by Shanghai Nuopu Biotechnology Co., Ltd., using Fmoc solid phase method, coupled with hemocyanin (KLH), and purified and identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS).

[0035] Results

[0036] The antigen epitope of sheep SRA was predicted by using online software SVMTriP (Table 2), and the regions of 138-240 were dominant antigenic determinants. The region of SRA (138-145) was used to prepare antigen to immunize animals, and the antibodies with high specificity were obtained.

[0037] Table 2 SRA antigen polypeptide prediction

[0038]

[0039] The conservation of sheep SRA antigen polypeptide was analyzed by comparing with the sequences of bovine, porcine, caprine and human, and the results are shown in Tables 2 and 3. Figure 1 Figure 2 It was found that the sheep SRA antigen polypeptide was completely consistent with the caprine, and the homologies with bovine, porcine and human were 87.5%, 75% and 62.5%, respectively. The SRA antibody prepared by the antigen could be used for the detection of caprine SRA protein, and could be used for the detection of bovine SRA protein.

[0040] Example 2

[0041] The SRA polyclonal antibody was prepared, and the specific steps were as follows:

[0042] Before the first immunization, the rabbit was subjected to ear vein blood collection, and the mouse was subjected to tail cutting blood collection, and the serum was used as a negative control of indirect ELISA.

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

[0044] Then, the coupled SRA antigen polypeptide was dissolved with 1×PBS, mixed with Freund's incomplete adjuvant at 1:1, emulsified on ice with an emulsifier, and subcutaneously injected into the rabbit and Kunming mouse for the immunization. The amount of injected antigen polypeptide was 250 μg per rabbit and 50 μg per Kunming mouse. On the seventh day after the end of the seventh immunization, the rabbit and mouse were subjected to blood collection. The rabbit was subjected to heart blood collection, and the mouse was subjected to enucleation blood collection. The collected whole blood was first placed at 37 ℃ for 30 min, and then placed at 4 ℃ overnight for serum separation. The serum was collected by centrifugation at a horizontal speed of 3000 r for 10 min, and stored at -80 ℃ in a 100 μL / tube. The rabbit-derived SRA polyclonal antibody and mouse-derived SRA polyclonal antibody were obtained.

[0045] Example 3

[0046] ​SRA polyclonal antibody titer analysis

[0047] Antibody titer was detected by indirect ELISA:

[0048] (1) Dilute the antigen polypeptide with coating solution, add 100 μL (antigen polypeptide 1 μg / well) per well, seal the plate film, and place at 4°C overnight;

[0049] (2) Discard the coating solution, block with 5% BSA at room temperature for 2 h (100 μL / well), discard the blocking solution, wash each well with 300 μL of PBST, and wash 3 times, then 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, after incubation, wash 3 times with PBST, and spin dry;

[0051] (4) Dilute the secondary antibody: HRP-goat anti-rabbit (1:10000) and HRP-goat anti-mouse (1:5000), 100 μL per well, react at 37°C for 25 min, after incubation, wash 3 times with PBST, and spin dry;

[0052] (5) Add TMB color developing solution, mix gently, react at 37°C for 10 min (turn blue) in the dark; add 2N concentrated sulfuric acid stop solution in the same proportion (turn yellow), and measure the OD value with an enzyme label instrument. 450

[0053] (6) Determine the antibody titer: if the OD 450 of the test well >0.1, and the OD 450 of the positive serum / the OD 450 of the negative serum >2.1, then it is determined to be positive, and the highest dilution factor of the positive serum is the serum antibody titer.

[0054] The results of the antibody titer determination by indirect ELISA showed that the antibody titer of the mouse-derived SRA was 10 2 As shown in Table 3, the antibody titer of the rabbit-derived SRA was more than 10 5 As shown in Table 4, it was shown that the mouse-derived and rabbit-derived SRA polyclonal antibodies had reactivity with the antigen polypeptide.

[0055] Table 3 Detection of mouse-derived SRA antibody titer

[0056]

[0057] Table 4 Detection of rabbit-derived SRA antibody titer

[0058]

[0059] Example 4

[0060] ​Indirect immunofluorescence (IFA) analysis:

[0061] (1) Cell crawling sheet: when CHO cells are transmitted to the fourth generation, the cells are digested with trypsin, and cell crawling sheets are added in a 12-hole plate. The cells are inoculated onto the crawling sheets and cultured at 37°C, 5% CO2 in a cell incubator.

[0062] (2) Transfection: after 24 h of cell adhesion, pcDNA3.1- MSR1 plasmid is transfected into CHO cells, and empty vector is transfected as a control, with 3 repeats in each group. 320 μL Opti-MEM medium is used to dilute 3 μg pcDNA3.1- MSR1 The mixture is mixed, 320 μL Opti-MEM medium is used to dilute 9 μL liposome nucleic acid transfection reagent, and incubated at room temperature for 5 min. The diluted plasmid DNA and liposome nucleic acid transfection reagent are mixed and incubated at room temperature for 20 min. 200 μL of the premixed solution is added to each well of the cells, and the cells are cultured at 37°C, 5% CO2 in a cell incubator. After 5 h of transfection, the medium is replaced with F12K medium containing 1% fetal bovine serum, and after 24 h of transfection, the medium is discarded and washed with PBS for 3 times, 5 min each time.

[0063] (3) Fixation: 4% paraformaldehyde is used for room temperature fixation for 15 min, and PBS is used for washing for 3 times, 5 min each time.

[0064] (4) Permeabilization: 0.2% Triton x-100 is used for 1 mL per well, and permeabilization is performed at room temperature for 10 min, and PBS is used for washing for 3 times, 5 min each time.

[0065] (5) Blocking: 2% BSA is used for room temperature blocking for 30 min; PBS is used for washing for 3 times, 5 min each time.

[0066] (6) Antibody incubation: rabbit SRA polyclonal antibody is diluted with PBS (1:100), and incubated at room temperature for 1 h. Goat anti-rabbit fluorescent secondary antibody is diluted with PBS (1:400), and incubated at room temperature in the dark for 50 min. PBS is used for washing for 3 times, 5 min each time.

[0067] (7) Staining and mounting: DAPI ready-to-use staining solution is added dropwise, and staining is performed at room temperature in the dark for 10 min. After mounting with mounting agent, the sample is observed and photographed under a fluorescence microscope.

[0068] IFA is used to detect the specificity of rabbit SRA polyclonal antibody. The results show that, compared with the cell sample transfected with empty vector, only the cells overexpressing SRA have fluorescent positive cells, such as SRA , and the rabbit SRA polyclonal antibody can specifically detect the cells overexpressing SRA. Figure 3 SRA ​​

[0069] Western blot analysis

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

[0071] (1) Sample preparation: the collected cell samples were added to the cell lysate RIPA containing 1% protease inhibitor, 10 μL per well for 96-well plate cells, 150 μL for each 25 mg of tissue, and the lysate was added to the tissue homogenizer after the tissue was cut into pieces, and homogenized on ice for 20 min, and shaken every 5 min to ensure complete lysis, 12000 r, 4 ℃ centrifugation for 10 min, take the supernatant, add 5×SDS PAGE Loading Buffer, boil in boiling water for 10 min.

[0072] (2) Protein gel preparation: assemble 1.0 mm gel preparation glass plate, fill with distilled water to detect whether it leaks, if not, dry the water, according to the One step PAGE gel preparation kit (10%) instruction manual to prepare concentrated gel and separation gel. Concentrated 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, cover the electrode line, pull out the comb vertically upwards, load the sample, and add 10 μL protein marker as molecular size indicator, connect the power supply 80 V constant voltage until the marker band starts to separate, adjust the voltage to 120 V constant voltage and continue electrophoresis for about 70 min, stop electrophoresis when the marker is completely separated at 50 kDa-70 kDa.

[0074] (4) Western blotting: measure the length and width of the gel, cut a NC membrane with the same length and width as the gel and 6 filter papers larger than the length and width of the gel, soak in 1×transfer solution, assemble in the order of: positive electrode-sponge-3 layers of filter paper-gel-membrane-3 layers of filter paper-sponge-negative electrode, remove air bubbles, assemble the transfer clamp, place it in the fully wet transfer tank, add enough transfer solution, add transfer ice bag, cover the cover, connect the power supply, use 300 mA constant current transfer for 70 min for SRA protein size detection, take out the membrane after transfer.

[0075] (5) Blocking: Blocking solution containing 5% skim milk powder was prepared with 1x TBST, and the transferred membrane was completely immersed in the blocking solution, and blocked at room temperature for 2 hours at low speed. The blocking solution was carefully poured out, and the membrane was washed with 1x TBST at high speed for four times, each time for 5 minutes.

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

[0077] (7) Exposure: In the darkroom, luminescent solution was prepared with ECL color developing luminescent kit, and A liquid was mixed with B liquid at 1:1, and added dropwise to the NC membrane for color development for 5 minutes, and exposed with a multifunctional imaging system.

[0078] The results show that the prepared rabbit anti-SRA antibody can specifically recognize SRA protein, as shown in Figure 4 .

[0079] With sheep alveolar macrophage protein samples, the rabbit SRA antibody was successfully detected to SRA protein, and the size was about 55Kda, as shown in Figure 5 .

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

[0081] It should be noted that when the present application claims involve numerical ranges, it should be understood that each numerical range has two endpoints and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0082] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

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

Claims

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

2. A method for preparing a polyclonal antibody against scavenger receptor A, characterized in that, Includes the following steps: Animals were immunized with a conjugate of hemocyanin and the antigenic polypeptide described in claim 1 as an immunogen to obtain scavenger receptor A polyclonal antibodies from the animals.

3. The preparation method according to claim 2, characterized in that, The antigenic polypeptide is linked to a cysteine ​​residue and then coupled to hemocyanin to obtain a complete antigen. The complete antigen was emulsified, and the rabbits were immunized with the emulsified complete antigen. Serum from 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 for immunizing rabbits is to immunize them every two weeks after the first immunization, for a total of 7 immunizations.

5. The preparation method according to claim 4, characterized in that, The initial immunization dose was 450 μg / animal to 500 μg / animal, and subsequent immunization doses were 200 μg / animal to 300 μg / animal.

6. The preparation method according to claim 3, characterized in that, Blood from rabbits containing the scavenger receptor A polyclonal antibody was collected, and the blood was allowed to stand at 4 °C for 12 h to 24 h to separate serum, thus obtaining the scavenger receptor A polyclonal antibody.

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

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