CN108359007A - Swine erythrocyte ECR1-like film combinations peptide fragment, polyclonal antibody, preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of porcine erythrocyte ECR1-like membrane-bound peptide antibody, and in particular relates to a porcine erythrocyte ECR1-like membrane-bound peptide, polyclonal antibody, preparation method and application. The amino acid sequence of the porcine erythrocyte ECR1-like membrane-bound peptide is shown in SEQ ID NO.1, which is ISANTVILFWFTCLFVQLY, and the corresponding nucleotide sequence is shown in SEQ ID NO.2, which is ata agt gcc aac acg gta atc cta ttc tgg ttt act tgt ctt ttt gtt caa ctatac. The invention successfully prepares the specific polyclonal antibody of the porcine erythrocyte ECR1-like membrane-bound peptide with immunological activity, and provides a research tool for further studying the molecular mechanism of porcine ECR1-like molecular immunology function.

Description

Porcine erythrocyte ECR1-like membrane-bound peptide, polyclonal antibody, preparation method and application

technical field

The invention belongs to the technical field of porcine erythrocyte ECR1-like membrane-bound peptide antibody, and in particular relates to a porcine erythrocyte ECR1-like membrane-bound peptide, polyclonal antibody, preparation method and application.

Background technique

Early studies on immune rosettes measured the rosette rates of C3b receptors and immune complexes on the erythrocytes of Yorkshire × Changbai and Jinhua hybrid pigs, and the average values were 8.14±1.25 and 4.64±0.84 respectively; the red blood cell immune function of Yunnan breed pigs, the C3bRR rate The ICR rate was 17.2±4.20, and the ICR rate was 11.2±3.27. There was no significant difference between the two (P>0.05). Researchers conducted a comparative study on the red blood cell immune function of black-and-white dairy cows, pigs, and Tibetan dogs in Xining area. The results proved that the red blood cell membranes of the three tested animals all had C3bR, and the C3bR rosette rate of pig red blood cells was 4.5±1.08%. The erythrocyte C3b receptor rosette (CbRR) and immune complex rosette (ICR) assays were used to confirm the presence of complement receptors on the cell surface of pigs such as Dahe pigs and Diqing Tibetan pigs. The C3bRR values were 14.87±2.21 and 21.25±3.12, respectively. From the above studies, it can be seen that the concept of red blood cell immune system is also applicable to pigs. According to the calculation of C3bRR and ICR rosette rate, it is found that there are differences in the number of red blood cell C3b in different breeds of pigs, so it can be inferred that the distribution of C3b in different breeds of pigs may also be different. . The study of the effect of natural infection of Eperythrozoon on porcine red blood cells shows that after pigs are naturally infected with Eperythrozoon, the activity of C3bR in porcine erythrocytes decreases, and the activity of SOD in erythrocytes also decreases with the increase of infection time, which in turn inhibits porcine erythrocytes from clearing immune complexes. The role of substances and pathogens. In summary, porcine erythrocytes also have immune function, and the rate of C3bR rosettes decreases after Eperythrozoon infection in pigs, suggesting that the two may be related. But in general, the detection index of porcine erythrocyte immune function is single and the sample data are limited, so the relevant mechanism of porcine erythrocyte immunity theory is still unclear.

The problem in the prior art is that the membrane-bound peptide sequence that plays a role in porcine erythrocyte ECR1-like is unclear, which affects the subsequent in-depth research on related issues such as the membrane distribution of ECR1-like and its impact on immune function.

Contents of the invention

The invention provides a porcine erythrocyte ECR1-like membrane-bound peptide, polyclonal antibody, preparation method and application, and provides a specific amino acid sequence of the membrane-bound peptide that plays a role in porcine erythrocyte ECR1-like. In-depth study of related issues such as membrane distribution status and its impact on immune function provides technical tools.

The present invention provides a porcine erythrocyte ECR1-like membrane-bound peptide, the amino acid sequence of the porcine erythrocyte ECR1-like membrane-bound peptide is shown in SEQ ID NO.1, which is ISANTVILFWFTCLFVQLY, and its corresponding nucleotide sequence is shown in SEQ ID NO.1 ID NO.2 shows ata agt gcc aac acg gta atc cta ttc tgg ttt act tgt cttttt gtt caa ctatac.

The invention provides a polyclonal antibody prepared from the porcine erythrocyte ECR1-like membrane binding peptide.

The present invention provides a method for preparing the polyclonal antibody of the porcine erythrocyte ECR1-like membrane-bound peptide, comprising the following steps:

S1, preparation of antigen

Artificially synthesize the amino acid sequence shown in SEQ ID NO.1 as an antigen;

S2, Preparation of polyclonal antibody

S21, Preparation of polyclonal antiserum:

Take 2 mL of complete Freund's adjuvant and 2 mL of antigen dissolved in PBS, emulsify it to obtain antigen emulsion, inject 0.5 mL of antigen emulsion into rabbits; Rabbits were boosted with the antigen emulsion; 10 days later, blood was collected from the heart of the rabbits, and rabbit serum was collected, which was the polyclonal antiserum;

S22, harvesting the polyclonal antibody in the polyclonal antiserum;

S23, Primary purification of antibody: Purify the polyclonal antibody with Sephadex A-50 to obtain primary purified antibody;

S24, secondary purification of antibody: Purify the primary purified antibody with a protein purification instrument to obtain the secondary purified antibody, which is a polyclonal antibody to porcine erythrocyte ECR1-like membrane-bound peptide.

The invention provides an application of the porcine erythrocyte ECR1-like membrane-bound peptide segment in preparing porcine erythrocyte immune function regulating reagents.

Compared with the prior art, the present invention provides a porcine erythrocyte ECR1-like membrane-bound peptide, polyclonal antibody, preparation method and application, which have the following beneficial effects:

Based on the results of human medical research, we took Landrace pigs as the research object and deeply analyzed the immune adhesion function of porcine erythrocytes. Pig erythrocytes can bind to sensitized GFP-E.coli by fluorescent immunocytochemical staining; pig erythrocytes can combine with one or more fresh rabbit serum sensitized GFP-E.coli by scanning electron microscopy and transmission electron microscopy Immunoadhesion, the tight combination of the two is not caused by abiotic factors.

The invention provides a porcine erythrocyte ECR1-like membrane-bound peptide segment, which is an important molecular basis for the ECR1-like molecule to stably exert the immune adhesion function. In-depth analysis of the biological information of the sequence has scientific significance for elucidating the regulation mechanism of porcine erythrocyte immune function. In order to further explore the biological functions related to porcine erythrocyte ECR1-like, the present invention uses rabbit immunization to prepare antiserum of porcine erythrocyte ECR1-like membrane-bound peptides, and uses yeast eukaryotic expression technology to express ECR1-like active membrane-bound peptides in vitro Peptides, laying a technical foundation for subsequent research on the distribution of ECR1-like membranes and their effects on immune function and other related issues.

Description of drawings

Figure 1 shows the score value of the 447-465 region of the TMpred analysis results;

Figure 2 is the analysis result of TMHMM tool;

Figure 3 is the chromatogram of polyclonal antibody purified by AKTApurifier;

Figure 4 is the SDS-PAGE detection result of porcine erythrocyte ECR1-like membrane-bound peptide polyclonal antibody;

Among them, lanes I and II are porcine erythrocyte ECR1-like membrane-bound peptide polyclonal antibodies, and lane M is maker;

Figure 5 is a microscopic view of antibody activity detection results;

Wherein Fig. 5A is the result of test group A, Fig. 5B is the result of test group B, and Fig. 5C is the result of test group C.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

The sources of experimental materials in the following examples are as follows:

(1) Experimental animals

Healthy Landrace pigs, healthy New Zealand white rabbits (Zhichao Farm, Beiwang Village, Taigu County), GFP-E.coli (produced by transferring a plasmid containing GFP into E.coli according to a conventional method).

Pichia pastoris strain X-33, expression plasmid pwPICZalpha, and Escherichia coli competent Trans10 cells (E.coli) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

(2) Main reagents and consumables

Reagents related to antibody preparation: Dylight488 goat anti-rabbit IgG and HRP goat anti-rabbit IgG were purchased from Wuhan Boster Company; IgG1 isotype antibody was purchased from Southern Biote; PBSB (0.1mol/L, pH=7.4PBS, 10mM glucose and 0.1% BSA), 0.9% NaCl, acetate buffer (60mmol/L, pH=4.0), 0.5mol/L NaOH, PB (0.1mol/L pH=6.5), carbonate buffer (0.5mol/L, pH=6.5), 9.5), 0.1mol/L HCl, Naphthalene's reagent, PBS (0.1mol/L, pH=7.4); carbonate buffer (0.5mol/L, pH=9.5), PBS (0.01mol/L, pH=9.5), PBS (0.01mol/L, pH= 7.4); Lymphocyte Separation Solution (Research Institute of Bioengineering, Chinese Academy of Medical Sciences); Heparin Anticoagulant Tube (Soleibao Biological Reagent Company), Lymphocyte Separation Solution (Shanghai Sangon Bioengineering Co., Ltd.), LB Solid Medium ( Beijing Kangwei Century Biotechnology Co., Ltd.), LB liquid medium (Beijing Kangwei Century Biotechnology Co., Ltd.), ampicillin sodium (Beijing Kangwei Century Biotechnology Co., Ltd.) potassium bisulfate (Tianjin Pengda Chemical Reagent Co., Ltd.) , basic fuchsin (Tianjin Pengda Chemical Reagent Co., Ltd.), sodium sulfite (Tianjin Pengda Chemical Reagent Co., Ltd.), Freund's complete adjuvant (Beijing Suolaibao Technology Co., Ltd.), octanoic acid (Chengdu Kelong Chemical Reagent Factory) , ammonium sulfate (Chengdu Kelong Chemical Reagent Factory), DEAE-Sephadex A-50 (Pharmacia, the United States); neutral resin (Shanghai Specimen Model Factory, China), FITC (Beijing Soleibao Technology Co., Ltd., batch number: 0633); 2mL, 1.5mL, 10mL EP tubes (Beijing Kangwei Reagent Co., Ltd.), 96-well opaque plate (black) (Beijing Kangwei Century Biotechnology Co., Ltd.), pipette tip (Thermo scientific company)

Reagents related to eukaryotic expression: SDS-PAGE gel preparation kits were purchased from Wuhan Boster Bioengineering Co., Ltd., EZNATM Gel Extraction Kit gel recovery kit, EZNATM Plasmid Mini Kit plasmid extraction kit were purchased from OMEGA Company of the United States, BCA Protein concentration assay kits were purchased from Shanghai Beyontian Biotechnology Co., Ltd. 50×TAE and high-sensitivity chemiluminescence detection kits were purchased from Beijing Kangwei Century Biotechnology Co., Ltd. Non-reducing protein loading buffer (NuPAGE LDS Sample Buffer (4X)) was purchased from Life Company of the United States, the reducing protein loading buffer was purchased from Beijing Soleibao Company, peptide N-glycosidase F (PNGase-F) was purchased from NEB Company of the United States, and restriction enzymes EcoR I, Xho I, Sac I, T4DNA Ligase, DNA Marker (DL2000) were all purchased from Dalian TakaRa Company, Zeocin and agar powder were purchased from Invitrogen Company, peptone and yeast extract powder were purchased from OXOID Company, imidazole, acid hydrolyzed casein, guanidine hydrochloride, Coomassie Brilliant Blue Protein Glue Fast Staining Solution was purchased from Beijing Suo Laibao Company, and other reagents were domestic analytical grade; centrifugal ultrafiltration tube (Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3membrane) was purchased from Millipore Company of the United States, and 3.5kD dialysis bag( 3) Purchased from US SPECTRUM Company, anion exchange resin (Poros 50 HQ) purchased from Applied Biosystems Company, and Ni-NTA Sefinose™ Resin purchased from Bio Basic Inc.

(3) Main instruments

Desktop acidity meter (pH211, Beijing Hana Keyi Technology Co., Ltd.), upright fluorescence microscope (BX51TF, Japan), centrifuge (TDL-50B, Shanghai Anting Scientific Instruments)); protein purifier (AKTApurifier UPC10, GE Healthcare) ; -86°C ultra-low temperature refrigerator (DW-HL338, Zhongke Meiling); adjustable pipettes (2.5 μL, 10 μL, 20 μL, 200 μL, 1000 μL; Eppendorf, Germany); electronic analytical balance (CP225D, Sartooeius); Ultrapure water generator (NW30VF type, Heal Force); vertical pressure steam sterilizer (LS-100HD type, Jiangyin Binjiang Medical Equipment Co., Ltd.); digital constant temperature water bath (HH-S6 type, Jintan City, Jiangsu Province) Jincheng Guosheng Experimental Instrument Factory); acidity meter (PH211 type; Beijing Hana Keyi Technology Co., Ltd.); constant flow peristaltic pump (BT100-2J type, Baoding Lange Constant Flow Pump Co., Ltd.); chromatography column (XK- 16 type, American GE company); gene introduction instrument (SCIENTZ-2C type, Ningbo Xinzhi Biotechnology Co., Ltd.); electric cup (165-2086 type, American BIO-ROD company); high-speed refrigerated centrifuge (2-16K type , U.S. Sigma Company); nucleic acid protein concentration analyzer (ND-1000 type, U.S. NanoDrop Company); agarose horizontal electrophoresis tank (DYCP-31E type, Beijing Liuyi Instrument Factory); PCR instrument (MyCycler type, U.S. Bio- rad company) agarose gel imaging system (JD-801 type, Jiangsu Jetta Technology Development Co., Ltd.) biochemical incubator (SPX-300-Ⅱ type, Shanghai Yuejin Medical Instrument Co., Ltd.) constant temperature shaking incubator (ZWY-2112B type, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); Rabbit Baoding.

A porcine erythrocyte ECR1-like membrane-bound peptide segment provided by the present invention has an amino acid sequence as shown in SEQ ID NO.1, which is ISANTVILFWFTCLFVQLY, and a corresponding nucleotide sequence as shown in SEQ ID NO.2, which is ata agt gccaac acg gta atc cta ttc tgg ttt act tgt ctt ttt gtt caa ctatac.

According to the porcine erythrocyte ECR1-like mRNA sequence obtained in our previous study, the amino acid sequence was translated according to the ECR1-like mRNA using the ORF Finder tool. The above sequences were analyzed using TMHMM and TMpred tools respectively,

Among them, the analysis result of TMpred tool is as follows: Among the predicted 469 amino acid residues, there are 9 regions belonging to the membrane binding region, including 4 predicted segments of inner-outer helix and 5 segments predicted by outer-internal helix. According to score 2000 The principle of approach shows that the highest score value in the 447-465 area is 1884/1579 respectively. Figure 1 shows the score value in the 447-465 area of the TMpred analysis results. Figure 2 shows the analysis results of the TMHMM tool. In Figure 2, the horizontal line at the top It is the outside extracellular region, the curve in the middle is transmembrane, the curve at the bottom and close to the abscissa is the inside intracellular region, and the columnar region in the ellipse represents the membrane-bound region with a very high prediction probability, distributed in the 447-465 region on the amino acid sequence.

Among them, the TMHMM tool analysis found that: the results show that a region exceeds the blue threshold line (the line closest to the abscissa), that is, the amino acid sequence of the 447-465 region is considered to be a membrane-binding region from a probability perspective, and the oval circle in Figure 2 indicates the region. Analysis of the remaining residues revealed that they were all extracellular free sites, as indicated by the arrows in Figure 2 . Combined with the comprehensive analysis of TMpred prediction results, the amino acid sequence in the 447-465 region was selected as the target fragment.

Comprehensive analysis and acquisition of the amino acid sequence of the extracellular tail region of porcine erythrocyte ECR1-like, using the Protein query vs. The sequence is: ISANTVILFWFTCLFVQLY, and the corresponding nucleotide sequence is ata agt gcc aacacg gta atc cta ttc tgg ttt act tgt ctt ttt gtt caa ctatac.

Based on the same inventive concept, the present invention also provides a method for preparing a polyclonal antibody to a porcine erythrocyte ECR1-like membrane-bound peptide, comprising the following steps:

S1, preparing antigen: artificially synthesize the amino acid sequence shown in SEQ ID NO.1 as an antigen, the specific operation is as follows:

The amino acid sequence shown as SEQ ID NO.1 was sent to Beijing Aoweisen Gene Technology Co., Ltd. to synthesize the target short peptide as an antigen for future use.

S2, Preparation of polyclonal antibody

S21, preparation of polyclonal antiserum: Take 2 mL of complete Freund's adjuvant and 2 mL of antigen dissolved in PBS, emulsify it to obtain antigen emulsion, inject 0.5 mL of antigen emulsion into rabbits; two weeks later, mix with Rabbits were boosted with antigen emulsion dissolved in Freund's incomplete adjuvant; 10 days later, blood was collected from the heart of the rabbits, and rabbit serum was collected, which was the polyclonal antiserum. The specific operation is as follows:

Take 2 mL of Freund's complete adjuvant and 2 mL of antigen dissolved in PBS (0.1 mol/L, pH=7.4), and emulsify it to obtain an antigen emulsion. Draw the antigen emulsion with a 1mL syringe, connect it with a 22G needle, and remove the air bubbles in the syringe. Put the rabbit on the safety rack, clean and disinfect the muscle area of the hind leg with 70% ethanol, inject 0.5 mL of antigen emulsion into the needle 1 cm deep. Two weeks later, rabbits were boosted with 1 mg of antigen emulsion miscible in Freund's incomplete adjuvant (2 mL of antigen emulsion was miscible in 2 mL of Freund's incomplete adjuvant), and the steps were the same as above. 10 days after the booster immunization, 20 mL of blood was collected from the heart of the rabbit. After standing the rabbit blood at room temperature for several hours, overnight at 4°C. Centrifuge at 5000×g for 15 min at 4°C to collect rabbit serum, which is the polyclonal antiserum.

S22. Harvest the polyclonal antibodies in the polyclonal antiserum: the polyclonal antiserum is diluted 4 times with acetate buffer (60 mmol/L, pH=4.0), and the pH is adjusted to 4.5. Add octanoic acid at room temperature at a ratio of 30 μL octanoic acid per 1 mL of diluent. Stir slowly for 30min. Centrifuge at 10,000×g for 30 min at 4°C, and take the supernatant. The supernatant was filtered through qualitative filter paper, 10 times the volume of PBS (0.1 mol/L, pH=7.4) was added to adjust the pH to 7.4, and it was left to stand on ice for 1 min. Add ammonium sulfate at a rate of 0.27 g of ammonium sulfate per milliliter of the mixture, and continue stirring for 30 minutes. Centrifuge at 5000×g for 5 min at 4°C to collect the precipitate. Add 2 mL of PBS to dissolve the protein precipitate, and centrifuge the solution at 15,000 g for 10 min at 4°C. The precipitate was discarded, the supernatant was polyclonal antibody (IgG), the supernatant was collected and set aside.

S23, Primary purification of antibody: Purify polyclonal antibody with Sephadex A-50 to obtain primary purified antibody, the specific operation is as follows:

Weigh 5g of DEAE-Sephadex A-50 (Sephadex A-50), suspend with 500mL ultrapure water, and let stand for 1h. Pour off the upper layer of fine particles. Add 75mL of 0.5mol/L NaOH solution, stir and let stand for 30min, drain and filter with Buchner funnel, wash with distilled water until neutral. Add 75mL of 0.1mol/L HCl, stir and let it stand for 30min, drain the Buchner funnel and filter, wash with distilled water until neutral. Soak A-50 in PB overnight and set aside. Fix the chromatography column on the burette rack, pour 0.1mol/L, pH=6.5 PB along the glass rod to 1/4 column bed height, pour the soaked DEAE-Sephadex A-50, and wait for it to settle naturally for 3 When -4cm, open the outlet clamp, continue to add DEAE-Sephadex A-50 to the desired height. Close the water outlet, and after the gel column has completely settled, put a layer of qualitative filter paper on the column surface, tighten the column cap, and connect the eluent bottle with a plastic tube. Open the water outlet clamp, balance the column bed with twice the column bed volume of 0.01mol/L pH=6.5PB, and control the water outlet speed at 10-15d/min. Detect the pH of the effluent, and close the water outlet when the pH of the effluent is consistent with that of the eluent. The dialyzed protein sample is added to the surface of the column bed along the tube wall, and the tube wall is washed with a small amount of eluent, and the sample volume is 1-2% of the column bed volume. Open the water outlet, and after the sample completely enters the column bed, elute with 0.1mol/L pH=6.5PB. The eluted fraction was collected in a test tube. Combine the uphill and downhill fractions of the eluate and concentrate to the desired volume. DEAE-Sephdex A-50 was washed with acid and alkali, added with 0.02g/100ml NaN ₃ , and stored at 4°C.

S24, secondary purification of antibody: Purify the primary purified antibody with a protein purification instrument to obtain the secondary purified antibody, that is, rabbit anti-pig porcine erythrocyte ECR1-like membrane-bound peptide polyclonal antibody, the specific operation is as follows:

The above-mentioned primary purified antibodies were purified using the AKTApurifier medium-pressure protein purification instrument. Start the instrument, and after the self-test is completed, start the medium-pressure protein purification system UNICORN5.31 program and enter the software control interface. Put A1 pipe of the instrument into 20mM sodium phosphate buffer (pH=7.0), put B1 pipe into 0.1M glycine-hydrochloric acid buffer (pH=2.7), click manual in the tool bar in the system control window, and select pump→pump wash purifier, select the A1 and B1 pipelines as ON, execute, and start cleaning the pipelines and medium pressure pumps. The high-pressure 1.5mL EP tubes were sequentially placed in the sample cup grooves of the Frac900 collector turntable, and 15 μL of 1M Tris-Hcl (PH=9.0) was added to each tube. Select pump→flow rate in manual, input flow rate 1mL/min, insert; select Alarm&mon→alarm pressure, set high alarm 0.3MPa, insert, execute. Carefully connect the purification column with the No. 1 tube of the sample loading loop according to the operating procedures, remove the plug at the lower end of the purification column, wait until the bottom of the purification column drips liquid and fill the upper port of the UV flow cell before connecting it to the UV flow cell. Fill the column, tubing and pump completely with loading buffer. Use the sample loading tube to slowly push the ascites after removal of impurities from the No. 3 port of the injection valve of the sample loading ring to complete the sample loading. Do not remove the sample tube after pushing it all in. In the software, set the affinity chromatography column type to HiTrap-protein_G_HP_1_mL, and set the volume to 0.962mL; set the column pressure safety range to 0.3MPa-0.00MPa, and set the flow rate to 1mL/min. Equilibrate the pipeline with buffer A, set the volume of the balance solution to 5mL; set the sample injection volume to 2mL; set the UV to zero, select Alarm&mon→autozero, the volume is 5mL; set the sample injection to 2.5mL, set Gradient 100{%B} , 5.00{base}, volume 12mL. Turn on the B pump, set the Pump wash Basicon, the volume is 12mL; set the flow rate to (1mL/min), and the volume is 12mL. Set the collection volume of Fractionation 900 to 1mL/tube in turn, and end the collection after collecting 10 tubes, and set End Method to end the collection procedure. Routinely clean the pipeline and medium-pressure pump according to the operating procedures, unload the chromatography column, prevent the interference of air bubbles during the whole process, and shut down the instrument routinely. Add the antibody in the collection tube to an ultrafiltration centrifuge tube, centrifuge at 4°C, 4,000r/min for 1h, add 2mL of PBS, centrifuge at 4°C, 4,000r/min for 1h, and dilute the purified antibody to 2mL with PBS, - Reserve at 20°C, the concentration to be tested.

Figure 3 is the chromatogram of the polyclonal antibody purified by AKTApurifier. When the sample begins to enter the chromatographic column, the UV flow cell monitors the breakthrough peak I, and the maximum peak is 570AU (wavelength 280nm); when the elution buffer begins to elute the antibody, the UV flow cell The elution peak II was monitored, and the Fractionation_900 started to collect the antibody at this time, and the maximum peak was 730AU (wavelength 280nm); the collection was stopped after the elution peak returned to the baseline.

S25, Determination of antibody concentration: completely dissolve the BSA protein standard, take 10 μL, and dilute to 250 μL with PBS (0.01 mol/L, pH=7.4), so that the final concentration is 0.2 mg/mL. Invert 5×G250 staining solution 3-5 times and mix well, take 2mL, add 8mL ultrapure water, and mix into 1×G250 staining solution (this staining solution can be stored at 4°C for one week). Add the diluted protein standards into 96-well plates at 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 12 μL, 16 μL, and 20 μL respectively, and do three repetitions for each gradient concentration, and use PBS (0.01 mol/ L, pH=7.4) to make up to 20 μL. The concentration of the antibody to be tested is made into three dilution gradients (1-fold dilution, 2-fold dilution, and 4-fold dilution), and each gradient is repeated three times. For the first gradient, 20 μL of the antibody to be tested is added to each well, and for the second gradient, Add 10 μL of antibody to each well, add 5 μL of antibody to each well in the third gradient, and make up the less than 20 μL with PBS. Diluted 1×G250 was added to all wells and left at room temperature for 3-5min. Put the 96-well plate into the microplate reader, mix and shake 3 times, set the wavelength to A595, and read the value. Draw a standard curve according to the absorbance value of the measured BSA protein standard substance, and calculate the concentration of the antibody to be tested. The purified antibody concentration was determined to be 9.0 mg/mL. Use PBS to adjust the antibody concentration to 0.2 mg/mL, aliquot into sterile EP tubes, and store at -80°C for later use.

S26, antibody SDS-PAGE detection:

Glue production: Prepare 10% separating gel according to Table 1, and pour the gel immediately after mixing. After filling the glue, add a layer of distilled water (can remove air bubbles and isolate air). When a broken line appears between the distilled water and the glue, slowly discard the water, and use filter paper to dry up the remaining distilled water. Pour 4% stacking gel on top of the prepared 10% separating gel. The formulations of the separating gel and stacking gel are shown in Table 1. Insert the comb quickly after filling, and the generation of air bubbles should be avoided during this process. The sample was mixed with the non-reducing protein loading buffer (NuPAGE LDS Sample Buffer (4X)) at a ratio of 5:1. After the sample was prepared, it was denatured in a metal bath at 95°C for 10 minutes. According to the calculation of the concentration of the secondary purified antibody determined, load 30 μg and 20 μL per well. Electrophoresis: stacking gel 80V, separating gel 120V, stop electrophoresis when bromophenol blue runs to the bottom of the gel. Prepare Coomassie Brilliant Blue staining solution according to the instructions, put the gel into a plate, add an appropriate amount of Coomassie Brilliant Blue staining solution, heat to boil, move to a horizontal shaker after 30 seconds, shake and cool to room temperature, discard the staining solution; Rinse with pure water, heat to boil, transfer to a horizontal shaker after 30 seconds, shake and cool to room temperature, repeat 3 to 4 times. Scan the gel: Use the MMAX scanner to scan the gel and acquire the image. Figure 4 shows the SDS-PAGE detection results of polyclonal antibodies to porcine erythrocyte ECR1-like membrane-bound peptides. Lanes I and II are polyclonal antibodies to porcine erythrocytes ECR1-like membrane-bound peptides, and lane M is maker. It can be seen in Figure 4 that there is no band in the antibody electrophoresis result, and the molecular weight of the monoclonal antibody conforms to the theoretical value of 98kDa.

Formulation of table 1 separating gel and stacking gel

S27, antibody activity detection:

Aseptically collect blood from the anterior vena cava of the test pigs, separate porcine red blood cells with porcine lymphocyte separation medium, and centrifuge and wash once at 2000r/min for 5min. Take the red blood cells from the bottom of the tube, resuspend them with 0.5% IgG-free BSA-Hank's buffer, let stand at room temperature for 15 minutes, wash once at 1500 r/min, 5 minutes. Add 0.1M glycine-Hank's, let stand at room temperature for 1h, centrifuge and wash at 1500r/min for 3min, resuspend with 0.5% IgG-free BSA-Hank's, observe and count the red blood cell morphology under a common microscope, and adjust the red blood cell concentration to 2.47×10 ⁷ /mL, spare.

Take 3 sterile 2Ml EP tubes and label them as group A, group B, and group C in sequence, and add 190 μL, 190 μL, and 198 μL of the above red blood cell suspension to each group in sequence. In group A, add 10 μL of secondary purified antibody to make the concentration of secondary purified antibody 10 μg/mL, incubate at 37°C for 1 h, shake intermittently, wash twice with Hank’s buffer at 1500 r/min, 5 min, and wash with 198 μL 0.5% IgG- freeBSA-Hank's resuspension. Add 2 μL of Dylight-488 goat anti-rabbit IgG secondary antibody to group A according to the concentration of secondary purified antibody suggested by the instructions, incubate at 37°C for 1 hour, shake intermittently, and protect from light throughout the incubation. After incubation, wash twice with Hank's buffer at 1500r/min for 5min, resuspend and smear under a fluorescence microscope to collect images. In group B, only 10 μL of Hank’s buffer was added as a control, and the smears were examined under a fluorescence microscope to collect images. In group C, mouse IgG1 isotype antibody (1:200) was added according to the concentration recommended in the instructions, and the later operation was the same as that of group A, and the smears were examined under a fluorescent microscope and images were collected.

Fig. 5 is a microscopic view of antibody activity detection results, wherein Fig. 5A is the result of test group A, Fig. 5B is the result of test group B, and Fig. 5C is the result of test group C. The erythrocytes in test group A were incubated with polyclonal antibodies and then incubated with Dylight488 goat anti-rabbit secondary antibody. Observation under a fluorescent microscope showed a small amount of green fluorescence on the surface of erythrocytes, indicating that the prepared polyclonal antibodies could specifically bind to porcine erythrocytes (Fig. 5A); red blood cells in group B were not incubated with antibodies, observed under a fluorescent microscope, no fluorescent spots were found (Figure 5B); group C were incubated with IgG1 isotype antibodies, and then incubated with Dylight488 goat anti-rabbit secondary antibody, observed under a fluorescent microscope , failed to find fluorescent spots on the surface of porcine erythrocytes (Fig. 5C).

The above results show that the present invention has successfully prepared immunologically active porcine erythrocyte ECR1-like membrane-bound peptide specific polyclonal antibody, which provides a research tool for further research on the molecular mechanism of porcine ECR1-like molecular immunology function.

It should be noted that, in order to avoid redundant description, the present invention describes preferred embodiments, but those skilled in the art can make additional changes and modifications to these embodiments once the basic inventive concept is known. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

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 equivalent technologies thereof, the present invention also intends to include these modifications and variations.

sequence listing

<120> Porcine erythrocyte ECR1-like membrane-bound peptide, polyclonal antibody, preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> PRT

<213> Artificial sequence

<400> 1

Ile Ser Ala Asn Thr Val Ile Leu Phe Trp Phe Thr Cys Leu Phe Val

1 5 10 15

Gln Leu Tyr

<210> 2

<211> 57

<212>DNA

<213> Artificial sequence

<400> 2

ataagtgcca acacggtaat cctattctgg tttacttgtc tttttgttca actatac 57

Claims (5)

1. A porcine erythrocyte ECR1-like membrane-bound peptide, characterized in that the amino acid sequence of the porcine erythrocyte ECR1-like membrane-bound peptide is shown in SEQ ID NO.1, which is ISANTVILFWFTCLFVQLY, and its corresponding nucleotide sequence As shown in SEQ ID NO.2, it is ata agt gcc aac acg gta atc cta ttc tgg ttt act tgt ctt tttgtt caa ctatac. 2. A polyclonal antibody made from the porcine erythrocyte ECR1-like membrane-bound peptide according to claim 1. 3. The preparation method of polyclonal antibody according to claim 2, is characterized in that, comprises the following steps: S1, preparation of antigen Artificially synthesize the amino acid sequence shown in SEQ ID NO.1 as an antigen; S2, Preparation of polyclonal antibody S21, Preparation of polyclonal antiserum: Take 2 mL of complete Freund's adjuvant and 2 mL of antigen dissolved in PBS, emulsify it to obtain antigen emulsion, inject 0.5 mL of antigen emulsion into rabbits; The antigen emulsion in the rabbit was boosted to immunize; 10 days later, blood was collected from the heart of the rabbit, and the rabbit serum was collected, which was the polyclonal antiserum; S22, harvesting the polyclonal antibody in the polyclonal antiserum; S23, Primary purification of antibody: Purify the polyclonal antibody with Sephadex A-50 to obtain primary purified antibody; S24, secondary purification of antibody: Purify the primary purified antibody with a protein purification instrument to obtain the secondary purified antibody, which is a polyclonal antibody to the porcine erythrocyte ECR1-like membrane-bound peptide. 4. The application of the porcine erythrocyte ECR1-like membrane-bound peptide according to claim 1 in the preparation of porcine erythrocyte immune function regulating reagents. 5. the application of a polyclonal antibody as claimed in claim 2 in the preparation of porcine erythrocyte immune function regulation reagent.