CN108359007B - Pig red blood cell ECR1-like membrane-bound peptide fragment, polyclonal antibody, preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of pig red blood cell ECR1-like membrane-bound peptide fragment antibodies, and particularly relates to a pig red blood cell ECR1-like membrane-bound peptide fragment, a polyclonal antibody, a preparation method and an application. The amino acid sequence of the membrane-bound peptide fragment of pig red blood cell ECR1-like is shown in SEQ ID NO.1 and is ISANTVILFWFTCLFVQLY, and the corresponding nucleotide sequence is shown in SEQ ID NO.2 and 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 red blood cell ECR1-like membrane-bound peptide segment with immunological activity, and provides a research tool for further researching the porcine ECR1-like molecular immunological function molecular mechanism.

Description

Pig red blood cell ECR1-like membrane-bound peptide fragment, polyclonal antibody, preparation method and application

Technical Field

The invention belongs to the technical field of pig red blood cell ECR1-like membrane-bound peptide fragment antibodies, and particularly relates to a pig red blood cell ECR1-like membrane-bound peptide fragment, a polyclonal antibody, a preparation method and an application.

Background

Early immune rosette studies determined the rates of C3b receptor and immune complex rosettes on approximately charxia x Changbai, Jinhua hybrid pig red blood cells, with mean values of 8.14 ± 1.25, 4.64 ± 0.84, respectively; the erythrocyte immune function of Yunnan breed pigs has the C3bRR rate of 17.2 +/-4.20 and the ICR rate of 11.2 +/-3.27, and the difference between the two rates is not significant (P is more than 0.05). Researchers make comparative studies on red blood cell immune functions of black and white cow, pig and Tibetan dog in Xining area, and the results prove that the red blood cell membranes of the three animals have C3bR, wherein the rosette rate of pig red blood cell C3bR is 4.5 +/-1.08%. The application of erythrocyte C3b receptor rosette (CbRR) and Immune Complex Rosette (ICR) tests proves that complement receptors exist on the cell surfaces of pigs such as Dahe pigs, Diqing pigs and the like, and the C3bRR is 14.87 +/-2.21 and 21.25 +/-3.12 respectively. From the above studies, it can be seen that the concept of the erythrocyte immune system is also applicable to pigs, and the distribution state of C3b in pigs of different breeds can be inferred to be different according to the difference of the number of the erythrocytes C3b in pigs of different breeds through calculating the florescence rate of C3bRR and ICR. Research on the influence of the natural infection of eperythrozoon on pig erythrocytes shows that after the pig is naturally infected with eperythrozoon, the activity of the pig erythrocytes C3bR is reduced, and the activity of erythrocyte SOD is also reduced along with the increase of infection time, so that the pig erythrocytes are inhibited from clearing immune complexes and pathogens. In conclusion, pig erythrocytes also have an immune function, and the C3bR rosette rate is reduced after the pig is infected with eperythrozoon, suggesting that the two may have a correlation. However, in general, the detection index of the immune function of pig red blood cells is single and sample data is limited, so the relevant mechanism of the pig red blood cell immune theory is not clear.

The prior art has the problem that the membrane-bound peptide segment sequence playing a role in pig red blood cell ECR1-like is not clear, and the subsequent ECR1-like membrane distribution state and the influence on the immune function and other related problems are influenced.

Disclosure of Invention

The invention provides a membrane-bound peptide segment of pig red blood cells ECR1-like, a polyclonal antibody, a preparation method and application, provides an amino acid sequence of the membrane-bound peptide segment which plays a role in the pig red blood cells ECR1-like, and provides a technical tool for the subsequent intensive research on relevant problems of ECR1-like membrane distribution state, influence on immune function and the like.

The invention provides a pig red blood cell ECR1-like membrane-bound peptide segment, wherein the amino acid sequence of the pig red blood cell ECR1-like membrane-bound peptide segment is shown as SEQ ID No.1 and is ISANTVILFWFTCLFVQLY, and the corresponding nucleotide sequence is shown as SEQ ID No.2 and is ata agt gcc aac acg gta atc cta ttc tgg ttt act tgt cttttt gtt caa cta tac.

The invention provides a polyclonal antibody prepared from the pig red blood cell ECR1-like membrane-bound peptide fragment.

The invention provides a preparation method of a polyclonal antibody of a porcine red blood cell ECR1-like membrane-bound peptide fragment, which comprises the following steps:

s1 preparation of antigen

Artificially synthesizing an amino acid sequence shown as SEQ ID NO.1 as an antigen;

s2 preparation of polyclonal antibody

S21, preparation of polyclonal antisera:

taking 2mL of Freund's complete adjuvant and 2mL of antigen dissolved in PBS, emulsifying to obtain antigen emulsion, and injecting 0.5mL of antigen emulsion into rabbits; after two weeks, the rabbits were boosted with antigen emulsion in Freund's incomplete adjuvant; after 10 days, collecting blood from the heart of the rabbit, and collecting rabbit serum, namely polyclonal antiserum;

s22, harvesting the polyclonal antibody in the polyclonal antiserum;

s23, primary antibody purification: purifying the polyclonal antibody by using sephadex A-50 to obtain a first-stage purified antibody;

s24, secondary antibody purification: and purifying the primary purified antibody by using a protein purification instrument to obtain a secondary purified antibody, namely the porcine red blood cell ECR1-like membrane-bound peptide fragment polyclonal antibody.

The invention provides application of the pig red blood cell ECR1-like membrane-bound peptide segment in preparation of a pig red blood cell immune function regulating reagent.

Compared with the prior art, the invention provides a porcine red blood cell ECR1-like membrane-bound peptide fragment, a polyclonal antibody, a preparation method and an application, and has the following beneficial effects:

based on the results of human medical research, Changbai pigs are taken as research objects, and the immunoadhesion function of the pig red blood cells is deeply analyzed. The pig red blood cells can be combined with sensitized GFP-E.coli through the observation of fluorescence immunocytochemical staining; the observation of a scanning electron microscope and a transmission electron microscope shows that the pig red blood cells can be subjected to immunoadhesion with one or more GFP-E.coli sensitized by fresh rabbit serum, and the close combination of the two is not caused by abiotic factors.

The invention provides a pig red blood cell ECR1-like membrane-bound peptide segment, an ECR1-like molecule stably plays an important molecular basis of an immunoadhesion function, and deep analysis of biological information of the sequence has scientific significance for explaining a regulation and control mechanism of the pig red blood cell immune function. In order to further discuss the relevant biological functions of the pig red blood cell ECR1-like, the invention prepares antiserum of the pig red blood cell ECR1-like membrane binding peptide segment by using a rabbit immunization method, and expresses the ECR1-like active membrane binding peptide segment in vitro by using a yeast eukaryotic expression technology, thereby laying a technical foundation for the subsequent research of relevant problems of ECR1-like membrane distribution state, influence on immune function and the like.

Drawings

FIG. 1 shows the score values of 447-465 region of TMpred analysis results;

FIG. 2 is a TMHMM tool analysis result;

FIG. 3 is a microscopic view of the results of antibody activity detection;

wherein A of FIG. 3 is the result of test group A, B of FIG. 3 is the result of test group B, and C of FIG. 3 is the result of test group C.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The experimental material sources in the following examples are as follows:

(1) laboratory animal

Healthy long white pigs, healthy new zealand white rabbits (the super farm of hamstrings king village, taigu county), GFP-e.

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

(2) Main reagent and consumable

Antibody preparation-related reagents: dylight488 goat anti-rabbit IgG and HRP goat anti-rabbit IgG were purchased from Dr. Wuhan; IgG1 isotype antibodies were 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/LpH ═ 6.5), carbonate buffer (0.5mol/L, pH 9.5), 0.1mol/LHCl, napthalene reagent, PBS (0.1mol/L, pH 7.4); carbonate buffer (0.5mol/L, pH 9.5), PBS (0.01mol/L, pH 7.4); lymphocyte separation fluid (research of bioengineering research of Chinese academy of medical sciences); heparin anticoagulant tube (songbao bio-reagent company), lymphocyte separation fluid (shanghai bio-engineering limited company), LB solid medium (beijing kang is century biotechnology limited company), LB liquid medium (beijing kang is century biotechnology limited company), ampicillin sodium (beijing kang is century biotechnology limited company), potassium hydrogen sulfate (tianjinpengda chemical reagent limited company), basic fuchsin (tianjinpengda chemical reagent limited company), sodium sulfite (tianjinpengda chemical reagent limited company), freunds complete adjuvant (beijing songbao technology limited company), octanoic acid (chengdong chemical reagent plant), ammonium sulfate (chengdong chemical reagent plant), DEAE-sephadex-50 (Pharmacia, usa); neutral resin (model factory of Shanghai province, China), FITC (Beijing Sorley Tech Co., Ltd., batch No. 0633); 2mL, 1.5mL, 10mLEP tubes (Beijing kang, reagent Co., Ltd.), 96-well opaque plate (Black) (Beijing kang, century Biotech Co., Ltd.), pipette tip (Thermoscientific Co., Ltd.)

Eukaryotic expression-related reagents: SDS-PAGE Gel preparation kits were purchased from WU.S. Bak & Dr bioengineering, Inc., E.Z.N.A.TM.gel Extraction Kit Gel recovery Kit, E.Z.N.A.TM.plasmid Mini Kit Plasmid Extraction Kit were purchased from U.S. OMEGA, BCA protein concentration determination Kit was purchased from Shanghai Binyun biotechnology, Inc., 50 XTAE, high sensitivity chemiluminescence detection Kit was purchased from Beijing kang, century Biotechnology, Inc., non-reducing protein loading Buffer (NuPAGE LDS Sample Buffer (4X)) was purchased from U.S. Life, reducing protein loading Buffer was purchased from Beijing Sorboard, peptide N-glycosidase F (PNGase-F) was purchased from U.S. NEB, restrictiveThe endonucleases EcoRI, XhoI, SacI, T4DNA Ligase, DNA Marker (DL2000) and the like are all purchased from Takara company, Zeocin and agar powder are purchased from Invitrogen company, peptone and yeast extract powder are purchased from OXOID company, imidazole, acid hydrolyzed casein, guanidine hydrochloride and Coomassie brilliant blue protein glue fast staining solution is purchased from Beijing Solebao company, and other reagents are all domestic analytically pure; centrifugal ultrafilter tubes (Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3 membrane) available from Millipore, Inc. of USA, 3.5kD dialysis bag

Available from SPECTRUM, USA, anion exchange resin (Poros 50 HQ) available from Applied Biosystems, Ni-NTA SefiniseTMResin available from Bio Basic Inc.

(3) Main instrument

A desktop acidimeter (pH211, beijing hannaco instruments science and technology ltd.), upright fluorescence microscope (BX51TF, Japan), centrifuge (TDL-50B, shanghai' ansin scientific instrument)); protein purifiers (AKTA purifier UPC10, GE Healthcare); ultra-low temperature refrigerator (DW-HL338 type, Mitsubishi of China family); adjustable pipettors (2.5. mu.L, 10. mu.L, 20. mu.L, 200. mu.L, 1000. mu.L; Eppendorf, Germany); electronic analytical balance (model CP225D, sartomeius corporation); an ultrapure water-making machine (model NW30VF, HealForce); a vertical pressure steam sterilizer (LS-100HD type, Jiangyin Binjiang medical equipment Co., Ltd.); digital display constant temperature water bath (HH-S6 type, Jincheng Guosheng laboratory instruments factory, Jiangsu Jintan city); acidimeters (PH211 type; Beijing Kanagracei science and technology Co., Ltd.); a constant flow peristaltic pump (BT100-2J, Baoding Lange constant flow pump, Inc.); a chromatography column (XK-16 type, GE, USA); gene transfer instrument (SCIENTZ-2C, Ningbo Xinzhi Biotech Co., Ltd.); electric rotary cup (type 165-2086, BIO-ROD, USA); high speed refrigerated centrifuge (model 2-16K, Sigma, USA); nucleic acid protein concentration analyzer (model ND-1000, NanoDrop, USA); agarose horizontal electrophoresis tank (model DYCP-31E, six instruments factories of Beijing); a PCR instrument (MyCycler model, U.S. Bio-rad) agarose gel imaging system (JD-801 model, Jiangsu Jetta science and technology development Co., Ltd.) a biochemical incubator (SPX-300-II model, Shanghai leap medical instruments Co., Ltd.) constant temperature shaking incubator (ZWY-2112B model, Shanghai Zhicheng analytical instruments manufacturing Co., Ltd.); rabbit retainer.

The amino acid sequence of the porcine red blood cell ECR1-like membrane-bound peptide segment is shown as SEQ ID NO.1 and is ISANTVILFWFTCLFVQLY, and the corresponding nucleotide sequence is shown as SEQ ID NO.2 and is ata agt gcc aac acg gta atc cta ttc tgg ttt act tgt ctt ttt gtt caa cta tac.

The amino acid sequence was translated from the ECR1-like mRNA using the ORF Finder tool based on the porcine red blood cell ECR1-like mRNA sequence obtained from our earlier study. The sequences were analyzed using TMHMM and TMpred tools, respectively,

wherein, the analysis result of the TMpred tool is as follows: of the 469 predicted amino acid residues, 9 regions in total belonging to the membrane binding region were found, wherein 4 segments were predicted for the inner-outer helix and 5 segments were predicted for the outer-inner helix, according to the score2000 approach principle, the score values of the 447-465 region were shown to be 1884/1579 at the highest, respectively, the score values of the 447-465 region of the TMpred analysis result are shown in FIG. 1, the score values of the 447-465 region of the TMpred analysis result are shown in FIG. 2, the TMHMM tool analysis result is shown in FIG. 2, the horizontal line at the uppermost position is the outside extracellular region, the curve at the middle position is transmurane, the curve at the lowermost position near the horizontal coordinate is the inside intracellular region, and the columnar regions within the oval circles indicate membrane binding regions with extremely high prediction probability and are distributed over the amino acid sequences of the 447-465 region.

Wherein, the TMHMM tool analyzes and finds that: the results show that a region exceeds the blue threshold line (the line closest to the abscissa), i.e., the amino acid sequences of the 447-465 region are probabilistically considered to be membrane-bound regions, the oval circles in FIG. 2 indicate regions. The remaining residues were analyzed and found to be extracellular free sites, as indicated by the arrows in FIG. 2. Combining with TMpred prediction result comprehensive analysis, selecting the amino acid sequence of the 447-465 region as a target fragment.

Comprehensively analyzing and obtaining the extracellular tail region amino acid sequence of the pig red blood cell ECR1-like, applying a Protein query vs. transformed database (tblastn) tool in NCBI, and supposing that the membrane-bound peptide fragment of the pig ECR1-like is a short peptide with 19 residues, and the sequence is as follows: ISANTVILFWFTCLFVQLY, and the corresponding nucleotide sequence is ata agt gcc aac acg gta atc cta ttc tgg ttt act tgt ctt ttt gtt caa cta tac.

Based on the same inventive concept, the invention also provides a preparation method of the polyclonal antibody of the porcine red blood cell ECR1-like membrane-bound peptide fragment, which comprises the following steps:

s1, preparation of antigen: an amino acid sequence shown in SEQ ID NO.1 is artificially synthesized and used as an antigen, and the specific operation is as follows:

the amino acid sequence shown as SEQ ID NO.1 is sent to Beijing Ovwison Gene science and technology Limited to synthesize the target short peptide as antigen for later use.

S2 preparation of polyclonal antibody

S21, polyclonal antiserum preparation: taking 2mL of Freund's complete adjuvant and 2mL of antigen dissolved in PBS, emulsifying to obtain antigen emulsion, and injecting 0.5mL of antigen emulsion into rabbits; after two weeks, the rabbits were boosted with antigen emulsion in Freund's incomplete adjuvant; after 10 days, blood is collected from the heart of the rabbit, and rabbit serum is collected, namely the polyclonal antiserum. The specific operation is as follows:

an antigen emulsion was obtained by emulsifying 2mL of Freund's complete adjuvant and 2mL of antigen dissolved in PBS (0.1mol/L, pH 7.4). The antigen emulsion was aspirated using a 1mL syringe, and a 22G needle was attached to remove air bubbles from the syringe. The rabbits were placed on a holding frame, cleaned and sterilized with 70% ethanol in the hind leg muscle area, and injected with 0.5mL of antigen emulsion 1cm deep. After two weeks, the rabbits were boosted with 1mg of antigen emulsion in Freund's incomplete adjuvant (2 mL of antigen emulsion in 2mL of Freund's incomplete adjuvant), as described above. After 10 days of booster immunization, 20mL of blood was collected from the heart of the rabbit. The rabbit blood was left at room temperature for several hours and then left overnight at 4 ℃. Centrifuging at 5000 Xg for 15min at 4 deg.C, and collecting rabbit serum to obtain polyclonal antiserum.

S22, harvesting polyclonal antibodies in the polyclonal antiserum: the polyclonal antiserum was diluted 4-fold with acetic acid buffer (60mmol/L, pH 4.0) and adjusted to pH 4.5. Caprylic acid was added at room temperature in a proportion of 30. mu.L caprylic acid per 1mL of the dilution. Slowly stir for 30 min. Centrifuging at 4 deg.C at 10000 Xg for 30min, and collecting supernatant. The supernatant was filtered through qualitative filter paper, added with 10 volumes of PBS (0.1mol/L, pH 7.4), adjusted to pH 7.4, and left on ice for 1 min. Ammonium sulfate was added in an amount of 0.27g per ml of the mixture, and the mixture was further stirred for 30 min. Centrifuging at 5000 Xg for 5min at 4 deg.C, and collecting precipitate. The protein precipitate was dissolved by adding 2ml PBS and the solution was centrifuged at 15000g for 10min at 4 ℃. And (4) discarding the precipitate, wherein the supernatant is polyclonal antibody (IgG), and collecting the supernatant for later use.

S23, primary antibody purification: purifying the polyclonal antibody by using sephadex A-50 to obtain a primary purified antibody, and specifically operating as follows:

5g of DEAE-SephadexA-50 (Sephadex A-50) was weighed, suspended in 500mL of ultrapure water, and allowed to stand for 1 hour. The fine particles in the upper layer are poured off. Adding 75mL0.5mol/LNaOH solution, stirring, standing for 30min, draining and filtering by a Buchner funnel, and washing by distilled water to be neutral. Adding 75ml of 0.1mol/LHCl, stirring, standing for 30min, draining and filtering by a Buchner funnel, and washing by distilled water to be neutral. A-50 was immersed in PB overnight for use. The chromatographic column is fixed on a burette stand, 0.1mol/L PB with the pH value of 6.5 is poured along a glass rod to the height of 1/4 column beds, the soaked DEAE-SephadexA-50 is poured, when the DEAE-SephadexA-50 naturally settles for 3-4cm, a water outlet clamp is opened, and DEAE-SephadexA-50 is continuously added to the required height. Closing the water outlet, placing a layer of qualitative filter paper on the cylindrical surface after the gel column is completely settled, screwing the cover of the column port, and connecting the elution liquid bottle with a plastic tube. The effluent clamp is opened, the column bed is balanced by 0.01 mol/LpH-6.5 PB of two times of the volume of the column bed, and the effluent speed is controlled to be 10-15 d/min. And detecting the pH of the effluent liquid, and closing the water outlet when the pH of the effluent liquid is consistent with that of the eluent. The dialyzed protein sample is applied to the surface of the bed along the tube wall, and the tube wall is washed with a small amount of eluent, the amount of the applied sample being 1-2% of the volume of the bed. The water outlet was opened and after the sample had completely entered the bed, it was eluted at 0.1mol/LpH ═ 6.5 PB. The eluted fractions were collected in a test tube. And combining the collected parts of the ascending section and the descending section of the eluent, and concentrating to the required volume. DEAE-Sephdex A-50 was washed by acid-base treatment, added with 0.02g/100ml NaN3, and stored at 4 ℃.

S24, secondary antibody purification: purifying the primary purified antibody by using a protein purification instrument to obtain a secondary purified antibody, namely a rabbit anti-pig erythrocyte ECR1-like membrane-bound peptide fragment polyclonal antibody, and specifically operating as follows:

the above primary purified antibody was purified using an AKTApurifier medium pressure protein purification apparatus. Starting the instrument, starting a UNICORN5.31 program of the medium-pressure protein purification system after the self-detection of the instrument is finished, and entering a software control interface. The instrument a1 tubing was placed in 20mM sodium phosphate buffer (PH 7.0), the B1 tubing was placed in 0.1M glycine-hydrochloric acid buffer (PH 2.7), the manual in the system control window was clicked, the pump → pump wash purifier was selected, the a1, B1 tubing was selected ON, execute, and cleaning of the tubing and the medium pressure pump was initiated. The 1.5mL EP tubes after the high pressure were placed in order in the cuvette wells of the Frac900 collector carousel, and 15 μ L of 1MTris-Hcl (PH 9.0) was added to each tube. Selecting pump → flow rate in manual, input flow rate 1mL/min, insert; alarm & mon → Alarm pressure is selected, high Alarm 0.3MPa, insert, execute is set. Carefully connecting the purification column with the No.1 position tube of the sample loading ring according to the operation specification, removing a plug at the lower end of the purification column, dripping liquid at the bottom end of the purification column, filling the upper port of the ultraviolet flow cell with the liquid, and then connecting the purification column into the ultraviolet flow cell to ensure that the sample loading buffer liquid is completely filled in the column, the pipeline and the pump. And (3) slowly pushing the ascites after impurity removal from a No. 3 port of the sampling ring Injection Valve by using a sampling tube to finish sampling. The sampling tube is not taken down after all the sampling tubes are pushed in. The type of the affinity chromatography column arranged in the piece is HiTrap-protein _ G _ HP _1_ mL, and the volume is 0.962 mL; setting the safety range of the column pressure to be 0.3MPa-0.00MPa and setting the flow rate to be 1 mL/min. Balancing the pipeline by using a buffer solution A, wherein the volume of the balancing liquid is set to be 5 mL; setting the sample injection volume to be 2 mL; the ultraviolet is zeroed, Alarm & mon → autozero, 5mL in volume is selected; the sample injection was set at 2.5mL, Gradient 100 {% B }, 5.00{ base }, volume 12 mL. Turning on the Pump B, and setting the Pump water basic to be 12mL in volume; the flow rate was set at (1mL/min) and the volume was set at 12 mL. The Fractionation 900 collection volumes were set to 1 mL/tube in order, 10 tubes were collected and the collection was completed, and the end method was set to complete the collection procedure. Cleaning the pipeline and the medium pressure pump conventionally according to the operation rules, dismounting the chromatographic column, preventing air bubble interference in the whole process, and closing the instrument conventionally. Adding the antibody in the collecting tube into an ultrafiltration centrifugal tube, centrifuging for 1h at 4 ℃ and 4,000r/min, adding 2mL PBS at 4 ℃ and 4,000r/min, centrifuging for 1h, diluting the purified antibody into 2mL with PBS, and keeping the antibody at-20 ℃ for later use to be measured.

S25, antibody concentration determination: the BSA protein standard was completely dissolved, and 10 μ L of the BSA protein standard was diluted to 250 μ L with PBS (0.01mol/L, pH 7.4) to a final concentration of 0.2 mg/mL. The 5 XG 250 staining solution was inverted 3-5 times and mixed, 2mL of which was taken and 8mL of ultrapure water was added to the resulting mixture to obtain a1 XG 250 staining solution (the staining solution was stored at 4 ℃ C. for one week). The diluted protein standard was added to a 96-well plate in an amount of 0. mu.L, 2. mu.L, 4. mu.L, 6. mu.L, 8. mu.L, 12. mu.L, 16. mu.L, and 20. mu.L, respectively, and each gradient was repeated in triplicate, and wells less than 20. mu.L were made up to 20. mu.L with PBS (0.01mol/L, pH 7.4). The concentration of the antibody to be tested was made in three dilution gradients (1-fold dilution, 2-fold dilution, 4-fold dilution), each gradient was repeated in three steps, the first gradient was made by adding 20. mu.L of the antibody to be tested per well, the second gradient was made by adding 10. mu.L of antibody per well, the third gradient was made by adding 5. mu.L of antibody per well, and finally less than 20. mu.L of PBS was used to make up. Diluted 1 XG 250 was added to all wells and left at room temperature for 3-5 min. And (3) putting the 96-well plate into an enzyme-labeling instrument, uniformly mixing and oscillating for 3 times, setting the wavelength A595, and reading. And drawing a standard curve according to the absorbance value of the BSA protein standard substance to be measured, and calculating the concentration of the antibody to be measured. The antibody concentration after purification was determined to be 9.0 mg/mL. The antibody was adjusted to a concentration of 0.2mg/mL using PBS, aliquoted in sterile EP tubes and kept at-80 ℃ until needed.

S26, antibody SDS-PAGE detection:

preparing glue: 10% of separation gel is prepared according to the table 1, and the gel is immediately filled after uniform mixing. After the glue is poured, a layer of distilled water (capable of removing air bubbles and isolating air) is added, when a broken line appears between the distilled water and the glue, the water is slowly discarded, and the residual distilled water is sucked dry by using filter paper. 4% of concentrated glue is poured on the prepared 10% of separation glue, the formula of the separation glue and the concentrated glue is shown in the table 1, and the comb is quickly inserted after the separation glue is filled, and the process should avoid the generation of air bubbles. The Sample was mixed with a non-reducing protein loading Buffer (NuPAGE LDS Sample Buffer (4X)) at a ratio of 5: 1, and the Sample was denatured for 10min at 95 ℃ using a metal bath. Based on the measured concentration of the secondary purified antibody, each well was loaded with 30. mu.g, 20. mu.L. Electrophoresis: gel 80V was concentrated, gel 120V was separated, and the electrophoresis was stopped when bromophenol blue ran to the bottom of the gel. Preparing a Coomassie brilliant blue staining solution according to the specification, putting the gel into a plate, adding a proper amount of the Coomassie brilliant blue staining solution, heating to boiling, moving to a horizontal shaking table after 30 seconds, shaking and cooling to room temperature, and removing the staining solution; rinsing with ultrapure water, heating to boil, transferring to a horizontal shaker after 30 seconds, shaking and cooling to room temperature, and repeating for 3 to 4 times. Scanning the gel: the gel was scanned using an MMAX scanner and images were acquired.

TABLE 1 formulation of the separation and concentration gums

S27, antibody activity assay:

the test pig is subjected to aseptic blood collection of the anterior vena cava, pig red blood cells are separated by using a pig lymphocyte separation medium, and the pig red blood cells are centrifuged and washed for 1 time at 2000r/min and 5 min. The tube bottom red blood cells are taken, resuspended by 0.5 percent IgG-free BSA-Hank's buffer solution, and are kept standing for 15min at room temperature, 1500r/min and washed for 1 time at 5 min. Adding 0.1M glycine-Hank's, standing at room temperature for 1h, centrifuging at 1500r/min for 3min, re-suspending with 0.5% IgG-free BSA-Hank's, observing and counting erythrocyte morphology under a common microscope, and adjusting erythrocyte concentration to 2.47 × 107/mL for use.

The 3 sterile 2mL EP tubes were labeled group A, group B, and group C in order, and 190. mu.L, and 198. mu.L of the above erythrocyte suspension were added to each group in order. In group A, 10. mu.L of the second purified antibody was added to a concentration of 10. mu.g/mL, incubated at 37 ℃ for 1 hour, shaken intermittently, centrifuged and washed 2 times with Hank's buffer 1500r/min for 5min, and resuspended in 198. mu.L of 0.5% IgG-free BSA-Hank's. Adding 2 mu of secondary purified antibody concentration into the group A according to the instruction suggestion, incubating for 1h at 37 ℃, intermittently shaking, and keeping the incubation away from light. After the incubation is finished, the cells are centrifugally washed for 2 times by using a Hank's buffer solution at 1500r/min for 5min, smeared under a fluorescence microscope after being resuspended, and then images are collected. In group B, only 10. mu. LHank's buffer was added as a control, and smear microscopy was performed under a fluorescence microscope to collect images. In group C, mouse IgG1 isotype antibody (1: 200) was added according to the instruction recommendations, the same group A was operated at the later stage, smear microscopy was performed under a fluorescence microscope, and images were collected.

FIG. 3 is a microscopic view of the results of antibody activity measurement, in which A of FIG. 3 is the results of test group A, B of FIG. 3 is the results of test group B, and C of FIG. 3 is the results of test group C. After the red blood cells of the group A are incubated with the polyclonal antibody, the red blood cells are incubated with Dylight488 goat anti-rabbit secondary antibody, and observation under a fluorescence microscope shows that a small amount of green fluorescence exists on the surfaces of the red blood cells, which indicates that the prepared polyclonal antibody can be specifically combined with the pig red blood cells (A in figure 3); group B erythrocytes were not incubated with the antibody and observed under a fluorescence microscope with no fluorescent spot (fig. 3B); group C was incubated with IgG1 isotype antibody, followed by Dylight488 goat anti-rabbit secondary antibody, and no fluorescent spots were found on the surface of porcine red blood cells when observed under a fluorescent microscope (FIG. 3C).

The results show that the specific polyclonal antibody of the porcine red blood cell ECR1-like membrane-bound peptide fragment with immunological activity is successfully prepared, and a research tool is provided for further researching the porcine ECR1-like molecular immunological function molecular mechanism.

It should be noted that the preferred embodiments of the present invention have been described for the purpose of preventing redundancy, but that additional variations and modifications to these embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such 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 such modifications and variations.

Sequence listing

<120> pig red blood cell ECR1-like membrane-bound peptide fragment, 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 (3)

1. The pig red blood cell ECR1-like membrane-bound peptide segment is characterized in that the amino acid sequence of the pig red blood cell ECR1-like membrane-bound peptide segment is shown as SEQ ID NO.1 and is ISANTVILFWFTCLFVQLY.

2. A polyclonal antibody prepared from the porcine red blood cells ECR1-like membrane-bound peptide of claim 1 as an antigen.

3. The method for producing a polyclonal antibody according to claim 2, comprising the steps of:

s1 preparation of antigen

Artificially synthesizing an amino acid sequence shown as SEQ ID NO.1 as an antigen;

s2 preparation of polyclonal antibody

S21, preparation of polyclonal antisera:

emulsifying 2mL of Freund's complete adjuvant and 2mL of antigen dissolved in PBS to obtain antigen emulsion, and injecting 0.5mL of antigen emulsion into rabbit; after two weeks, the rabbits were boosted with antigen emulsion in Freund's incomplete adjuvant; after 10 days, collecting blood from the heart of the rabbit, and collecting rabbit serum, namely polyclonal antiserum;

s22, harvesting the polyclonal antibody in the polyclonal antiserum;

s23, primary antibody purification: purifying the polyclonal antibody by using sephadex A-50 to obtain a first-stage purified antibody;

s24, secondary antibody purification: and purifying the primary purified antibody by using a protein purification instrument to obtain a secondary purified antibody, namely the porcine red blood cell ECR1-like membrane-bound peptide fragment polyclonal antibody.

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