Broad-spectrum affinity peptide ligand for influenza vaccine isolation and purification and application thereof

Abstract

The application discloses a broad-spectrum affinity peptide ligand for influenza vaccine separation and purification and application thereof. The broad-spectrum affinity peptide ligand contains an amino acid sequence which is combined with the conserved structural units 130-loop, 150-loop and 190-helix of the hemagglutinin receptor binding site of an influenza virus, and the amino acid sequence is R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 , R 1 is any one of glutamic acid, arginine, histidine and proline, R 2 is tryptophan, R 3 is any one of tyrosine and phenylalanine, R 4 is glycine, R 5 is any one of tryptophan, tyrosine and phenylalanine, R 6 is any one of glutamic acid, histidine, glutamine and tyrosine, R 7 is any one of tryptophan and phenylalanine, and R 8 is proline. The broad-spectrum affinity peptide ligand has high broad-spectrum affinity, and the broad-spectrum affinity chromatography medium can obtain the influenza vaccine with high purity and yield, and has great potential in application to the separation and purification process of the influenza vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccine separation and purification, and in particular to a broad-spectrum affinity peptide ligand for influenza vaccine separation and purification and application thereof. BACKGROUND

[0002] Vaccination is still the best strategy for preventing influenza in groups such as the elderly and children. Current influenza vaccine preparation systems mainly include chicken embryo cells and mammalian cells. In the process of preparing influenza vaccines, ultracentrifugation, gel filtration chromatography, and ion exchange chromatography are still the most commonly used unit operations in the separation and purification thereof. However, these methods are not only laborious, time-consuming, and energy-consuming, but also have poor selectivity and limited processing capacity. Moreover, the ultracentrifugation process is very difficult to scale up. Therefore, developing a broad-spectrum affinity chromatography technology is undoubtedly an important method to overcome the above problems.

[0003] Affinity chromatography technology has been widely used in antibody purification processes. However, there are few successful cases in the purification of viruses such as vaccines and lentiviruses. This is mainly due to the large particle size, complex structure, and large number of mutants of virus particles, which brings great challenges to the development of affinity chromatography ligands.

[0004] Hemagglutinin (HA) is an important and abundant glycoprotein on the surface of influenza viruses, and thus is an important target for designing small molecule drugs and affinity ligands for vaccine purification. Liang Guizhao et al. have successively disclosed two patents (CN201010540985.9 and CN201010541077.1) for obtaining hemagglutinin protein affinity peptides of influenza A H1N1 virus and influenza A H3N2 virus by using phage display technology. Teruhiko et al. reported that polypeptide LVRPLAL was obtained as a peptide ligand against influenza virus by using phage display technology combined with molecular docking (Bioorganic & Medicinal Chemistry, 2016, 24(5): 1106-14.). However, the above-mentioned affinity peptide molecules and peptide ligands do not have broad-spectrum properties. The high variability of HA antigens is an important reason for the long-term problem of influenza. Due to the lack of a broad-spectrum affinity ligand suitable for different serotypes of influenza vaccines, the application of affinity chromatography technology in the existing influenza vaccine preparation process has been greatly limited. Therefore, it is urgent to develop a new type of broad-spectrum affinity ligand for influenza vaccines and to synthesize a broad-spectrum affinity chromatography medium therefrom. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is that there is a lack of a broad-spectrum affinity ligand with excellent performance in the current purification process of influenza vaccine, so that the need for vaccine production of different serotypes of influenza virus cannot be effectively met; in view of the above problems, the present application provides a broad-spectrum affinity peptide ligand for separation and purification of influenza vaccine, and a broad-spectrum affinity chromatography medium synthesized with the broad-spectrum affinity ligand as the broad-spectrum affinity ligand and the application thereof, and a preparation method of the broad-spectrum affinity chromatography medium is provided.

[0006] A broad-spectrum affinity peptide ligand contains an amino acid sequence that binds to the conserved structural units 130-loop, 150-loop and 190-helix of the receptor binding site of influenza virus hemagglutinin.

[0007] The amino acid sequence is R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ;

[0008] wherein R 1 is any one of glutamic acid, arginine, histidine and proline, R 2 is tryptophan, R 3 is any one of tyrosine and phenylalanine, R 4 is glycine, R 5 is any one of tryptophan, tyrosine and phenylalanine, R 6 is any one of glutamic acid, histidine, glutamine and tyrosine, R 7 is any one of tryptophan and phenylalanine, and R 8 is proline.

[0009] As an embodiment parallel thereto, the amino acid sequence of the broad-spectrum affinity peptide ligand is PWFGGGYGGEWP, HWFGGGYGGQWP or RWYGGGWGGHFP.

[0010] A broad-spectrum affinity chromatography medium comprises a solid phase matrix and the above-mentioned broad-spectrum affinity peptide ligand coupled to the solid phase matrix.

[0011] The solid phase matrix is a mercaptopyridylized solid phase matrix.

[0012] A preparation method of a broad-spectrum affinity chromatography medium comprises:

[0013] Obtaining a broad-spectrum affinity peptide ligand and a solid phase matrix;

[0014] The solution containing the broad-spectrum affinity peptide ligand is mixed with the solid phase matrix to obtain the broad-spectrum affinity chromatography medium through a coupling reaction.

[0015] The coupling reaction process is: introducing an amino acid group used for coupling at the C-terminal of the broad-spectrum affinity peptide ligand, and preparing a solution containing the broad-spectrum affinity peptide ligand using a cross-linking buffer; performing pyridylthiolation on the solid phase matrix, mixing the pyridylthiolated solid phase matrix with the solution containing the broad-spectrum affinity peptide ligand, and reacting in a water bath shaker.

[0016] The amino acid group used for coupling is three glycines and one cysteine sequentially connected at the C-terminal of the broad-spectrum affinity peptide ligand;

[0017] And / or, the process of performing pyridylthiolation on the solid phase matrix is:

[0018] (1) Activation: sequentially washing and activating the solid phase matrix using deionized water and an activating substance, and obtaining an activated solid phase matrix after the reaction.

[0019] (2) Thiolation: performing a first reaction on the activated solid phase matrix using a phosphate buffer and a sodium thiosulfate solution, then sequentially adding a sodium bicarbonate solution and a dithiothreitol-dissolved ethylenediaminetetraacetic acid solution for a second reaction, and sequentially washing with the sodium bicarbonate and ethylenediaminetetraacetic acid solution to obtain a thiolated solid phase matrix;

[0020] (3) Pyridylation: washing the thiolated solid phase matrix, then sequentially adding an acetone washing solution and a dithiopyridine solution, mixing and reacting, and finally washing and drying to obtain a pyridylthiolated solid phase matrix.

[0021] In the activation step: the activating substance is dimethyl sulfoxide, epichlorohydrin, and a sodium hydroxide solution, wherein the dimethyl sulfoxide is added in an amount of 0.4-2 times the mass of the solid phase matrix, the epichlorohydrin is added in an amount of 0.2-1 times the mass of the solid phase matrix, the sodium hydroxide solution is added in an amount of 0.2-1 times the mass of the solid phase matrix, the volume concentration of the dimethyl sulfoxide is 20%-100%, and the mass concentration of the sodium hydroxide solution is 0.5-1 mol / L; the activation reaction time is 3-6 h.

[0022] In the thiolation step, the concentration of the phosphate buffer is 0.1-0.5 mol / L, and the pH value is 6-7; the concentration of the sodium thiosulfate solution is 0.5-2 mol / L; the adding amount of the phosphate buffer and / or the sodium thiosulfate solution is 0.4-2 times the mass of the activated solid phase matrix; the time of the first reaction is 3-24 h; the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L, the concentration of the ethylenediaminetetraacetic acid solution is 0.5-1 mmol / L, and the adding amount of dithiothreitol in the ethylenediaminetetraacetic acid solution is 0.5-1 g / L; the adding amount of the two solutions is 0.5-1 times the mass of the activated solid phase matrix; and the time of the second reaction is 0.5-1.5 h.

[0023] In the pyridylation step, the acetone cleaning solution is an acetone solution with a volume concentration of 50%-70%, and the acetone cleaning solution contains 0.5-1 mmol / L ethylenediaminetetraacetic acid and 10-50 mmol / L sodium bicarbonate; the mass concentration of the dithiodipyridine solution is 0.1-0.5 mol / L; the adding amount of the acetone cleaning solution is 1-2 times the mass of the thiolated solid phase matrix, and the adding amount of the dithiodipyridine solution is 2-4 times the mass of the thiolated solid phase matrix; the reaction time is 1-3 h; and in the final cleaning step, an acetone solution with a volume concentration of 50%-70% and an ethylenediaminetetraacetic acid solution with a concentration of 0.5-1 mmol / L are used for cleaning.

[0024] The application of the above broad-spectrum affinity chromatography medium in the separation and purification of an influenza vaccine.

[0025] The separation and purification process is as follows: flowing a biological raw material liquid containing an influenza vaccine component into the above broad-spectrum affinity chromatography medium, collecting an eluted component after the adsorption, cleaning and elution steps, and obtaining an influenza vaccine.

[0026] The adsorption buffer used in the adsorption process is a 0.01-0.05 mol / L PB buffer with a pH of 5.0-6.5 and containing 0-0.3 mol / L NaCl; and the low-pH elution buffer used in the elution process is a 0.01-0.10 mol / L Gly-HCl buffer with a pH of 2.5-4.0.

[0027] The technical scheme of the present application has the following advantages:

[0028] 1. The present application provides a broad-spectrum affinity peptide ligand which has high affinity with the receptor binding site of influenza virus hemagglutinin protein and binds to the conserved structural units 130-loop, 150-loop and 190-helix of the receptor binding site of influenza virus hemagglutinin; based on the receptor binding site of influenza virus hemagglutinin protein, a polypeptide library is constructed by using the de novo design strategy, and the broad-spectrum affinity peptide ligand which can bind to the conserved structural units 130-loop, 150-loop and 190-helix of the receptor binding site of influenza virus hemagglutinin is obtained by four rounds of molecular simulation screening; the broad-spectrum affinity peptide ligand can specifically bind to hemagglutinin protein and inactivated influenza vaccine, effectively overcoming the problem that the existing chromatography ligand cannot be applied to HA antigens with high variability, thereby resulting in poor broad-spectrum affinity.

[0029] 2. The present application provides a broad-spectrum affinity peptide ligand which can effectively bind to the conserved structural units 130-loop, 150-loop and 190-helix of the receptor binding site of influenza virus hemagglutinin, and the amino acid sequence structure of the broad-spectrum affinity peptide ligand is R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ; it has been verified that the broad-spectrum affinity peptide ligand can specifically bind to hemagglutinin protein and inactivated influenza vaccine and has broad-spectrum affinity; when it is used as a broad-spectrum affinity ligand to synthesize a broad-spectrum affinity chromatography medium, influenza vaccine can be harvested with high purity and yield, and the broad-spectrum affinity chromatography medium has great potential for application in the separation and purification process of influenza vaccine.

[0030] 3. The present application provides a broad-spectrum affinity chromatography medium, a broad-spectrum affinity peptide ligand which has high affinity with the receptor binding site of influenza virus hemagglutinin protein is coupled with a thio-pyridyl solid phase matrix, such as agarose gel medium, to prepare a broad-spectrum affinity chromatography medium for the separation and purification of influenza vaccine; the broad-spectrum affinity chromatography medium has the advantages of good universality, good specificity, simple medium preparation and low process cost.

[0031] 4. The present application provides the application of the broad-spectrum affinity chromatography medium in the present application in the separation and purification of influenza vaccine, which can efficiently separate and purify influenza vaccine components from complex raw material liquid and hardly adsorb individual impurities; under relatively high yield, the purity of the separated vaccine can reach more than 92%, even more than 95%, and the separation effect is very significant. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0033] Figure 1 is the acquisition process diagram of the broad-spectrum affinity peptide ligand in the embodiment 1 of the present application;

[0034] Figure 2 is the amino acid positioning result diagram of different docking regions in step (1) of the embodiment 1 of the present application; wherein, a is the amino acid positioning result of 130-loop region, b is the amino acid positioning result of 150-loop region, and c is the amino acid positioning result of 190-helix region;

[0035] Figure 3 is the docking conformation diagram of different amino acid residues in step (1) of the embodiment 1 of the present application; wherein, d is the docking conformation of E, W and P in 130-loop region, e is the docking conformation of Y in 150-loop region, and f is the docking conformation of P, W and F in 190-helix region;

[0036] Figure 4 is the docking scoring distribution diagram of polypeptide library molecules in step (2) of the embodiment 1 of the present application; wherein, a is the docking scoring distribution of Autodock vina, b is the docking scoring distribution of HADDOCK, and c is the docking scoring distribution of ROSETTAFlexPepDock;

[0037] Figure 5 is the diagram of the results of molecular dynamics simulation and MM / PBSA calculation in step (3) of the embodiment 1 of the present application;

[0038] Figure 6 is the microscale thermophoresis result diagram of the top three polypeptides and hemagglutinin protein in step (4) of the embodiment 1 of the present application;

[0039] Figure 7 is the chromatography result diagram of PWFSep-6FF separating hemagglutinin protein from ovalbumin and the SDS-PAGE analysis diagram of affinity chromatography elution result in the embodiment 2 of the present application;

[0040] Figure 8 is the chromatography result diagram of PWFSep-4FF separating inactivated influenza A vaccine from Vero cell supernatant and the SDS-PAGE analysis diagram of affinity chromatography elution result in the embodiment 2 of the present application. DETAILED DESCRIPTION

[0041] Example 1

[0042] A broad-spectrum affinity peptide ligand is obtained as shown in Figure 1 , specifically as follows:

[0043] (1) Construction of polypeptide library

[0044] The receptor binding site of hemagglutinin protein is used as a template, and an amino acid positioning method is used to generate short peptide fragments. The 19 amino acids excluding Cys are docked at the conserved structural units 130-loop, 150-loop and 190-helix of the hemagglutinin receptor binding site; the binding free energy of the amino acid and the corresponding region is used as a standard for screening, Figure 2 wherein a-c are the binding free energies of the amino acids in the three docking regions, and those lower than -2.5 kcal / mol are listed as candidate amino acids.

[0045] As shown in Figure 2 a, in addition to W, E, H, Q, R, Y, F, P and L also meet the screening standard, and then according to the binding position of the above 9 candidate amino acids in the 130-loop region, they are divided into three groups: EHQYR, LWF and P, from which 15 combinations of tripeptide fragments can be obtained; as shown in Figure 3 d, E, W and P are used as representatives to show the binding positions of the three groups of amino acids in the 130-loop region. In the same way, as shown in Figure 2 b, the amino acids meeting the standard in the 150-loop region are F, W and Y; as shown in Figure 3 e, the three amino acids are all bound between Trp152 and Leu153. As shown in Figure 2 c, the amino acids meeting the standard in the 190-helix region are D, E, H, R, N, W, F, P, I and Y, which are divided into DEHNIPR, W and FY three groups according to the binding position, from which 14 combinations of tripeptide fragments can be obtained; Figure 3 f, P, W and F are used as representatives to show the binding positions of the three groups of amino acids in the 190-helix region.

[0046] Glycine is selected as the linker between short peptide fragments. If the distance between the C / N terminal of an amino acid residue and the N / C terminal of another residue is less than (i.e. the total length of one peptide bond and two amino acid main chains), then an amino acid residue can be inserted between the two residues; if the distance between them is between and , then two amino acid residues can be inserted between them; if the distance is greater than Then 3 or more residues can be inserted. The maximum distance between the amino acids in the 130-loop region and the 150-loop region is Thus two glycines can be inserted. The maximum distance between the 150-loop amino acids and the 190-helix amino acids is Thus three glycines can be inserted. Thus far, the basic model of the polypeptide has been determined, and there are 630 peptide ligands in the peptide library for the next screening process.

[0047] (2) Molecular docking screening of polypeptide library

[0048] The first round of screening uses Autodock vina for semi-flexible docking, as shown in Figure 4 a, the binding free energy of the peptide ligand and the hemagglutinin protein is in the range of -5.8 to -11.3 kcal / mol. The peptide ligand with a binding free energy lower than -8.7 kcal / mol is selected for the next round of screening, and thus 314 polypeptides are screened. In order to further explore the affinity of the peptide ligand and the design site, the second round of screening uses HADDOCK for local semi-flexible docking, as shown in Figure 4 b, the binding free energy of the polypeptide and the RBS is between -71.8 and -110.1 kJ / mol. For the convenience of screening, the HADDOCK score of the positive control of the broad-spectrum antibody C05 of the influenza A virus is -91.8 kJ / mol, and those below this value are listed as candidate peptide ligands, and thus 114 peptide ligands are screened. Further determine the structure of the peptide ligand as R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ; wherein R 1 is any one of glutamic acid, arginine, histidine and proline, R 2 is tryptophan, R 3 is any one of tyrosine and phenylalanine, R 4 is glycine, R 5 is any one of tryptophan, tyrosine and phenylalanine, R 6 is any one of glutamic acid, histidine, glutamine and tyrosine, R 7 is any one of tryptophan and phenylalanine, R 8 is proline.

[0049] Neither vina nor HADDOCK is a semi-flexible molecular docking, which cannot consider the impact of protein side chain flexibility. ROSETTA FlexPepDock docking program can consider the flexibility of polypeptide and the side chain flexibility of the receptor protein, and is usually used to optimize the binding conformation between the peptide ligand and the target protein. If the complex results of the target protein and its ligand are known, ROSETTA FlexPepDock can achieve high-precision simulation of the complex. In the present application, the interface energy score (I_sc) is used to evaluate the affinity between the 114 peptide ligands obtained in the last round and HA. I_sc includes hydrogen bonding, Lennard-Jones attraction and repulsion energy, lazaris-jarplus solvation energy and salt bridge interaction, etc. In order to improve the accuracy of docking, three parallel dockings were performed for each peptide ligand and protein, and the docking results were shown in Fig. c, and finally the top ten polypeptides were obtained for the next step of molecular dynamics simulation screening. The sequences of the top ten polypeptides are shown in SEQ No. 1-10, and the results of three parallel dockings of the top ten polypeptides are shown in Table 1. Figure 4 c, and finally the top ten polypeptides were obtained for the next step of molecular dynamics simulation screening. The sequences of the top ten polypeptides are shown in SEQ No. 1-10, and the results of three parallel dockings of the top ten polypeptides are shown in Table 1.

[0050] Table 1

[0051]

[0052] (3) Molecular dynamics simulation and MM / PBSA free energy calculation

[0053] In order to further detect the affinity of polypeptide and hemagglutinin protein and the microcosmic molecular behavior of polypeptide-hemagglutinin complex in solution, 100 ns of molecular dynamics simulation was performed on the 10 peptide ligand-hemagglutinin complexes obtained by molecular docking screening. The conformation of the 10 polypeptides and hemagglutinin protein complex obtained by ROSETTA FlexPepDock was used as the initial conformation, and the simulation was performed using software GROMACS2022.2 under constant temperature and pressure and periodic boundary conditions, using Amber99sb-ildn all-atom force field, placing the complex in a box of 12.3 nm x 4.75 nm x 3.8 nm, using TIP3P water model, then adding Na + and Cl -; then energy minimization was performed to remove the collision and incorrect geometry between atoms in the system; next, the restrained dynamics equilibration under the regular (NVT) ensemble for 1000 ps and the isothermal-isobaric (NPT) ensemble for 1000 ps, and finally, the unrestrained MD simulation for 100 ns were performed. All hydrogen bonds were constrained using the LINCS algorithm, and the integration step was 2 fs. The electrostatic interactions were calculated using the Particle-mesh Ewald (PME) method, with a cutoff of 1.2 nm, and the non-bonded interaction cutoff was 1.2 nm was updated every 10 steps. The simulation temperature was controlled at 295 K using the V-rescale temperature coupling method, and the pressure was controlled at 1 bar using the Parrinello-Rahman barostat method. After the simulation, the last 20 ns of the conformation was selected for the free energy calculation using the MM / PBSA method.

[0054] First, the minimum distance and the average number of atomic contacts between the polypeptides and the hemagglutinin protein during the simulation were calculated. The results of the minimum distance calculation are shown in Figure 5 , and the results of the average number of atomic contacts calculation are shown in Figure 5 . It was found that, except for HWFGGGWGGRWP, the minimum distance between the other nine polypeptides and the hemagglutinin protein was between 0.17-0.28 nm, and the contact number was stable at about 2000. In addition, as shown in Figure 5 c, the MM / PBSA results showed that, except for HWFGGGWGGRWP, the binding free energy of the other nine peptide ligands and the hemagglutinin protein was negative, indicating that the peptide ligand and the hemagglutinin protein were tightly bound during the entire molecular dynamics simulation, proving that the nine peptide ligands were likely to be effective broad-spectrum peptide ligands.

[0055] (4) Microcalorimetric mobility affinity characterization

[0056] The top three broad-spectrum peptide ligands, PWFGGGYGGEWP, HWFGGGYGGQWP, and RWYGGGWGGHFP, selected by MM / PBSA were characterized for affinity.

[0057] Specifically, the Monolith NT.115 microthermal mobility instrument of the Nano Temper Company is applied to determine the binding constant between the polypeptide and the protein. The His-HA is labeled by using the Nano Temper His-Tag labeling kit RED-tris-NTA, 100 μL of the HA protein (200 nmol / L) and 100 μL of the dye (100 nmol / L) are mixed, and the sample is incubated at room temperature for 30 minutes, centrifuged at 4°C, 15000g for ten minutes, and the supernatant is taken to a new tube. In this experiment, the final concentration of HA is 50 nmol / L, and the gradient dilution method is used to obtain 16 different concentrations from 100 μmol / L of the broad-spectrum affinity peptide ligand. The HA protein and the broad-spectrum affinity peptide ligand solution are mixed in a volume ratio of 1:1 to obtain 20 μL of the mixed solution, and the sample is taken by the capillary tube and detected on the machine. The signal is measured at medium power and 40% LED power, and the fluorescence change with the concentration is detected from high concentration to low concentration, and a series of concentration-fluorescence curves are obtained. The MO.Control and MO.Affinity Analysis software are used for fitting to obtain the dissociation constant K d of the broad-spectrum affinity peptide ligand and the HA protein. As shown in Figure 6 , the affinity of the PWFGGGYGGEWP to the three different influenza A virus HA proteins is stable at 0.58-0.73 μmol / L, the HWFGGGYGGQWP is stable at 0.94-2.01 μmol / L, and the RWYGGGWYYHFP is stable at 2.19-7.27 μmol / L; it is sufficient to prove that the affinity of the broad-spectrum affinity peptide ligand screened in the application to different influenza A virus hemagglutinin proteins is relatively high.

[0058] Example 2

[0059] A broad-spectrum affinity chromatography medium containing a broad-spectrum affinity peptide ligand, the highest affinity PWFGGGYGGEWP is selected for subsequent chromatographic separation and purification experiments, and the acquisition process and experimental process of the broad-spectrum affinity chromatography medium are as follows:

[0060] (1) Obtain a pyridyl thiol solid phase matrix

[0061] Sepharose TM6Fast Flow(IMAC chromatography packing) was washed with deionized water and DMSO and dried, 3 g of the medium was weighed and 6 mL of dimethyl sulfoxide (DMSO), 3 mL of epichlorohydrin (ECH) and 6 mL of 1 mol / L NaOH were sequentially added, and the water bath shaker was incubated at 25°C and 170 rpm for 3 h. Then, deionized water was washed and dried, and 6 mL of 0.5 mol / L PB (pH 6.3) and 3 mL of 2 mol / L Na2S2O3 were sequentially added. Incubation was carried out under the same conditions for 6 h. After the reaction was completed, deionized water was used to wash and dry, and 3 mL of 0.2 mol / L NaHCO3 and 3 mL of 1 mmol / L EDTA containing 0.5 g of dithiothreitol (DTT) were sequentially added, and the water bath shaker was continued to incubate under the same conditions for 30 min. After the reaction was completed, 0.2 mol / L NaHCO3, 1 mmol / L EDTA and acetone cleaning solution (60% acetone solution containing 1 mmol / L EDTA and 20 mmol / L NaHCO3) were sequentially used to clean, and then 5 mL of acetone cleaning solution and 10 mL of 0.3 mol / L 2,2'-dithiodipyridine solution were sequentially added, and incubation was carried out for 1 h. After the reaction was completed, the medium was sequentially washed with 60% acetone solution, 1 mmol / L EDTA and 0.1 mol / L Tris-HCl to obtain a thio-pyridylated solid phase matrix.

[0062] (2) Obtaining of broad-spectrum affinity chromatography medium

[0063] Three glycines and a cysteine were introduced at the C-terminus of the broad-spectrum affinity peptide ligand as a coupling amino acid group, and the method of introducing three glycines and a cysteine at the C-terminus of the polypeptide belongs to the routine means of those skilled in the art, which will not be described here.

[0064] 0.1 mol / L Tris-HCl was used as a cross-linking buffer, 20 mg of the above-mentioned broad-spectrum affinity peptide ligand containing a coupling amino acid group was reacted with 0.6 g of thio-pyridylated solid phase matrix in a water bath shaker for 1.5 h, and after the reaction was completed, an appropriate amount of cysteine was added and incubated under the same conditions for 30 min to block the unreacted active groups of PWFGGGYGGEWP. After the reaction was completed, the medium was named PWFSep-6FF.

[0065] (3) Obtaining of broad-spectrum affinity chromatography medium for virus isolation and purification experiment

[0066] The solid phase matrix used for the virus isolation and purification experiment was Sepharose TM 4Fast Flow, and the remaining steps were the same as steps (1) and (2) above. The finally synthesized broad-spectrum affinity chromatography medium was named PWFSep-4FF.

[0067] (4) Affinity chromatography experiment

[0068] HA and inactivated influenza vaccine were purified from ovalbumin (OVA) and Vero cell supernatant, respectively, on a Tricorn 5 / 50 column packed with 1 mL of broad-spectrum affinity chromatography medium. The adsorption buffer was 20 mmol / L PBS buffer (100 mmol / L NaCl, pH 6.0). HA and OVA were dissolved in the adsorption buffer and incubated together for 2 h, and the final concentrations of HA and OVA were 1 mg / mL and 2 mg / mL, respectively. The inactivated influenza vaccine and Vero cell supernatant were dissolved in the adsorption buffer and incubated together for 2 h, and the final concentrations of the inactivated influenza vaccine and Vero cell supernatant were 1 mg / mL and 2 mg / mL, respectively.

[0069] Specifically, after the Tricorn 5 / 5 column was filled with PWFSep-6FF medium and equilibrated with the adsorption buffer for at least 10 column volumes, the sample (OVA+HA) was loaded at a flow rate of 0.5 mL / min, eluted with 0.8 mL / min of 0.1 mol / L Gly-HCl (pH 3.0), and detected by a UV-900 detector at an absorbance of 280 nm. PWFSep-6FF had almost no adsorption of the single impurity, but produced an elution peak after the mixture was passed through, and by analyzing the elution components by SDS-PAGE, the hemagglutinin protein was successfully separated and purified, and the results are shown in FIGS. 1a and 1b, where a is a chromatography result, and b is an electrophoresis result. Figure 7

[0070] Figure 7 In a of FIG. 1, W represents effluent, E represents elution, and R represents regeneration. Figure 7 In b of FIG. 1, M represents a protein marker, F represents a feedstock, W represents a washing fraction, and E represents an eluted fraction. By measuring the protein concentration and gray scale analysis, the recovery rate of the hemagglutinin protein was 88.3%-92.2%, and the purity was 92.3%-95.7%.

[0071] ​The inactivated influenza vaccine purification experiment was carried out by using the sample (Vero cell supernatant + inactivated influenza vaccine) at the same time, except that the medium was PWFSep-4FF and the adsorption buffer was changed to 20 mmol / L PBS buffer (300 mmol / L NaCl, pH 6.0), and all other steps were the same as above. The results of the inactivated influenza vaccine purification experiment showed that under the new medium and new adsorption conditions, PWFSep-4FF had almost no adsorption on the single impurity, but the mixture was passed in, and then the elution peak was generated. Through SDS-PAGE analysis of the elution components, the inactivated influenza vaccine was successfully separated and purified, and the results are shown in Figure 8 Fig. 1, wherein a is a chromatography result diagram, and b is an electrophoresis result diagram. Figure 8 In Fig. 1b, M represents a protein marker, F represents a feedstock, FT represents a washing fraction, and E represents an eluted fraction. Through determination of the protein concentration and gray scale analysis, the recovery rate of the hemagglutinin protein was 68.3%-72.2%, and the purity was 95.9%-97.2%.

[0072] Therefore, the above experiments further show that the peptide ligand PWFGGGYGGEWP can effectively purify the influenza A vaccine, and it is a broad-spectrum affinity peptide ligand with high affinity. The affinity chromatography experiment proves that the broad-spectrum affinity chromatography medium coupled with the broad-spectrum affinity peptide ligand PWFGGGYGGEWP in the present application can obtain the influenza vaccine with high purity and yield, thereby proving that the broad-spectrum affinity chromatography medium has great potential for application in the separation and purification process of the influenza vaccine.

[0073] Obviously, the above examples are only examples for clearly illustrating, but not limitation to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A broad-spectrum affinity peptide ligand, characterized in that, It has an amino acid sequence of conserved structural units 130-loop, 150-loop, and 190-helix that bind to the influenza virus hemagglutinin receptor binding site; the amino acid sequence of the broad-spectrum affinity peptide ligand is: PWFGGGYGGEWP, HWFGGGYGGQWP, or RWYGGGWGGHFP.

2. A broad-spectrum affinity chromatography medium, characterized in that, It includes a solid matrix and a broad-spectrum affinity peptide ligand coupled to the solid matrix as described in claim 1.

3. The broad-spectrum affinity chromatography medium according to claim 2, characterized in that, The solid matrix is ​​a thiol-pyridylated solid matrix.

4. The method for preparing the broad-spectrum affinity chromatography medium according to claim 2 or 3, characterized in that, include: Obtaining broad-spectrum affinity peptide ligands and solid-phase matrices; A broad-spectrum affinity chromatography medium is obtained by mixing a solution containing a broad-spectrum affinity peptide ligand with a solid matrix and then coupling the mixture.

5. The preparation method according to claim 4, characterized in that, The coupling reaction process is as follows: an amino acid group for coupling is introduced at the C-terminus of the broad-spectrum affinity peptide ligand, and a solution containing the broad-spectrum affinity peptide ligand is prepared using a cross-linking buffer; the solid matrix is ​​thiopyridinated, and the thiopyridinated solid matrix is ​​mixed with the solution containing the broad-spectrum affinity peptide ligand and reacted in a water bath shaker.

6. The preparation method according to claim 5, characterized in that, The amino acid groups used for coupling are three glycines and one cysteine, which are sequentially linked to the C-terminus of the broad-spectrum affinity peptide ligand. And / or, the process of thiol-pyridinization of the solid matrix is ​​as follows: (1) Activation: The solid matrix is ​​washed and activated sequentially with deionized water and activating substance to obtain an activated solid matrix after the reaction; (2) Thiohydration: The activated solid matrix was reacted first with phosphate buffer and sodium thiosulfate solution, and then sodium bicarbonate solution and ethylenediaminetetraacetic acid solution containing dithiothreitol were added sequentially for the second reaction. The matrix was washed with sodium bicarbonate and ethylenediaminetetraacetic acid solution to obtain the thiohydrated solid matrix. (3) Pyridation: Clean the thiolized solid matrix, then add acetone cleaning solution and dithiodipyridine solution in sequence, mix and react, and finally clean and dry to obtain the thiol-pyrided solid matrix.

7. The preparation method according to claim 6, characterized in that, In the activation step: the activating substances are dimethyl sulfoxide, epichlorohydrin, and sodium hydroxide solution. The amount of dimethyl sulfoxide added is 0.4-2 times the mass of the solid matrix, the amount of epichlorohydrin added is 0.2-1 times the mass of the solid matrix, and the amount of sodium hydroxide solution added is 0.2-1 times the mass of the solid matrix. The volume concentration of dimethyl sulfoxide is 20%-100%, and the mass concentration of sodium hydroxide solution is 0.5-1 mol / L. The activation reaction time is 3-6 h. In the thiolization step: the concentration of phosphate buffer is 0.1-0.5 mol / L, and the pH value is 6-7; the concentration of sodium thiosulfate solution is 0.5-2 mol / L; the amount of phosphate buffer and / or sodium thiosulfate solution added is 0.4-2 times the mass of the activated solid matrix; the first reaction time is 3-24 h; the concentration of sodium bicarbonate solution is 0.1-0.2 mol / L, the concentration of ethylenediaminetetraacetic acid solution is 0.5-1 mmol / L, and the amount of dithiothreitol added to the ethylenediaminetetraacetic acid solution is 0.5-1 g / L; the amount of both solutions added is 0.5-1 times the mass of the activated solid matrix; the second reaction time is 0.5-1.5 h. In the pyridination step: the acetone cleaning solution is a 50%-70% acetone solution, containing 0.5-1 mmol / L ethylenediaminetetraacetic acid and 10-50 mmol / L sodium bicarbonate; the dithiopyridine solution has a mass concentration of 0.1-0.5 mol / L; the amount of acetone cleaning solution added is 1-2 times the mass of the thiolized solid matrix, and the amount of dithiopyridine solution added is 2-4 times the mass of the thiolized solid matrix; the reaction time is 1-3 h; in the final cleaning step, a 50%-70% acetone solution and a 0.5-1 mmol / L ethylenediaminetetraacetic acid solution are used for cleaning.

8. The use of the broad-spectrum affinity chromatography medium as described in claim 2 or 3 in the isolation and purification of influenza vaccines containing hemagglutinin protein.

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