A human enterovirus 71 VP1 protein-specific binding polypeptide and its preparation method and application

Abstract

The present invention provides a human enterovirus 71 VP1 protein-specific binding polypeptide, its preparation method and application, and belongs to the field of molecular biology technology. The amino acid sequence of the polypeptide is HNWMWYASLPDR. In response to the problem that existing hand, foot and mouth disease diagnostic and therapeutic drugs are not specific, the present invention uses the important capsid protein VP1 of human enterovirus 71 as the target molecule, and obtains a polypeptide by biopanning a phage display random 12-peptide library. The phage ELISA experiment shows that the polypeptide can specifically recognize and bind to the EV71 virus VP1 protein, has a small molecular weight, is easy to chemically synthesize, and has low immunogenicity. The successful preparation of this polypeptide has good application prospects in the research and development of products such as hand, foot and mouth disease diagnosis, treatment, and prevention.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a human enterovirus 71 VP1 protein-specific binding polypeptide, a preparation method and an application thereof. Background Art

[0002] Human enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus belonging to the genus Enterovirus A in the family Picornaviridae. It is one of the main pathogens causing hand-foot-and-mouth disease (HFMD), the primary symptoms of which are fever, diarrhea, and herpangina. Unlike typical HFMD, infection with certain EV71 strains can cause severe HFMD and lead to serious neurological complications such as aseptic meningitis, brainstem encephalitis, poliomyelitis-like paralysis, and neurogenic pulmonary edema, with high mortality and disability rates. EV71 is a widespread disease and has become a global public health concern.

[0003] EV71 has three genotypes: A, B, and C. Its genome contains approximately 7,500 nucleotides and encodes a polyprotein consisting of 2,193 amino acids. This polyprotein can be hydrolyzed into three precursor proteins: P1, P2, and P3. These precursor proteins are then cleaved by endogenous enzymes into four structural proteins: VP1, VP2, VP3, and VP4, and seven non-structural proteins: 2A, 2B, 2C, 3A, 3B, 3C, and 3D. While VP4 is embedded within the viral capsid, the remaining three structural proteins are exposed on the surface of the virus particle, directly exposed to the host immune system. VP1, a key capsid protein of EV71, determines the virus's genotype and is highly susceptible to amino acid sequence changes. It is a key determinant of virulence and a direct cause of pulmonary edema in patients. Therefore, VP1 has become a key focus of scientific research.

[0004] Since hand, foot and mouth disease (HFMD) first appeared in my country, research and development of antiviral drugs and vaccines targeting EV71 has been ongoing to prevent the spread of the virus and reduce the risk of HFMD outbreaks. Between December 2015 and May 2017, three HFMD vaccines were approved for marketing in my country by the China Food and Drug Administration: the EV71 human diploid cell vaccine from the Institute of Medical Biology, Chinese Academy of Medical Sciences, the inactivated EV71 vaccine from Beijing Sinovac Biotech Co., Ltd., and the inactivated EV71 vaccine from the Wuhan Institute of Biological Products. Vaccination has significantly reduced the chances of hand, foot and mouth disease outbreaks in my country, but there are still some problems that need to be overcome: (1) The lack of vaccination coverage and the fact that not everyone responds well to existing vaccines make it necessary to develop new drugs to diagnose and treat EV71 virus, especially in the event of large-scale infection; (2) EV71 virus is neurotropic, and its specific mechanism of action is still unclear, and whether the neurological diseases caused by severe cases can be avoided; (3) EV71 virus can cut proteins involved in immunity to avoid immune responses, and whether this problem can be overcome is also a difficulty; (4) The virus has an incubation period, and in most cases the best treatment period has been missed when the disease occurs. If the virus can be detected during the incubation period and targeted prevention and treatment can be carried out, it will not only reduce the harm to the patient but also reduce the cost of treatment.

[0005] Therefore, the preparation of a polypeptide that can specifically recognize VP1 protein is of great significance for the detection of EV71 virus and the development of therapeutic drugs. Summary of the Invention

[0006] The present invention provides a polypeptide that can specifically recognize and bind to the VP1 protein of the EV71 virus, and the amino acid sequence of the polypeptide is HNWMWYASLPDR.

[0007] The present invention also provides a method for preparing the polypeptide, comprising the following steps:

[0008] S1. Preparation of human enterovirus 71 VP1 protein: using the nucleotide sequence shown in SEQ ID No. 3 as a template, prepare the VP1 protein with the amino acid sequence shown in SEQ ID No. 4;

[0009] S2. Biopanning: VP1 protein and GST protein are added to a well plate, and E. coli strains and a culture medium containing tetracycline are inoculated and cultured; the residual liquid is poured off, a blocking solution is added, and the plate is washed several times with a washing solution; a phage solution is added for amplification, purification, and elution to obtain eluted phage; the eluted phage solution is added to an E. coli culture for amplification, and an amplified eluate is separated; a second and third round of screening are repeated to obtain screened positive plaques;

[0010] S3. Amplify positive phages: Dilute the E. coli culture, pick several well-separated blue plaques and inoculate them for amplification; then centrifuge and collect the supernatant to obtain the amplified positive phages;

[0011] S4. Determination of DNA sequence encoding phage-displayed polypeptides, deduction of amino acid sequence, and analysis of amino acid sequence homology;

[0012] S5. Perform ELISA to identify the positive phages screened out.

[0013] In an optional embodiment, step S1 includes the following steps:

[0014] S1-1, synthesizing the cDNA of the EV71 virus VP1 protein as shown in SEQ ID No. 3, amplifying the nucleotide sequence of the EV71 virus VP1 protein, and ligating the amplified sequence into the pFastBac1 vector to obtain the recombinant vector VP1-pFastBac1;

[0015] S1-2, transforming the recombinant vector VP1-pFastBac1 into Escherichia coli DH10Bac to obtain a baculovirus shuttle vector expressing VP1 protein, and selecting two shuttle vectors to transfect Sf9 cells to produce first-generation virus P1;

[0016] S1-3. The cell culture supernatant, cell lysis supernatant, and resuspended cell lysis precipitate of Sf9 cells in step S1-2 were added to a sample loading buffer, mixed evenly, boiled, and centrifuged. The supernatant was subjected to 10% SDS-PAGE gel electrophoresis to prepare a dry gel with a clear background for storage; the dry gel was subjected to Western Blot purification to obtain a P1 virus stock solution;

[0017] S1-4, select P1 virus stock with a multiplicity of infection of 1 to 10 to infect Sf9 cells, determine the optimal multiplicity of infection, and select the P1 virus stock with the optimal multiplicity of infection for expansion culture to obtain the second-generation virus P2;

[0018] S1-5. The supernatant of the lysate of Sf9 cells infected with P2 was collected and the VP1 protein was purified using His60 Ni Superflow Resin. The column was eluted in stages with PBS buffer containing 30 mmol / L, 50 mmol / L, 200 mmol / L, and 300 mmol / L imidazole. The proteins eluted in each concentration gradient were collected and analyzed by SDS-PAGE gel electrophoresis.

[0019] S1-6. Perform Western Blot analysis on the gel after SDS-PAGE analysis in step S1-5. The amino acid sequence of the purified VP1 protein obtained is shown in SEQ ID No. 4.

[0020] In an optional embodiment, step S2 includes the following steps:

[0021] S2-1. Add 200 μL of VP1-His solution and GST-His solution diluted with 0.1 mol / L NaHCO3 solution to two wells of a 96-well plate, respectively, and incubate at 4°C overnight. Inoculate 10 mL of E. coli into 20 mL of LB medium containing tetracycline and culture at 37°C. Remove the 96-well plate and remove any residual liquid. Add 200 μL of TBS blocking solution and incubate at 4°C for 2 h. Then wash several times with TBST buffer, removing any residual liquid after each wash.

[0022] S2-2, take 200 μL diluted with TBST buffer and contain 2×10 11 Add the pfu phage solution to the GST-His-coated wells blocked in step S2-1 for pre-adsorption and incubate at room temperature for 1 hour. Aspirate the pre-adsorbed liquid and transfer it to the blocked VP1-His-coated wells for binding and incubate at room temperature for 1 hour. Pour off the liquid to remove unbound phage and wash several times with TBST buffer, removing all residual liquid each time. Then elute with 100 μL of elution buffer and quickly add neutralization solution to obtain an eluted phage solution.

[0023] S2-3. Measure the titer of 1 μL of the eluted phage solution from step S2-2, add the remaining eluted phage solution to 20 mL of E. coli culture at an OD of 0.2-0.3, and culture at 37°C with shaking for 4.5 hours. Centrifuge the mixture at 4°C and 10,000 rpm for 10 minutes, remove the supernatant, and centrifuge again. Remove the 80 v / v% supernatant from the second centrifugation, add 1 / 6 of the supernatant volume of PEG8000 / NaCl solution, and let it stand at 4°C overnight.

[0024] S2-4. Take out the liquid obtained in step S2-3 and centrifuge it at 10,000 rpm at 4°C for 15 minutes, remove the supernatant; repeat the centrifugation once, and aspirate the residual supernatant; resuspend it with 1 mL of TBS buffer, centrifuge it at 10,000 rpm at 4°C for 5 minutes, remove the supernatant, add 1 / 6 of the supernatant volume of PEG8000 / NaCl solution, and bathe it on ice for 1 hour; then centrifuge it at 10,000 rpm at 4°C for 15 minutes, remove the supernatant; repeat the centrifugation once more, aspirate the residual supernatant; resuspend the precipitate with 200 μL of TBS buffer containing 0.01 w / v% NaN3, and centrifuge it at 10,000 rpm at 4°C for 1 minute. The obtained supernatant is the amplified eluate, and the first round of screening is completed;

[0025] S2-5. Recoat one well of a 96-well plate with 200 μL of VP1-His and 200 μL of GST-His protein solutions, respectively, and perform the second and third rounds of screening according to the methods in steps S2-1 to S2-4. In the second and third rounds of screening, the concentrations of VP1-His protein are 50 mg / L and 20 mg / L, respectively, and the concentration of Tween in the TBST buffer is 0.5 v / v%.

[0026] S2-6. Take 1 μL of the eluted product after the third round of screening for titer determination. Randomly pick 50 well-separated plaques from the plate with less than 100 plaques in the titer determination for subsequent amplification and purification.

[0027] In an optional embodiment, step S3 includes the following steps:

[0028] S3-1. Dilute the overnight culture of E. coli ER2738 with LB medium at a volume ratio of 1:100 and dispense 1 mL / tube into several 50 mL culture tubes.

[0029] S3-2, dip 50 well-separated blue plaques into the culture tubes, and culture at 37°C with shaking for 4.5 hours to complete amplification;

[0030] S3-3. Centrifuge the phage culture liquid amplified in step S3-2 for 30 seconds; take the supernatant and centrifuge again, and then take 80% of the supernatant of the second centrifugation to obtain the amplified positive phage.

[0031] In an optional embodiment, step S4 includes the following steps:

[0032] S4-1, taking the supernatants of several positive phages obtained after amplification in step S3 and performing DNA sequencing;

[0033] S4-2. Based on the DNA sequencing results in step S4-1, the amino acid sequence of each phage-presented peptide is deduced, and all amino acid sequences are compared with the amino acid sequences of proteins or polypeptides of known sequences for homology to confirm the types of polypeptides presented by the several positive phages.

[0034] In an optional embodiment, step S5 includes the following steps:

[0035] S5-1. Coat a 96-well ELISA plate with 100 mg / L VP1-His protein solution (200 μL per well). Set up a 100 mg / L GST-His and 1 w / v% BSA-coated well for each VP1-His-coated well as a negative control. Incubate overnight at 4°C.

[0036] S5-2, pour off the coating solution, add blocking solution, and block at 4°C for 2 hours; then pour off the blocking solution and wash several times with TBST buffer, removing all residual liquid after each wash;

[0037] S5-3. According to the sequencing results of step S4, several purified positive phages were added to the VP1-His, GST-His and BSA coated wells respectively. The titer of positive phage in each well was 1×10 9 pfu, incubated at room temperature for 2 h; washed several times with TBST buffer, added 100 μL of HRP-labeled mouse anti-M13 phage mAb to each well, incubated at room temperature for 1 h; washed several times with TBST buffer, developed with OPD, and measured the absorbance at 490 nm; the concentration of the HRP-labeled mouse anti-M13 phage mAb was 0.1 μg / mL.

[0038] The polypeptide capable of specifically recognizing and binding to the EV71 virus VP1 protein prepared by the above method can be used to prepare products for detecting EV71 virus and / or prepare drugs for treating EV71 virus.

[0039] The beneficial effects of the present invention are as follows: the present invention prepares a human enterovirus 71 VP1 protein-specific binding polypeptide, which is a new polypeptide that specifically blocks EV71. It can specifically recognize VP1 protein, has a small molecular weight, is easy to chemically synthesize, and has low immunogenicity. It has good potential application value in the fields of rapid detection of EV71 virus, research and development of new and efficient specific drugs for the treatment of hand, foot and mouth disease, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The figure shows the results of SDS-PAGE and WB identification of EV71 virus VP1 protein;

[0041] Figure 2This is the result of SDS-PAGE analysis of the purification of EV71 virus VP1 protein;

[0042] Figure 3 This is the identification diagram of the purification results of EV71 virus VP1 protein by Western Blot method;

[0043] Figure 4 It is the absorbance value of the EV71 virus VP1 protein-specific binding polypeptide prepared by the present invention at 490nm. DETAILED DESCRIPTION

[0044] The following is a clear and complete description of the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Any equivalent transformation or substitution made by those skilled in the art based on the following embodiments is within the scope of protection of the present invention.

[0045] The sources or main components of the reagents and biological materials used in the following examples are as follows:

[0046] pFastBac1 vector: Pujian Biotechnology (Wuhan) Technology Co., Ltd.;

[0047] Escherichia coli DH10Bac: Pujian Biotechnology (Wuhan) Technology Co., Ltd.;

[0048] Sf9 cells: Pujian Biotechnology (Wuhan) Technology Co., Ltd.;

[0049] His60 Ni Superflow Resin: 10 mL, Pujian Biotechnology (Wuhan) Technology Co., Ltd.

[0050] TBS buffer: containing 20 mmol / L Tris-HCl buffer, 150 mmol / L NaCl, pH = 7.5;

[0051] TBST buffer: contains the above-mentioned TBS buffer and 0.05 v / v% Tween 20, unless otherwise specified;

[0052] PBS buffer: 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH = 7.4;

[0053] Escherichia coli ER2738: New England Biolab, Ph.D.-12 TM ;

[0054] HRP-labeled mouse anti-M13 phage mAb: New England Biolab;

[0055] Goat anti-mouse IgG-HRP: Abcam;

[0056] RIPA lysis buffer: Beyotime Biotechnology Co., Ltd., P0013;

[0057] 2x loading buffer: Beyotime Biotechnology Co., Ltd., product number P0015B;

[0058] PEG-8000 / NaCl: Contains 20w / v% PEG-8000, 2.5mol / L NaCl.

[0059] Example 1

[0060] The present invention provides a method for preparing a polypeptide specifically binding to the human enterovirus 71 VP1 protein, comprising the following steps:

[0061] Expression and purification of S1 and EV71 virus VP1 proteins

[0062] S1-1, EV71 virus VP1 protein nucleotide sequence amplification

[0063] The cDNA encoding the EV71 VP1 protein (nucleotide sequence shown in SEQ ID No. 3) was synthesized and amplified by PCR to obtain a DNA fragment encoding the VP1 protein. This DNA fragment was then ligated into the pFastBac1 vector to generate the recombinant vector VP1-pFastBac1. To facilitate purification, a His-tag was added to the C-terminus of the VP1 protein amino acid sequence. This process was performed by Pujian Biotechnology (Wuhan) Technology Co., Ltd.

[0064] S1-2, transforming the recombinant vector VP1-pFastBac1 into Escherichia coli DH10Bac to obtain a baculovirus shuttle vector expressing VP1 protein, and selecting two shuttle vectors to transfect Sf9 cells to produce first-generation virus P1;

[0065] S1-3. Collect the infected cells in S1-2 and centrifuge at 1000 rpm for 5 minutes, collect the cell culture supernatant and cell pellet respectively, add RIPA lysis buffer to resuspend the cell pellet, repeatedly pipette to lyse the cells, centrifuge at 1200 rpm for 10 minutes, collect the cell lysis supernatant and cell lysis pellet respectively; take 30 μL of cell culture supernatant, cell lysis supernatant and cell lysis pellet resuspended in TBS buffer, add 30 μL of 2x loading buffer and mix well. Boil in boiling water for 5 minutes, centrifuge at 12000rpm for 5 minutes, take 10 μL supernatant and load it on 10w / v% separation gel and 6w / v% stacking gel. After loading, the voltage is 90V for about 20 minutes. After the sample enters the separation gel, the voltage is increased to 110V for about 1.5 hours, and it is stained with 0.5w / v% Coomassie Brilliant Blue R-250 for 2 hours. After decolorization until the background is clear, dry gel is prepared for storage; after the end of SDS-PAGE, the gel is removed and, according to the Bio-Rad product instructions, the gel is placed close to the cathode side and the nitrocellulose (NC) membrane is placed close to the anode side. Electrophoresis is performed at a constant voltage of 100V for 1 hour in pre-cooled transfer buffer (25mmol / LTris, 192mmol / L Glycine, 20v / v% methanol) to transfer the protein to the NC membrane; after the end of electrophoresis, the NC membrane is removed and washed with TBST buffer (containing 20mmol / L HCl, pH = 7.5) for 1 hour. After washing with Tris-HCl buffer (150 mmol / L NaCl, 0.05 v / v% Tween20), the membrane was immersed in blocking solution (TBST buffer containing 2 w / v% BSA) at 37°C for 1 hour, washed with TBST buffer 3 times at room temperature, 5 minutes each time, mouse anti-His monoclonal antibody (diluted with TBS buffer, final concentration of 0.1 μg / mL) was added, incubated at 37°C for 1 hour, washed 3 times with TBST buffer, goat anti-mouse IgG-HRP was added, incubated at 37°C for 1 hour, washed 3 times with TBST buffer, and then washed 3 times with TBS buffer (20 mmol / L Tris-HCl, 150 mmol / L NaCl, pH = 7.5). The NC membrane was immersed in the color developing solution, developed for an appropriate time in the dark at room temperature, and rinsed with distilled water to terminate the reaction. The identification results are shown in FIG. Figure 1 As shown; Figure 1 In the middle, the left picture is the result of SDS-PAGE gel electrophoresis, and the right picture is the result of Western Blot identification; Medium represents cell culture medium, NPE represents the supernatant of Sf9 cell lysis, DPE represents the precipitate of Sf9 cell lysis, MW represents the protein molecular weight marker, Indicates negative control, 1 indicates clone 1, 2 indicates clone 2; + indicates positive control (other proteins with His tags). Figure 1 As can be seen in the figure, a large amount of VP1 protein is present in the lysis supernatant of clone 2;

[0066] S1-4. Infect Sf9 cells with P1 virus stocks at multiplicities of infection (MOI) of 1, 5, and 10, respectively. Determine the optimal MOI of infection to be 1. Infect Sf9 cells at this MOI and culture at 28°C for 24 hours to obtain the second-generation virus P2.

[0067] S1-5, using His60 Ni Superflow Resin was used to purify VP1: infected cells in S1-4 were collected, centrifuged at 1000 rpm for 5 min, the cell pellets were collected, the cell pellets were resuspended with RIPA lysis buffer, the cells were repeatedly blown and lysed, and centrifuged at 1200 rpm for 10 min. The cell lysate supernatant was collected, the column and the supernatant were fully combined and incubated for 2 h; the unadsorbed sample was washed away with 20 column volumes of equilibration buffer (PBS buffer at pH = 7.5) at a flow rate of 1-2 mL / min until the effluent was spotted with Coomassie Brilliant Blue G250 and no blue color was shown; the protein adsorbed on the column was eluted in stages with PBS buffer containing 30 mmol / L, 50 mmol / L, 200 mmol / L, and 300 mmol / L imidazole for about 5-10 column volumes, the proteins eluted in each concentration gradient were collected, and the eluate was analyzed by SDS-PAGE (separation gel concentration was 10 w / v%, stacking gel concentration was 6 w / v%). The results are shown in FIG. Figure 2 As shown; Figure 2 In the figure, MW indicates protein molecular weight marker; IN indicates the loaded sample; FT indicates the through peak; W1 to W3 indicate the collected washing solutions 1 to 3, and E1 to E9 indicate the collected eluates 1 to 9. Figure 2 It can be seen that VP1 protein with good purity appeared in the eluates collected from No. 3 to No. 9;

[0068] S1-6. Western Blot analysis of purified VP1 protein

[0069] After the SDS-PAGE in step S1-5 is completed, the gel is removed and, according to the product instructions of Bio-Rad, the gel is placed near the cathode side and the nitrocellulose (NC) membrane is placed near the anode side, and electrophoresis is performed at a constant voltage of 100 V for 1 hour in pre-cooled transfer buffer (containing 25 mmol / L Tris, 192 mmol / L Glycine, and 20 v / v% methanol) to transfer the protein to the NC membrane. After the electrophoresis is completed, the NC membrane is removed and washed with TBST buffer (containing 20 mmol / L Tris-HCl, 150 mmol / L After washing with 1% NaCl, 0.05v / v% Tween20, pH=7.5), the membrane was immersed in blocking solution (TBST washing solution containing 2w / v% BSA) at 37°C for 1 hour, washed with TBST buffer 3 times at room temperature, each time for 5 minutes, and mouse anti-His monoclonal antibody (diluted with TBS, final concentration of 0.1μg / mL) was added respectively, incubated at 37°C for 1 hour, washed again with TBST buffer 3 times, and goat anti-mouse IgG-AP was added, incubated at 37°C for 1 hour, washed again with TBST buffer 3 times, and then washed 3 times with TBS buffer (containing 20mmol / L Tris-HCl, 150mmol / L NaCl, pH=7.5). The NC membrane was immersed in color developing solution, developed for an appropriate time at room temperature in the dark, and rinsed with distilled water to terminate the reaction; the identification results are as follows: Figure 3 As shown, Figure 3 MW in the middle indicates protein molecular weight marker, and 2 μg indicates the loading amount; Figure 3 As can be seen from the figure, the VP1 protein was well purified. The amino acid sequence of the VP1 protein is shown in SEQ ID No. 4.

[0070] S2. Bio-panning

[0071] S2-1. Add 200 μL of VP1-His protein solution and GST-His protein solution diluted with 0.1 mol / L NaHCO3 (pH = 8.6) to two wells of a 96-well plate, respectively, with the protein concentration of each being 100 μg / mL; place in a humidified chamber with gentle shaking and incubate at 4°C overnight; pre-inoculate 10 mL (for titer measurement) of Escherichia coli ER2738 into 20 mL of LB medium (for phage amplification) containing tetracycline (main components include: The cells were incubated at 37°C in a 96-well plate (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride). The 96-well plate was removed, the liquid was poured off, and the plate was inverted on a clean paper towel to gently remove any residual liquid. 200 μL of TBS buffer containing 1 w / v% BSA was added and the cells were incubated at 4°C for 2 h. The cells were then washed six times with TBST buffer (TBS + 0.1 v / v% Tween), each time inverting the 96-well plate on a clean paper towel to gently remove any residual liquid.

[0072] S2-2, dilute 200 μL of 2x10 11 pfu phage (New England Biolab, Ph.D.-12 TM The solution of the phage display peptide library was added to the blocked GST-His coated wells for pre-adsorption and incubated at room temperature for 1 hour. The pre-adsorbed liquid was aspirated and transferred to the blocked VP1-His coated wells for binding and incubated at room temperature for 1 hour. The liquid was poured off to remove unbound phages, and the plate was washed 10 times with TBST buffer. Each time, the 96-well plate was inverted on a clean paper towel to gently remove any residual liquid. The bound phages were eluted with 100 μL of elution buffer (0.2 mol / L Glycine-HCl, 10 g / L BSA, pH = 2.2) and quickly neutralized with neutralization buffer (1 mol / L Tris-HCl, pH = 9.1) to obtain an eluted phage solution.

[0073] S2-3. Take 1 μL of the eluted phage solution obtained in step S2-2 to measure the titer, add the remaining eluted phage solution to 20 mL of ER2738 culture at an OD of 0.2-0.3, and culture at 37°C with vigorous shaking for 4.5 hours to amplify; pour the mixture of amplified phage and E. coli ER2378 into a centrifuge tube, and centrifuge at 10,000 rpm for 10 minutes at 4°C; transfer the supernatant to a new centrifuge tube, centrifuge again using the same method, and take 80 v / v% of the supernatant and add it to a new centrifuge tube; add 1 / 6 of the supernatant volume of PEG8000 / NaCl to the centrifuge tube, and let it stand at 4°C overnight;

[0074] S2-4. The next day, take out the PEG8000 / NaCl precipitated liquid from step S2-3, centrifuge it at 10,000 rpm for 15 min at 4°C, and carefully pour off the supernatant; centrifuge it again quickly in the same way, and use a micropipette to remove the remaining supernatant; use 1 mL Resuspend the pellet in TBS buffer, transfer the resuspension to a small centrifuge tube, and centrifuge at 10,000 rpm for 5 minutes at 4°C to precipitate any remaining cells; transfer the supernatant to a new small centrifuge tube, add 1 / 6 the volume of the supernatant, and incubate on ice for 1 hour; centrifuge at 10,000 rpm for 15 minutes at 4°C, carefully discard the supernatant, and centrifuge again quickly using the same method. Aspirate any remaining supernatant with a micropipette; resuspend the pellet in 200 μL of TBS buffer containing 0.01 w / v% NaN3, and centrifuge at 10,000 rpm for 1 minute to precipitate any insoluble material; transfer the supernatant to a new centrifuge tube, which is the amplified eluate; take 1 μL of the eluate for titer measurement, and store the remaining liquid at 4°C;

[0075] S2-5. Recoat one well of a 96-well plate with 200 μL of VP1-His protein solution and 200 μL of GST-His protein solution, respectively. Perform the second and third rounds of screening according to the methods in steps S2-1 to S2-4. To improve the stringency of peptide screening, the concentration of GST-His protein in the second and third rounds of screening was the same as that in the first round of screening, while the concentration of VP1-His protein was reduced to 50 mg / L and 20 mg / L, respectively. The concentration of Tween in the TBST buffer was increased to 0.5 v / v%.

[0076] S2-6. The eluted product after the third round of bio-panning does not need to be amplified. Take 1 μL of the eluted product for titer determination, and store the remaining eluted product at 4°C. Randomly pick 50 well-separated plaques from the plate with less than 100 plaques in the titer determination for subsequent amplification and purification. At this time, it should be noted that the culture time of the picked plaques should not exceed 18 hours.

[0077] S3. Amplification of positive phages

[0078] S3-1. Dilute the overnight culture of Escherichia coli ER2738 with LB medium at a volume ratio of 1:100 and dispense into 50 50 mL culture tubes at 1 mL / tube;

[0079] S3-2. Use a sterile toothpick to pick up 50 well-separated blue plaques obtained in step S2-6 and place them in the above culture tubes. Cultivate with vigorous shaking at 37°C for 4.5 hours to amplify.

[0080] S3-3. Pour the phage culture liquid amplified in step S3-2 into a centrifuge tube and centrifuge for 30 seconds; transfer the centrifugal supernatant to a new centrifuge tube, centrifuge again using the same method, and take 80 v / v% of the supernatant, which is the amplified positive phage; the amplified positive phage can be stored at 4°C for several weeks. For long-term storage, add sterile glycerol to a final concentration of 50 v / v% and store at -20°C.

[0081] S4. Sequence determination of phage-displayed peptides

[0082] S4-1. Phage single-stranded DNA sequence determination

[0083] Take 10 μL of each of the 50 phage supernatants amplified in step S3 and send them to Suzhou Genewise Biotechnology Co., Ltd. for DNA sequencing;

[0084] S4-2. Amino acid sequence deduction and amino acid homology analysis of phage-displayed peptides

[0085] Based on the DNA sequencing results, the amino acid sequence of the peptide presented by each phage was deduced, and the amino acid sequences of 50 different clones were compared; the results showed that the 50 phage clones presented a total of 12 polypeptide sequences, and these 12 polypeptide sequences had no homology with known protein or polypeptide sequences, and were newly discovered polypeptide sequences.

[0086] S5. Phage ELISA identification

[0087] S5-1. Coat three wells of a 96-well enzyme-linked microtiter plate with 100 mg / L VP1-His protein solution (200 μL per well). Simultaneously, set up a negative control well coated with 100 mg / L GST-His and 1 w / v% BSA for each VP1-His-coated well. Incubate overnight at 4°C.

[0088] S5-2. Pour off the coating solution and add blocking solution (containing 5 mg / L BSA, 0.1 mol / L NaHCO3, pH = 8.6) and block at 4°C for 2 h. Pour off the blocking solution and wash six times with TBST buffer. Each time, invert the 96-well plate on a clean paper towel to gently remove any residual liquid.

[0089] S5-3, according to the sequence analysis results in step S4, 12 purified positive phages were added to the corresponding wells, and each phage was added to 3 VP1-His, 3 GST-His and 3 BSA coated wells (1×10 9 pfu / well) at room temperature for 2 h; washed 6 times with TBST buffer, added 100 μL HRP-labeled mouse anti-M13 phage mAb to each well (diluted with TBST, final concentration of 1 μg / mL), incubated at room temperature for 1 h; washed 6 times with TBST buffer, developed with OPD (o-phenylenediamine), and measured the absorbance at 490 nm. The results are shown in Figure 2. Figure 4 shown; from Figure 4 It can be seen that the screened polypeptide has a high specificity for the EV71 virus VP1 protein and can recognize and bind to the EV71 virus VP1 protein; the amino acid sequence of the polypeptide is shown in SEQ ID No.1, and the nucleotide sequence is shown in SEQ ID No.2.

[0090] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It will be apparent to anyone skilled in the art that various modifications and variations of the present invention are possible. Any simple equivalent variations and modifications made in accordance with the scope of protection of the present invention and the contents of the specification are intended to be included within the scope of protection of the present invention. Sequence Listing <110> Hubei University of Arts and Science <120> A human enterovirus 71 VP1 protein-specific binding polypeptide and its preparation method and application <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <400> 1 His Asn Trp Met Trp Tyr Ala Ser Leu Pro Asp Arg 1 5 10 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <400> 2 cataattgga tgtggtatgc gtctttgccg gatagg 36 <210> 3 <211> 942 <212> DNA <213> Artificial Sequence <400> 3 ggatccgccg ccaccatggg tgaccgtgtc gctgacgtca tcgaaagctc catcggtgac 60 agcgtgtccc gtgctctgac tcaggctcta ccagcaccca caggccagaa cacacaggtg 120 agcagtcatc gactggatac aggcaaggtt ccagcactcc aagctgctga aattggagca 180 tcatcaaatg ctagtgacga gagcatgatt gagacacgct gtgttcttaa ctcgcacagt 240 acagctgaga ccactcttga tagtttcttc agcagggcgg gattagttgg agagatagat 300 ctccctctta agggcacaac taacccaaat ggttatgcca actgggacat agacataaca 360 ggttacgcgc aaatgcgtag aaaggtagag ctattcacct acatgcgctt tgatgcagag 420 ttcacttttg ttgcgtgcac acccaccggg gaagttgtcc cacaattgct ccaatatatg 480 tttgtgccac ctggagcccc taagccagat tctagggaat cccttgcatg gcaaaccgcc 540 accaacccct cagtttttgt caagctgtca gaccctccag cgcaggtttc agtgccattc 600 atgtcacctg cgagtgctta ccaatggttt tatgacggat atcccacatt cggagaacac 660 aaacaggaga aagatcttga atacggggca tgtcctaata acatgatggg cacattctca 720 gtgcggacag tggggacctc caagtccaag tacccattag tggttaggat ttacatgaga 780 atgaagcacg tcagggcgtg gatacctcgc ccgatgcgca accagaacta cctgttcaaa 840 gccaacccaa attatgctgg caactccatt aagccaactg gtgccagtcg cgctgccatc 900 actactctgg gttcccacca ccaccaccat cactgactcg ag 942 <210> 4 <211> 306 <212> PRT <213> Artificial Sequence <400> 4 Met Gly Asp Arg Val Ala Asp Val Ile Glu Ser Ser Ile Gly Asp Ser 1 5 10 15 Val Ser Arg Ala Leu Thr Gln Ala Leu Pro Ala Pro Thr Gly Gln Asn 20 25 30 Thr Gln Val Ser Ser His Arg Leu Asp Thr Gly Lys Val Pro Ala Leu 35 40 45 Gln Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala Ser Asp Glu Ser Met 50 55 60 Ile Glu Thr Arg Cys Val Leu Asn Ser His Ser Thr Ala Glu Thr Thr 65 70 75 80 Leu Asp Ser Phe Phe Ser Arg Ala Gly Leu Val Gly Glu Ile Asp Leu 85 90 95 Pro Leu Lys Gly Thr Thr Asn Pro Asn Gly Tyr Ala Asn Trp Asp Ile 100 105 110 Asp Ile Thr Gly Tyr Ala Gln Met Arg Arg Lys Val Glu Leu Phe Thr 115 120 125 Tyr Met Arg Phe Asp Ala Glu Phe Thr Phe Val Ala Cys Thr Pro Thr 130 135 140 Gly Glu Val Val Pro Gln Leu Leu Gln Tyr Met Phe Val Pro Pro Gly 145 150 155 160 Ala Pro Lys Pro Asp Ser Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr 165 170 175 Asn Pro Ser Val Phe Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser 180 185 190 Val Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly 195 200 205 Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr Gly 210 215 220 Ala Cys Pro Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly 225 230 235 240 Thr Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr Met Arg Met 245 250 255 Lys His Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn Gln Asn Tyr 260 265 270 Leu Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile Lys Pro Thr 275 280 285 Gly Ala Ser Arg Ala Ala Ile Thr Thr Leu Gly Ser His His His His 290 295 300 His His 305

Claims

1. A polypeptide specifically binding to the VP1 protein of human enterovirus 71, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. The nucleotide encoding the human enterovirus 71 VP1 protein-specific binding polypeptide according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID No.

2.

3. The method for preparing a polypeptide specifically binding to human enterovirus 71 VP1 protein according to claim 1, characterized in that: The following steps are involved: S1. Preparation of human enterovirus 71 VP1 protein: using the nucleotide sequence shown in SEQ ID No. 3 as a template, prepare the VP1 protein with the amino acid sequence shown in SEQ ID No. 4; S2. Biopanning: VP1 protein and GST protein are added to a well plate, and E. coli strains and a culture medium containing tetracycline are inoculated and cultured; the residual liquid is poured off, a blocking solution is added, and the plate is washed several times with a washing solution; a phage solution is added for amplification, purification, and elution to obtain eluted phage; the eluted phage solution is added to an E. coli culture for amplification, and an amplified eluate is separated; a second and third round of screening are repeated to obtain screened positive plaques; S3. Amplify positive phages: dilute the E. coli culture, pick several well-separated blue plaques and inoculate them for amplification; then centrifuge and collect the supernatant to obtain the amplified positive phages; S4. Determination of DNA sequence encoding phage-displayed polypeptides, deduction of amino acid sequence, and analysis of amino acid sequence homology; S5. Perform ELISA to identify the positive phages screened out.

4. The preparation method according to claim 3, characterized in that Step S1 includes the following steps: S1-1, synthesizing the cDNA of the EV71 virus VP1 protein as shown in SEQ ID No. 3, amplifying the nucleotide sequence of the EV71 virus VP1 protein, and ligating the amplified sequence into the pFastBac1 vector to obtain the recombinant vector VP1-pFastBac1; S1-2, transforming the recombinant vector VP1-pFastBac1 into Escherichia coli DH10Bac to obtain a baculovirus shuttle vector expressing VP1 protein, and selecting two shuttle vectors to transfect Sf9 cells to produce first-generation virus P1; S1-3. The cell culture supernatant, cell lysis supernatant, and resuspended cell lysis precipitate of Sf9 cells in step S1-2 were added to the sample loading buffer, mixed evenly, boiled, and centrifuged. The supernatant was subjected to 10% SDS-PAGE gel electrophoresis to prepare a dry gel with a clear background for storage; the dry gel was subjected to Western Blot purification to obtain the P1 virus stock solution; S1-4, select P1 virus stock with a multiplicity of infection of 1-10 to infect Sf9 cells, determine the optimal multiplicity of infection, and select the P1 virus stock with the optimal multiplicity of infection for expansion culture to obtain the second-generation virus P2; S1-5. The supernatant of the lysate of Sf9 cells infected with P2 was collected and the VP1 protein was purified using His60 Ni Superflow Resin. The column was eluted in stages with PBS buffer containing 30 mmol / L, 50 mmol / L, 200 mmol / L, and 300 mmol / L imidazole. The proteins eluted in each concentration gradient were collected and analyzed by SDS-PAGE gel electrophoresis. S1-6. Perform Western Blot analysis on the gel after SDS-PAGE analysis in step S1-5 to obtain the amino acid sequence shown as SEQ ID No.

4.

5. The preparation method according to claim 3, characterized in that Step S2 includes the following steps: S2-1. Add 200 μL of VP1-His solution and GST-His solution diluted with 0.1 mol / L NaHCO3 solution to two wells of a 96-well plate, respectively, and incubate at 4°C overnight. Inoculate 10 mL of E. coli into 20 mL of LB medium containing tetracycline and incubate at 37°C. Remove the 96-well plate and remove any residual liquid. Add 200 μL of TBS blocking solution and incubate at 4°C for 2 h. Then wash several times with TBST buffer, removing any residual liquid after each wash. S2-2, take 200 μL diluted with TBST buffer and contain 2×10 11 Add the pfu phage solution to the GST-His-coated wells blocked in step S2-1 for pre-adsorption and incubate at room temperature for 1 hour. Aspirate the pre-adsorbed liquid and transfer it to the blocked VP1-His-coated wells for binding. Incubate at room temperature for 1 hour. Pour off the liquid to remove unbound phage and wash several times with TBST buffer, removing all residual liquid each time. Then elute with 100 μL of elution buffer and quickly add neutralization solution to obtain an eluted phage solution. S2-3. Measure the titer of 1 μL of the eluted phage solution from step S2-2, add the remaining eluted phage solution to 20 mL of E. coli culture at an OD of 0.2-0.3, and culture at 37°C with shaking for 4.5 hours. Centrifuge the mixture at 4°C and 10,000 rpm for 10 minutes, remove the supernatant, and centrifuge again. Remove the 80 v / v% supernatant from the second centrifugation, add 1 / 6 of the supernatant volume of PEG8000 / NaCl solution, and let it stand at 4°C overnight. S2-4. Take out the liquid obtained in step S2-3 and centrifuge it at 10,000 rpm at 4°C for 15 minutes, remove the supernatant; repeat the centrifugation once, and aspirate the residual supernatant; resuspend it with 1 mL of TBS buffer, centrifuge it at 10,000 rpm at 4°C for 5 minutes, remove the supernatant, add 1 / 6 of the supernatant volume of PEG8000 / NaCl solution, and bathe it on ice for 1 hour; then centrifuge it at 10,000 rpm at 4°C for 15 minutes, remove the supernatant; repeat the centrifugation once more, aspirate the residual supernatant; resuspend the precipitate with 200 μL of TBS buffer containing 0.01 w / v% NaN3, and centrifuge it at 10,000 rpm at 4°C for 1 minute. The obtained supernatant is the amplified eluate, and the first round of screening is completed; S2-5. Coat one well of each 96-well plate with 200 μL of VP1-His and 200 μL of GST-His protein solutions, respectively. Perform the second and third rounds of screening according to the methods in steps S2-1 to S2-4. In the second and third rounds of screening, the concentrations of VP1-His protein are 50 mg / L and 20 mg / L, respectively, and the concentration of Tween in TBST buffer is 0.5 v / v. S2-6. Take 1 μL of the eluted product after the third round of screening for titer determination. Randomly pick 50 well-separated plaques from the plate with less than 100 plaques in the titer determination for subsequent amplification and purification.

6. The preparation method according to claim 3, characterized in that Step S3 includes the following steps: S3-1. Dilute the overnight culture of E. coli ER2738 with LB medium at a volume ratio of 1:100 and dispense 1 mL / tube into several 50 mL culture tubes. S3-2, dip 50 well-separated blue plaques into the culture tubes, and culture at 37°C with shaking for 4.5 hours to complete amplification; S3-3. Centrifuge the phage culture liquid amplified in step S3-2 for 30 seconds; take the supernatant and centrifuge again, and then take 80% of the supernatant of the second centrifugation to obtain the amplified positive phage.

7. The preparation method according to claim 3, characterized in that Step S4 includes the following steps: S4-1, taking the supernatants of several positive phages obtained after amplification in step S3 and performing DNA sequencing; S4-2. Based on the DNA sequencing results in step S4-1, the amino acid sequence of each phage-presented peptide is deduced, and all amino acid sequences are compared with the amino acid sequences of proteins or polypeptides of known sequences for homology to confirm the types of polypeptides presented by the several positive phages.

8. The preparation method according to claim 3, characterized in that Step S5 includes the following steps: S5-1. Coat a 96-well ELISA plate with 100 mg / L VP1-His protein solution (200 µL per well). Set up a 100 mg / L GST-His and 1 w / v% BSA-coated well for each VP1-His-coated well as a negative control. Incubate overnight at 4°C. S5-2, pour off the coating solution, add blocking solution, and block at 4°C for 2 hours; then pour off the blocking solution and wash several times with TBST buffer, removing all residual liquid after each wash; S5-3. According to the sequencing results of step S4, several purified positive phages were added to the VP1-His, GST-His and BSA coated wells respectively. The titer of positive phage in each well was 1×10 9 pfu, incubated at room temperature for 2 h; washed several times with TBST buffer, added 100 μL of HRP-labeled mouse anti-M13 phage mAb to each well, incubated at room temperature for 1 h; washed several times with TBST buffer, developed with OPD, and measured the absorbance at 490 nm; the concentration of the HRP-labeled mouse anti-M13 phage mAb was 0.1 μg / mL.

9. Use of the polypeptide according to claim 1 in the preparation of a product for detecting EV71 virus.

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