Polypeptide with binding affinity to hpv18 e6 protein and application thereof

Abstract

The present application provides a polypeptide with binding affinity to HPV18 E6 protein, taking the Z domain of staphylococcal protein A as a scaffold, randomly mutating the surface amino acid residues to simulate antibody binding sites, constructing a mutant library through phage display technology, screening the library through affinity with HPV18 E6 as a target antigen, and finally obtaining a polypeptide with high affinity to HPV18 E6 through a large number of screening work. Through in-depth research, the present application first discloses a polypeptide with binding affinity to the E6 protein of HPV18. In addition, the present application also provides the diagnostic or therapeutic use of the polypeptide as a drug or molecular targeting reagent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly to a polypeptide with binding affinity to HPV18E6 protein and application thereof. BACKGROUND

[0002] Cervical cancer is the fourth most common cancer in women worldwide and the fourth leading cause of cancer death, with 604,000 new cases and 342,000 deaths in 2020. The occurrence and development of cervical cancer are highly related to persistent infection of human papillomavirus (HPV), among which the most common are high-risk serotypes HPV16 and HPV18. HPV16 accounts for about 62.6% of cervical tumors, and HPV18 is associated with about 37% of cervical adenocarcinoma and 12% of cervical squamous cell carcinoma.

[0003] HPV is a double-stranded DNA virus without envelope. The virus expresses early proteins such as E1, E2, E4, E5, E6 and E7, and late proteins such as major and minor capsid proteins L1 and L2. Integration of viral DNA into the host genome for viral replication is essential for the carcinogenic process induced by HPV. Genomic instability caused by viral oncoproteins E6 and E7 may be a key factor in triggering integration, which accelerates the occurrence of double-strand breaks in viral genomes and host DNA. Notably, HPV18E6, as a non-cellular oncoprotein, is not expressed in normal cells; E6 protein mainly causes degradation of tumor suppressor gene P53 by binding to cellular ubiquitin ligase (E6AP), which blocks cell cycle arrest or apoptosis, and also leads to increased DNA damage and telomerase activation, which contributes to immortalization and cancer development. Therefore, HPV18E6 oncoprotein is one of the ideal targets for HPV18-related tumor molecular diagnosis and targeted therapy.

[0004] Currently, cervical cancer is mainly diagnosed by detecting HPV DNA, but its diagnostic specificity is poor. Early cervical cancer has a good prognosis and can be cured by surgery, radiotherapy or comprehensive treatment, while advanced cervical cancer is mainly treated by local radiotherapy to improve the 5-year survival rate of patients. Therefore, it is necessary to study specific diagnostic methods and new treatment regimens.

[0005] Based on the above description, there is still an urgent need in the art to study new drugs or new methods for targeted treatment of HPV infection and related tumors to improve the current clinical situation. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a polypeptide with binding affinity to HPV18E6 protein and application thereof.

[0007] In a first aspect of the present application, there is provided a polypeptide having binding affinity to HPV18 E6 protein, wherein the polypeptide is a polypeptide having 12-20 amino acid variations based on the amino acid sequence of Z domain of Staphylococcal protein A, and the amino acid sequence of the polypeptide having binding affinity to HPV18 E6 protein is shown in any one of SEQ ID NO: 2-4.

[0008] In a preferred embodiment, the polypeptide having binding affinity to HPV18 E6 protein has a KD value of 1 x 10 -3 M to 1 x 10 -6 M.

[0009] In another aspect of the present application, there is provided a targeting molecule targeting HPV18 E6, wherein the targeting molecule comprises the polypeptide as described above, and a conjugate linked to the polypeptide, wherein the conjugate comprises, but is not limited to, a cysteine residue, a polypeptide tag, a drug inhibiting HPV18 E6, or a detectable label, wherein the detectable label comprises, but is not limited to, a fluorescent label, an enzyme, biotin, or a radioisotope.

[0010] In another aspect of the present application, there is provided an isolated polynucleotide encoding the polypeptide having binding affinity to HPV18 E6 protein as described above.

[0011] In another aspect of the present application, there is provided a polynucleotide encoding the targeting molecule targeting HPV18 E6, and wherein the conjugate is a peptide, and the polypeptide tag comprises, but is not limited to, a His tag, an HA tag, a Myc tag, a Flag tag.

[0012] In another aspect of the present application, there is provided a recombinant vector comprising the polynucleotide as described above.

[0013] In another aspect of the present application, there is provided a host cell comprising the recombinant vector as described above, or comprising or having integrated into its genome the polynucleotide as described above.

[0014] In another aspect of the present application, there is provided use of the polypeptide having binding affinity to HPV18 E6 protein or the targeting molecule targeting HPV18 E6 for the manufacture of a medicament for treating a disease infected by HPV18 E6 virus or a tumor positive for HPV18 E6 expression; or for the manufacture of a detection reagent for detecting a disease infected by HPV18 E6 virus; or for the manufacture of a diagnostic reagent for diagnosing a disease infected by HPV18 E6 virus or a tumor positive for HPV18 E6 expression.

[0015]

[0016] ​In another aspect of the present application, the targeting molecule targeting HPV18 E6, the conjugate is an anti-tumor drug or a drug inhibiting HPV18 E6 virus, and the polypeptide having binding affinity to HPV18 E6 protein or the targeting molecule targeting HPV18 E6 is used for treating HPV18 E6 virus infection disease or HPV18 E6 expression positive tumor.

[0017] In another preferred embodiment, the targeting molecule targeting HPV18 E6, the conjugate is a detectable label such as a fluorescent label or an enzyme, and the polypeptide having binding affinity to HPV18 E6 protein or the targeting molecule targeting HPV18 E6 is used for diagnosing HPV18 E6 virus infection disease or HPV18 E6 expression positive tumor.

[0018] In another aspect of the present application, a pharmaceutical composition is provided, which comprises the polypeptide having binding affinity to HPV18 E6 protein or the targeting molecule targeting HPV18 E6 as described above; and a pharmaceutically acceptable carrier.

[0019] In another aspect of the present application, a kit for diagnosing HPV18 E6 virus infection disease or HPV18 E6 expression positive tumor is provided, which comprises the targeting molecule targeting HPV18 E6 protein and a detection reagent for detecting the targeting molecule, the targeting molecule comprising a polypeptide tag or a detectable label.

[0020] In another aspect of the present application, a kit for diagnosing HPV18 E6 virus infection disease or HPV18 E6 expression positive tumor is provided, which comprises the polypeptide having binding affinity to HPV18 E6 protein, the targeting molecule targeting HPV18 E6 or the pharmaceutical composition.

[0021] In a preferred embodiment, the polypeptide having binding affinity to HPV18 E7 protein is further included.

[0022] In a preferred embodiment, the HPV18 E6 expression positive tumor comprises cervical cancer, head and neck tumor or external genitalia tumor.

[0023] Further aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein and the associated drawings and description of specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to make the technical solutions of the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or the prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings obtained according to these drawings without creative labor are still within the scope of the present application.

[0025] Figure 1 , each Z HPV18E6 and Z WT Comparison of sequences;

[0026] Figure 2 , SDS-PAGE detection and Western Blot identification of HPV18 E6 recombinant protein (A. HPV18 E6 recombinant protein plasmid map; B. HPV18 E6 recombinant protein SDS-PAGE gel electrophoresis result. M: Protein Marker; 1: E. coli BL21 (DE3); 2: pET21a (+) / E. coli BL21 (DE3); 3: pET21a (+) / HPV18 E6 / E. coli BL21 (DE3) before induction; 4. pET21a (+) / HPV18 E6 / E. coli BL21 (DE3) after induction; 5. pET21a (+) / HPV18 E6 / E. coli BL21 (DE3) after purification; C. Western Blot identification analysis of HPV18 E6 recombinant protein. M: Protein Marker; 6: pET21a (+) / HPV18 E6 / E. coli BL21 (DE3) before induction; 7. pET21a (+) / HPV18 E6 / E. coli BL21 (DE3) after purification);

[0027] Figure 3 , helper phage M 13 Titer determination of K07 and determination of library capacity of three rounds of panning (A. Titer determination of helper phage M 13 Titer determination of K07 and determination of library capacity of three rounds of panning (A. Titer determination of helper phage M

[0028] Figure 4 , ELISA detection of monoclonal affinity in the third library;

[0029] Figure 5 , p ET21a(+) / TAT-Z HPV18E6 Recombinant plasmid construction (A. pET21a (+) / TAT-Z HPV18E6 Recombinant plasmid map; B.p ET21a(+) / TAT-Z HPV18E6 Schematic diagram of construction of recombinant plasmid;C. p ET21a(+) / TAT-Z HPV18E6 Sequencing identification of recombinant plasmid;

[0030] Figure 6 , TAT-Z HPV18E6 Protein expression and identification (A. TAT-Z HPV18E6 SDS-PAGE electrophoresis identification of purified protein; B. TAT-Z HPV18E6 Western Blot identification of protein; M: Protein Marker; 1: TAT-Z HPV18E6 : 4; 2: TAT-Z HPV18E6 : 59 ； 3: TAT-Z HPV18E6 : 352; 4: TAT-Zwt)

[0031] Figure 7 , TAT-Z HPV18E6 Kinetic / affinity analysis diagram of protein with target protein HPV18E6

[0032] Figure 8 , TAT-Z HPV18E6 ELISA detection results of protein with HPV18E6 (A. Different concentrations of Z HPV18E6 ELISA analysis of protein with HPV18E6; B. TAT-Z HPV18E6 ELISA analysis of protein (final concentration 200 ng / μL) with HPV18E6);

[0033] Figure 9 , IP identification TAT-Z HPV18E6 Binding specificity with HPV18E6 (A. Western Blot short exposure results; B. Western Blot long exposure results; M: Protein Marker; 1. Capture protein is TAT-Z HPV18E6 :4; 2. Capture protein is TAT-Z HPV18E6 :59; 3. Capture protein is TAT-Z HPV18E6 :352; 4. Capture protein is TAT-Zwt);

[0034] Figure 10 , Dylight755-TAT-Z HPV18E6 Fluorescent protein identification diagram (A. Coomassie brilliant blue staining diagram; B. Fluorescent imaging diagram; 1. Dylight755-TAT-Z HPV18E64; 2. Dylight755-TAT-Z HPV186 59; 3. Dylight755-TAT-Z HPV186 352; 4. Dylight755-TAT-Zwt

[0035] Figure 11 Dylight755-TAT-Z HPV18E6 affibody in tumor-bearing nude mice model imaging figure (A. HeLa 229 (HPV18+) tumor-bearing nude mice model imaging figure; B. SiHa (HPV18-) tumor-bearing nude mice model imaging figure);

[0036] Figure 12 TAT-Z HPV18E6 / 7 Effect on HPV cell proliferation (A-C. Effect of TAT-Z HPV18E6 59, TAT-Z HPV18E7 228, TAT-Zwt protein and TAT-Z HPV18E6 59, TAT-Z HPV18E7 Effect of TAT-Z HPV18E6 228 on HeLa 229, SiHa, C33A cell proliferation; D-F. Columnar chart of effect on HeLa 229, SiHa, C33A cell proliferation after incubating the protein for 48h). DETAILED DESCRIPTION

[0037] The present inventors have made an in-depth study and first disclosed a polypeptide having binding affinity to E6 protein of human papillomavirus type 18 (HPV18). The present application also provides diagnostic or therapeutic use of the polypeptide as a drug or molecular targeting reagent.

[0038] As used herein, the "polypeptide having binding affinity to HPV18 E6" refers to a polypeptide obtained by performing 10-20 amino acid variations on the amino acid sequence of the Z segment of staphylococcal protein A, and the polypeptide can specifically bind to HPV18 E6 with little or no non-specific binding.

[0039] As used herein, the "polypeptide of the present application", "polypeptide having binding affinity to HPV18 E6", "HPV18 E6 binding polypeptide", "TAT-Z HPV18E6 affibody polypeptide", "TAT-Z HPV18E6 affibody", "TAT-Z HPV18E6 ", "affibody protein", "affibody recombinant protein", "TAT-Z HPV18E6 recombinant protein" can be used interchangeably.

[0040] The application will be further described below in connection with specific examples.

[0041] Example 1, Expression and immunogenicity of HPV18 E6 recombinant protein

[0042] 1. Prokaryotic expression, purification and identification of HPV18 E6 recombinant protein.

[0043] 1) Prokaryotic expression of HPV18 E6 protein:

[0044] A. First active: inoculate the bacteria liquid synthesized by the company into 10 mL LB-Amp medium at a ratio of 1:100 (100 μL), and incubate at 37°C constant temperature oscillation overnight;

[0045] B. Second active: inoculate the 10 mL bacteria liquid grown overnight into 500 mL LB-Amp medium at a ratio of 1:50 (10 mL), and incubate at 37°C oscillation until the absorbance A 600 is 0.6-0.8, add 5 mL IPTG to make the final concentration 1 mMOL / L, and continue to incubate at 37°C for 6 h;

[0046] C. Collect the 500 mL bacteria liquid in two 50 mL centrifuge tubes, centrifuge at 4°C 12000 rpm for 5 min, discard the supernatant, collect the bacteria, and repeat the step until the bacteria liquid is completely centrifuged;

[0047] D. Wash the collected bacteria precipitate once with pre-cooled PBS (30 mL), centrifuge at 4°C 12000 rpm for 5 min, discard the supernatant, and collect the bacteria.

[0048] E. Prepare a small sample of bacteria: centrifuge the small sample of bacteria before and after induction, discard the supernatant, add 40 μL 8M urea and 10 μL 5x Loadding buffer, shake and mix, then heat denature at 100°C metal bath for 10 min, and store at -20°C for SDS-PAGE electrophoresis analysis;

[0049] F. Ultrasonic crushing of bacteria: wash the ultrasonic rod with primary water for 5 min, resuspend the collected bacteria with 8M urea, and then put it into a pre-cooled glass beaker for ice bath ultrasonic. Ultrasonic parameters: total working time 30 min, single ultrasonic time 3 s, interval 3 s, working temperature 4°C, working power 250 w;

[0050] G. After ultrasonic, collect the bacteria liquid, put it into two original centrifuge tubes, centrifuge at 4°C 12000 rpm for 30 min in a pre-cooled ultracentrifuge; filter the supernatant of the bacteria liquid with a 0.45 μm filter membrane to remove excess impurities and bacterial fragments, and collect them in a sterile 50 mL centrifuge tube.

[0051] 2) pET21a(+) Purification and renaturation of HPV18 E6 protein:

[0052] ① pET21a(+) Purification of HPV18 E6 protein:

[0053] A. Add 5 mL of nickel chelate affinity chromatography gel (Ni-NTA Agarose) to the affinity chromatography column, place it at 4°C, and after the nickel is completely precipitated, use 50 mL of column equilibration buffer (pH=8) to equilibrate the nickel column;

[0054] B. Pour in the protein supernatant, adjust the drop rate to 4s / drop through the speed regulator until the supernatant is hung up; use 30 mL of column equilibration buffer (pH=8) to wash the nickel column;

[0055] C. Prepare imidazole eluent with a concentration of 10 mmoL / L, 50 mmoL / L, 100 mmoL / L, 150 mmoL / L, and 200 mmoL / L using column equilibration buffer (pH=8), adjust the drop rate to 3s / drop, and elute the protein; after washing the nickel column with 30 mL of column equilibration buffer (pH=8), finally seal the column with 20% anhydrous ethanol;

[0056] D. Identify the purity of the protein: take 40 μL of the eluted protein at different concentrations of imidazole and add it to the labeled EP tube, respectively add 10 μL of 5xLoading Buffer, mix well, and then heat denature in a metal bath for 10 min, and store at -20°C for SDS-PAGE electrophoresis and Western BLot analysis;

[0057] ② pET21a(+) Renaturation of HPV18 E6 protein:

[0058] A. Place the dialysis bag with a molecular weight cutoff of 3.5kD in 50 mL of 1M EDTA·2Na solution, heat and boil for 30 min; then thoroughly rinse with deionized water;

[0059] B. Place the purified protein into the dialysis bag, clamp it tightly with the labeled dialysis bag clamp, place it in 4M and 2M urea for two hours each, and then place it in PBS overnight for dialysis to remove excess imidazole;

[0060] C. After dialysis, divide the protein solution into new 15 mL centrifuge tubes and store them at -80°C for later use;

[0061] D. Protein concentration determination: Add 285 μL / well of CBB solution to the enzyme label strip, take 15 μL of protein solution, mix gently, place on the enzyme label instrument, select absorbance 490 nm-630 nm reading, calculate the protein concentration according to the protein concentration determination standard curve.

[0062] 3) SDS-PAGE electrophoresis analysis:

[0063] A. Preparation of 15% SDS-PAGE electrophoresis gel: proportionally configure 5 mL of 15% separation gel, quickly inject the separation gel into the glass plate, cover the top layer with isopropyl alcohol, and place it in a 37°C oven for 40 min; configure 3 mL of 5% concentrated gel, discard the isopropyl alcohol, quickly inject the separation gel into the glass plate, insert the loading comb, and place it in a 37°C oven for 30 min, and take it out after the concentrated gel solidifies;

[0064] B. Place the prepared electrophoresis gel in the electrophoresis tank, fill the electrophoresis tank with SDS-PAGE electrophoresis buffer, carefully pull out the loading hole comb, add 10 μL of protein sample and 4 μL of standard protein pre-dye Marker, and connect the power supply for electrophoresis. Electrophorese at 80V for 35-40 min, compress the bromophenol blue into a thin line, the standard protein pre-dye Marker starts to separate, the 70kDa red band starts to appear, adjust the voltage to 120V, and electrophorese to the bottom of the separation gel to end the electrophoresis;

[0065] C. Place the gel in the Coomassie brilliant blue staining solution, shake on the shaker for about 45 min; recover the staining solution, add Coomassie brilliant blue destaining solution for 3h of elution; take a photo to analyze the position of the recombinant protein band.

[0066] 4) Western BLot identification:

[0067] ① SDS-PAGE gel electrophoresis: the method is the same as above;

[0068] ② Membrane transfer

[0069] A. Cut the PVDF membrane to be slightly larger than the gel size, activate it in methanol for 90 s; according to the order of cathode-filter paper-gel-membrane-filter paper-anode, place it in the membrane transfer buffer, remove the air bubbles;

[0070] B. Install the membrane transfer tank, put in the clamp, add pre-cooled membrane transfer buffer, cover the lid, connect the power supply, and transfer the membrane at a constant current of 250mA for 1 h;

[0071] C. Blocking: after the membrane transfer is completed, place the membrane in the incubation box, add blocking solution, and block at 37°C for 2 h;

[0072] D. Incubate the first antibody: dilute the His mouse monoclonal antibody with the first antibody diluent at a ratio of 1:5000, add it to the incubation box, and incubate it at 4°C overnight;

[0073] E. Incubate the second antibody: discard the first antibody, wash the membrane with TBST 5 times, 5 min each time; dilute the Goat-anti-Mouse-IgG(H+L)-HRP with 5% milk (dissolved in TBST) at a ratio of 1:5000, add it to the incubation box, and incubate it at 37°C for 2 hours on a shaker;

[0074] F. Exposure: after incubation, discard the second antibody, wash the membrane with TBST 5 times, 5 min each time; follow the instructions of the ECL color developing kit to prepare the color developing solution in the dark, and use the exposure instrument to display the chemiluminescence results.

[0075] The pET21a(+) / HPV18 E6 recombinant plasmid synthesized by the company was transformed into the E. coLi.BL21(DE3) expression strain, and IPTG was induced for 6h. After nickel column purification, the protein expression and purification effect were identified by SDS-PAGE protein gel electrophoresis. The results showed that after induction and purification, there was a clear protein band at a molecular weight of about 18.0 kDa (A), which was consistent with the theoretical predicted value; further identification by Western Blot showed a single band at 18.0 kDa (B). It is shown that the HPV18 E6 recombinant protein in this study is successfully expressed and purified in the E. coli expression system. Figure 2 Figure 2

[0076] Example Two, Screening and Expression of HPV18 E6 Binding Affibody Molecules

[0077] 1. Phage (M13K07) amplification:

[0078] 1) Phage (M13K07) amplification:

[0079] A. Take the phage with measured titer and inoculate it into TG1 bacterial liquid in the logarithmic growth phase, and incubate it at 37°C on a shaker for 2h;

[0080] B. Transfer the above bacterial liquid to 500mL of 2YT liquid medium, continue to shake culture at 37°C for lh; then add kanamycin aqueous solution to a final concentration of 70µg / ml (i.e. add 35mg of kanamycin), continue to shake culture overnight (12-16h);

[0081] C. Centrifuge at 5000g, 4°C for 15min to collect the supernatant, add 1 / 5 volume of PEG / NaCl, and stand in ice bath for lh;​​

[0082] D. 10000rpm, 4℃ centrifuge 15min, discard the supernatant; dissolve the precipitate in 30 mL 2YT buffer solution, filter sterilization with 0.22µm membrane, store at 4℃ and can be used for more than half a year.

[0083] 2) Determine the titer of amplified helper phage

[0084] A. Take the amplified phage M 13 K07 stock solution 110µl into the EP tube, and then add 90µl 2YT medium to the subsequent EP tube, take 10µl M 13 K07 to the next tube, mix well with pipette, and then perform dilution by ratio;

[0085] B. Add 500µl TG1 bacterial solution with OD value of 0.6-0.8 to each EP tube, mix well, and then incubate in a 37℃ constant temperature incubator at 1500rpm for 20min;

[0086] C. Take out the EP tube, add 3mL of top agar to each, mix well, and then spread on 2YT solid medium, incubate overnight in a 37℃ incubator after complete solidification;

[0087] D. Calculate the titer of amplified helper phage according to the number and growth of plaque on the plate.

[0088] 3) Pre-washing stage:

[0089] A. Randomly select one of the phage affibody libraries constructed and identified in the laboratory in the early stage, inoculate it into 9mL 2YT-AG medium, place it in a 37℃ constant temperature water bath for 30min, and then place it in a 37℃ constant temperature shaking incubator for 1.5h at 250rpm;

[0090] B. Take out the centrifuge tube, add 100µL M 13 K07 helper phage to make its final concentration 1×10 12 pfu / mL, place it in a 37℃ constant temperature water bath for 30min, and then place it in a 37℃ constant temperature shaking incubator for 30min at 250rpm;

[0091] C. Take out the centrifuge tube, centrifuge at 1500rpm for 10min, discard the supernatant, add 10mL 2YT-AKG medium, and gently resuspend the precipitate with a pipette, place it in a 37℃ constant temperature shaking incubator for overnight incubation at 250rpm (note that the whole process is sterile operation);

[0092] D. TG1 bacterial liquid was inoculated in 10 mL 2YT medium at a ratio of 1:100, and placed in a 37°C constant temperature shaking incubator at 250 rpm for overnight culture;

[0093] E. Protein coating: wash the immunization tube with 1xPBS (sterile) 3 times, and irradiate with ultraviolet light for 30 min. Add 2 mL of HPV18E6 protein with a final concentration of 100 μg / mL, and coat overnight.

[0094] 4) First round of washing:

[0095] A. Discard the protein in the immunization tube, and wash the immunization tube with sterile 1xPBS along the tube wall 3 times, and absorb the residual liquid with filter paper;

[0096] B. Add 3 mL of 5% skimmed milk (diluted with PBS) to the immunization tube, and block in a 37°C constant temperature incubator for 2 h;

[0097] C. Centrifuge the primary library cultured overnight at 4°C at 1500 rpm for 30 min, and slowly absorb the supernatant (containing phages) into a new sterile 50 mL centrifuge tube;

[0098] D. Add 2 mL of PEG8000 / NaCl at a ratio of 1:5, and mix gently, and vertically ice bath for 45 min (phage chromatography);

[0099] E. After centrifugation at 12000 rpm for 30 min, slowly discard the supernatant in the super-clean bench, and after absorbing the residual liquid with filter paper, add 4 mL of 2YT and mix gently;

[0100] F. Add 6 mL of 5% skimmed milk at a ratio of 3:2, mix gently, and stand at room temperature for 30 min;

[0101] G. Discard the blocking solution in the immunization tube, and wash the immunization tube with sterile 1xPBS along the tube wall 3 times, and absorb the residual liquid with filter paper;

[0102] H. Add 2 mL of phage supernatant prepared in step F to the immunization tube, and culture in a 37°C constant temperature shaking incubator at 100 rpm for 30 min, and stand in a 37°C constant temperature incubator for 1.5 h;

[0103] I. Discard the liquid in the immunization tube, and wash the immunization tube with sterile 1xPBS along the tube wall 3 times, and absorb the residual liquid with filter paper;

[0104] J. Add 2 mL of TG1 bacterial solution in logarithmic growth phase (Step D) to each tube, and incubate in a 37°C constant temperature incubator at 250 rpm for 1.5 h;

[0105] K. At this time, the HPV18E6 protein primary affibody library is obtained in the immunization tube. Mix the bacterial solution and 30% glycerol at a ratio of 1:1, and store in sterile 1.5 mL EP tubes at -20°C for later use;

[0106] L. Take 100 μL of the primary library stock solution, dilute it by 10 0 -10 8 times, and evenly spread it on SOB-AG plates. Incubate at 37°C overnight, and observe the growth of colonies on the plates the next day. The first round of panning is complete.

[0107] 5) Second and third rounds of panning: Repeat the first panning step, and use the HPV18E6 protein primary affibody library obtained in the previous round of panning to perform the experiment, to obtain the HPV18E6 protein secondary and tertiary affibody libraries.

[0108] 6) Indirect ELISA screening of affibody molecules with high affinity for HPV18E6:

[0109] A: Take 30 μL of the HPV18E6 protein tertiary affibody library obtained from the above experiment, dilute it, and spread it on 2YT-AG plates. Incubate at 37°C overnight.

[0110] B. Randomly pick single colonies from the plates, place them in sterile 1.5 mL EP tubes containing 800 μL of 2YT-AG medium, and label them. Incubate in a 37°C constant temperature incubator at 250 rpm for 4 h.

[0111] C. Take 100 μL of the bacterial solution from the EP tube, and add it to a new sterile 1.5 mL EP tube with the corresponding number. Add 700 μL of 2YT-M medium (dilute M 13 K07 to a final concentration of 2.5×10 10 pfu / mL), and incubate at 37°C in a constant temperature incubator at 250 rpm for 3 h.

[0112] D. Centrifuge at 1500 rpm for 20 min, slowly discard the supernatant, and add 800 μL of 2YT-AKG medium (bacteriophages are resistant to Kana) to mix well. Incubate at 37°C in a constant temperature incubator at 250 rpm overnight.

[0113] E. Dilute HPV18E6 protein to a final concentration of 10 μg / mL with coating buffer, add 100 μL to each well of a 96-well microplate, and coat overnight at 4°C.

[0114] F. Remove the microplate, wash it three times with 1×PBST, pat it dry, add 200μL of skim milk diluted with 5% PBST to each well, and incubate at 37℃ for 2 h.

[0115] G. Remove the blocked microplate, wash it 3 times with 1×PBST and pat it dry. Centrifuge the bacterial culture from step D at 1500 rpm for 20 min. Add 100 μL of the supernatant to each well according to the number and incubate it in a constant temperature incubator at 37℃ for 2 h.

[0116] H. Remove the enzyme-labeled plate after the reaction, wash it 5 times with 1×PBST, pat it dry, and dilute the secondary antibody M at a ratio of 1:15000. 13 Add 100 μL of HRP to each well and incubate at 37°C for 2 h.

[0117] I. Remove the microplate after incubation, wash it three times with 1×PBST, pat it dry, add 100μL / well TMB II colorimetric solution in the dark, and incubate it in a constant temperature incubator at 37℃ for 10min;

[0118] J. Remove the microplate after color development and add 50 μL (10% H2SO4) stop solution to each well to terminate the reaction;

[0119] L. Read OD using an enzyme-linked immunosorbent assay (ELISA) reader 450 Select OD 450 Single-clone bacterial cultures with a value greater than 0.5 were sent to Hangzhou Qingke Co., Ltd. for sequencing.

[0120] Determination of amplified auxiliary phage titers: when diluted 10... 14 At concentrations above a certain level, only a few clearly defined phage plaques are visible on the plate. Figure 3 A), therefore it is inferred that the amplified helper phage M 13 K07 titer is 10 14 The above information can be used for subsequent washing experiments.

[0121] Affibody molecules with affinity for the target protein HPV18 E6 were screened from a previously constructed random affibody library using phage display library technology. After screening, the volumes of the first, second, and third-level libraries were measured, and the results showed that the first-level library had a volume of 10. 6 The secondary storage capacity is 10. 9 The third-level storage capacity is 10. 7 ( Figure 3B). After dilution and plating, single clones were randomly selected from the plate and the OD values were measured by indirect ELISA 450 Readings were roughly judged for their affinity to the target protein, and 119 OD values were selected from 480 single clones 450 Readings were roughly judged for their affinity to the target protein, and 119 OD values were selected from 480 single clones Figure 4 After sequencing, 48 non-repetitive sequences with correct amino acid sequences and no stop codons were obtained. Then, OD 450 Readings were roughly judged for their affinity to the target protein, and 119 OD values were selected from 480 single clones HPV18E6 :4, TAT-Z HPV18E6 :59, TAT-Z HPV18E6 :352, and the amino acid sequence is shown in SEQ ID NO: Figure 1

[0122] 2. p ET21a(+) / TAT-Z HPV18E6 Construction of recombinant plasmid

[0123] 1) Primer design: The primer sequence is SEQ ID NO: 5-6, the upstream contains a BamH I restriction site, the downstream contains a Hind III restriction site, and the homologous bases at both ends of the site facilitate homologous recombination.

[0124] 2) Acquisition of target gene: The BamH I-affibody-Hind III target fragment of about 200 bp was amplified from the single clone liquid with correct sequencing and OD 450 higher than 1.5 before, using upstream and downstream primers, and the PCR reaction system and temperature are shown in Tables 1-2.

[0125]

[0126]

[0127] 3) Linearization of vector: The plasmid was digested with restriction enzymes BamH I and Hind III pET21(+) , and the vector was obtained by gel recovery and purification, as shown in Table 3.

[0128]

[0129] 4) Recombination reaction: The specific use amount of linearized vector and insert fragment is referred to the following formula,

[0130] ​Optimal amount of cloning vector used = [0.02 x number of cloning vector base pairs] ng (0.03 pmol)

[0131] Optimal amount of insert used = [0.04 x number of insert base pairs] ng (0.06 pmol)

[0132]

[0133] 5) Transformation:

[0134] A. Add all the above enzyme ligation products into 100 μL E. coli. BL21(DE3) competent cells, ice bath for 30 min;

[0135] B. After ice bath, heat shock at 42°C for 90 s, then ice bath for 2-3 min. Add 700 μL LB medium without Amp in the clean bench, shake culture at 37°C for 45 min;

[0136] C. Centrifuge the above bacterial solution at 5000 rpm for 5 min, discard the supernatant and only keep 50 μL medium. Resuspend the bacterial solution and spread on LB(Amp) plate, incubate in 37°C constant temperature incubator overnight.

[0137] 6) The next day, pick single colony and after PCR verification, select positive colony to send to company for sequencing.

[0138] The single colony vector obtained by phage display library technology is pcantAB5E, which cannot be used for prokaryotic expression. Therefore, we cloned the DNA sequence encoding three affibody genes into the pET21a(+) vector prepared and verified in the laboratory by molecular cloning technology, and added HA and His tags at the C terminal (convenient for subsequent experiments) Figure 5 A), which were completely correct after sequencing alignment, indicating that the recombinant plasmid pET21a(+) / TAT-Z HPV18E6 Affibody was successfully constructed Figure 5 B).

[0139] 3. pET21a(+) / TAT-Z HPV18E6 Prokaryotic expression and purification of protein

[0140] The method is the same as the first part. TAT-Z HPV18E6 protein was prepared by prokaryotic expression, and the target protein was purified by nickel column.

[0141] 4. pET21a(+) / TAT-Z HPV18E6 WB verification of protein

[0142] 1) Tris-Tricine-SDS-PAGE electrophoresis:

[0143] A. Prepare Tris-Tricine-SDS-PAGE gel according to the above formula;

[0144] B. Correctly install the Tris-Tricine-SDS-PAGE gel into the electrophoresis tank, fill the inner tank with 1× anode buffer, pour a sufficient amount of 1× cathode buffer into the outer tank, slowly pull out the comb, cover the electrophoresis tank cap, turn on the power, and perform pre-electrophoresis at 30V for 10 min.

[0145] C. Take out the prepared bacterial sample (TAT-Z) HPV18E6: 4, 59, 352 and Zwt), add 15 μL of protein sample to each well, and add 3 μL of protein marker at the same time;

[0146] F. Electrophoresis conditions: 30V for 1 hour, then adjust to 100V for 2.5 hours, turn off the power when the gel reaches the bottom of the separating gel.

[0147] 2) Western Blot:

[0148] The transfer process was the same as in Part 1; after completion, the membrane was transferred to a 0.2% glutaraldehyde solution diluted with TBST and fixed at room temperature in the dark for 30 min; after washing the membrane 5 times with TBST, it was blocked, incubated with antibody, and then exposed to light.

[0149] The correct recombinant plasmid p ET21a(+) / TAT-Z HPV18E6 Transform to E. coli Prokaryotic expression was performed in BL21 (DE3) cells, induced with 1M IPTG for 6 hours, and purified using His-tagged affinity chromatography. Analysis was performed by Tris-Tricine-SDS-PAGE gel electrophoresis. The results showed a relatively single protein band at approximately 7 kDa, consistent with the predicted size, indicating successful protein purification. Figure 6 A). Further verification was performed using Western blotting experiments, which showed that a single band appeared at a molecular weight of approximately 7 kDa after incubation with the His mouse monoclonal antibody. Figure 6 B), indicating that TAT-Z HPV18E6 The protein was successfully expressed with high purity, and further experimental research can be carried out.

[0150] Example 3: In vivo and in vitro targeted binding study of HPV18E6 to affibody molecules

[0151] (I) In vitro targeted binding study of HPV18E6 to affibody molecules

[0152] 1. TAT-Z HPV18E6 Binding affinity with HPV18E6 at the molecular level

[0153] 1) Surface plasmon resonance (SPR) technique for identifying binding affinity:

[0154] A. Preparation of HPV18E6 and TAT-Z HPV18E6 And Zwt protein, the method is the same as in Part II;

[0155] B. Place the Series S Sensor Chip CM7 in the SPR analyzer, wash the chip twice with EDC / NHS buffer, and activate the chip;

[0156] C. Dilute the target protein HPV18E6 with SPR-Loading Buffer reagent and place it in an SPR analyzer to couple and immobilize it onto the chip channel;

[0157] D. Dilute TAT-Z with HBS-EP Buffer HPV18E6 4. TAT-Z HPV18E6 :59、TAT-Z HPV18E6 :352 and wild-type Zwt were prepared to a final concentration of 200 μg / ml. The experimental histones were serially diluted and flowed through the chip to detect the affinity of the experimental histones for the target protein HPV18E6.

[0158] Wash the chip three times with E. glycine-HCl, add PBS to the chip surface, and store at 4°C.

[0159] F. Biacore T200 Evaluation Software analyzes experimental data.

[0160] To verify TAT-Z HPV18E6 To investigate the binding affinity of the affinity protein to HPV18E6, we used SPR, a protein-protein interaction assay that allows for precise molecular-level verification. The results showed that the resonance signal increased in a concentration-dependent manner. Figure 7 (A, B, C), and all affinities showed good binding affinity to the HPV18E6 recombinant protein; however, when TAT-Zwt flowed through the chip, no effective resonance signal could be detected. Figure 7 D). Furthermore, kinetic BIAcore analysis indicates that TAT-Z HPV18E6 4. TAT-Z HPV18E6 :59 and TAT-Z HPV18E6 The dissociation equilibrium constant (KD) of 352 is 1.663 × 10 -4mol / L, 3.761 × 10 - 6 mol / L and 2.803×10 -5 The dissociation constant of TAT-Zwt is 6.27 × 10⁻⁶ mol / L. -2 mol / L (Table 5). Z obtained after screening HPV18E6 The affinity for the target protein HPV18E6 reached the μM level, while Zwt showed almost no binding affinity to HPV18E6. This indicates that the screened affibody has a high affinity for the target protein HPV18E6 and can be used for subsequent experimental studies.

[0161]

[0162] 2) Indirect ELISA for determining binding affinity:

[0163] A. Encapsulation and sealing are the same as in Part Two;

[0164] B. Remove the blocked ELISA plate, wash it three times with 1×PBST, pat it dry, and then add TAT-Z. HPV18E6 4. TAT-Z HPV18E6 :59、TAT-Z HPV18E6 :352 and TAT-Zwt were diluted to 250µg / mL with PBS and then serially diluted. They were added to microplates with three replicates for each group. HPV18E6 mouse serum antibody was set as a positive control. The plates were incubated at 37°C for 2 hours.

[0165] C. Remove the incubated ELISA plate, wash it 3 times with 1×PBST and pat it dry. Add His mouse monoclonal antibody (1:5000) to the experimental group and incubate at 37℃ for 2 hours.

[0166] D. The incubation, color development, termination reaction, and reading of the secondary antibody are the same as in Part II.

[0167] TAT-Z was detected at the molecular level using an indirect ELISA method. HPV18E6 The binding affinity of the protein to HPV18 E6. Experimental results show that the protein's affinity increases with TAT-Z. HPV18E6 The increase in TAT-Z with increasing protein concentration indicates that... HPV18E6 The binding affinity of the protein to HPV18E6 gradually increased; while the OD of the control protein TAT-Zwt increased. 450 The value did not change significantly with changes in TAT-Zwt protein concentration, and the OD 450 The values ​​are all low, indicating that TAT-Zwt has almost no affinity for HPV18E6. Figure 8 A). Z HPV18E6OD value of the protein was diluted to 250 μg / mL 450 TAT-Zwt < TAT-Z HPV18E6 :4 < TAT-Z HPV18E6 :352 < TAT-Z HPV18E6 :59, and finally through statistical analysis, the Z HPV18E6 OD value of the protein 450 value was statistically different compared with TAT-Zwt (P < 0.05), indicating that the affinity of TAT-Z Figure 8 B), which showed that the affinity of TAT-Z HPV18E6 protein to HPV18E6 was TAT-Z HPV18E6 :4 < TAT-Z HPV18E6 :352 < TAT-Z HPV18E6 :59.

[0168] 2. TAT- Z HPV18E6 The binding affinity of TAT-Z

[0169] Immunoprecipitation (IP) identified the binding specificity of TAT-Z HPV18E6 to HeLa229 cells:

[0170] A. HeLa229 cell total protein extraction: place a 10 cm cell culture dish on ice, wash the infected cells with pre-cooled PBS twice; add 400 μL IP lysis buffer and lyse for 15 min on ice; use a cell scraper to scrape the adherent cell fragments into the lysis buffer, vortex to mix, and continue to lyse for 15 min; centrifuge at 13000 rpm for 15 min at 4°C; discard the precipitate and transfer the supernatant to a centrifuge tube;

[0171] B. Divide the protein supernatant into 4 groups and add 8 μg of TAT-Z HPV18E6 :4, TAT-Z HPV18E6 :59, TAT-Z HPV18E6 :352 and TAT-Zwt to capture the target protein, slowly bind at 4°C for 6 h, then add 1 μg of His-tag mouse monoclonal antibody as the binding antibody for affibody, and slowly bind at 4°C overnight;

[0172] C. The next day, add 30 μL of fully resuspended Protein A+G Agarose to each group and slowly bind at 4°C for 2 h;

[0173] D. Centrifuge at 2500 rpm for 5 min, discard the supernatant, and wash the precipitate with pre-cooled PBS 5 times, centrifuge at 2500 rpm for 5 min;

[0174] E. After the last wash, discard the supernatant, add 30 μL of 1x SDS-PAGE loading buffer, resuspend the pellet;

[0175] F. The metal bath was heated at 100 °C for 10 min, and after a brief high-speed centrifugation, it was used for subsequent experiments.

[0176] G. The SDS-PAGE electrophoresis and membrane transfer steps were the same as in the first part. The primary antibody was incubated with HPV18E6 monoclonal antibody (1:1000), and the subsequent steps were the same as in the first part.

[0177] IP experiments are commonly used to isolate individual proteins, facilitating the study of their characteristics, expression, or activation or modification state. To further confirm that TAT-Z HPV18E6 As the captured protein when capturing proteins, the captured protein is the target protein HPV18E6, we further select IP experiments for analysis and verification. First, use TAT-Z HPV18E6 or TAT-Zwt (as a negative control, containing His-tag) as the capture antibody for the natural target protein HPV18E6, adsorb and elute the target protein captured by each group, i.e., HeLa229 cell total protein, and then perform WB detection. The results show that when the three TAT-Z HPV18E6 proteins are used as capture antibodies, the captured proteins have obvious bands at a relative molecular mass of 17 kDa, indicating that the captured proteins can be recognized by the detection antibody; while the control group TAT-Zwt does not have obvious bands ( Figure 9 ). Further prove that TAT-Z HPV18E6 protein can specifically recognize and bind to target protein HPV18 E6.

[0178] (II) In vivo target binding study of HPV18E6 binding affibody molecules

[0179] 1. Establishment of tumor-bearing nude mouse model

[0180] A. Preparation of nude mice: divide the nude mice into groups, 5 in each group, and raise them in SPF-level environment IVC cages to adapt to the new environment. Before inoculating cells, mark the ear tags and record them;

[0181] B. Cell inoculation: digest the well-conditioned cells, centrifuge at 1000 g for 5 min, discard the supernatant, and resuspend the cells with the corresponding basal medium. Adjust the cell density to 2x10 7 6 / mL, transfer to 1.5 mL EP tubes, wrap with tin foil paper, and irradiate under ultraviolet light for 30 min to pass through the gold SPF barrier system. Inoculate 100 μL of cell suspension at the right scapular position of the nude mouse;

[0182] C. Regularly change feed, mouse cage, supplement water, observe the physiological condition of nude mice every three days, and when the tumor grows to 200-300mm 3 , take out the SPF barrier system, and carry out subsequent experiments.

[0183] 2. Dlight755 fluorescently labeled TAT-Z HPV18 E6 protein and identification of fluorescent protein

[0184] A. After centrifugation of Dylight755 fluorescent dye powder at 4°C and 12000 rpm for 10 min, dissolve in 1 mL DMF solution;

[0185] B. Prepare TAT-Z HPV18E6 and TAT-Zwt protein according to the experimental method of part two, adjust the final concentration to 500 μg / mL, take 1 mL in 1.5 mL brown EP tube respectively, add 100 μL Dylight755 fluorescent dye, mix well by blowing, and then place in 4°C refrigerator overnight for coupling;

[0186] C. After taking out the coupled protein (named Dylight755- TAT-Z HPV18E6 , Dylight755- TAT-Zwt), dialyze to remove the fluorescent dye that has not been successfully coupled, take 10 μL protein to perform SDS-PAGE gel electrophoresis analysis in the dark;

[0187] D. Place the SDS-PAGE gel in CRi Maestro 2.10 near-infrared small animal live imaging instrument for observation and photography.

[0188] 3. Dylight755- TAT-Z HPV18E6 , Dylight755-Zwt in vivo fluorescence imaging of tumor-bearing nude mice

[0189] A. Anesthesia of tumor-bearing nude mice: anesthetize the mice taken out of the barrier system with appropriate amount of isoflurane for subsequent experiments;

[0190] B. Inject 100 μL of coupled fluorescent recombinant protein through the tail vein in the dark;

[0191] C. Turn on the near-infrared small animal live imaging instrument, select 671-705 nm excitation light filter, 750 nm emission light filter, and 810-830 nm wavelength exposure 5000 ms every 10 nm, and take pictures of nude mice injected with fluorescent recombinant protein at 0 min, 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 24 h, 48 h, respectively, to obtain the fluorescence imaging of the protein in vivo;

[0192] D. The CRi Maestro 2.10 near-infrared in vivo imaging instrument was used to quantify the image information.

[0193] Dylight755 fluorescent dye was coupled with TAT-Z HPV18E6 wt recombinant proteins were fluorescently labeled, and after electrophoresis on a 15% SDS-PAGE gel in the dark, the results were observed under a small animal in vivo imaging instrument. A clear single red fluorescent band appeared at a position of about 10 kD, indicating successful coupling. Figure 10 B), and the imaged SDS-PAGE was stained with Coomassie blue, showing a blue result at the same position. Figure 10 A). In summary, the Dlight755 fluorescently labeled protein was successful.

[0194] To observe the in vivo biodistribution and metabolism of Dylight755-TAT-Z HPV18E6 proteins in HeLa 229 and SiHa tumor-bearing nude mouse models. Dylight755-TAT-Z HPV18E6 :4, Dylight755-TAT-Z HPV186 :59, Dylight755-TAT-Z HPV186 :352 and Dylight755-TAT-Zwt proteins were injected from the tail vein, and the fluorescence distribution at 0h, 5min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h after injection was recorded on a mouse in vivo imaging instrument. The tumor / skin fluorescence intensity signal ratio was statistically analyzed. In the HeLa 229 xenograft tumor-bearing nude mouse model, we observed that the fluorescence signal at the tumor site was significantly stronger than that in the skin 30min after injection of the protein. The peak was reached at 2h after injection. Dylight755-TAT-Z HPV186 :59, Dylight755-TAT-Z HPV18E6 :352 could be maintained in the tumor site for more than 8h, while Dylight755-TAT-Z HPV18E6 :4 had no obvious fluorescence signal in the tumor site. Figure 11 A, B). In the SiHa xenograft tumor-bearing nude mouse model, we observed that the fluorescent protein non-specifically aggregated in the tumor tissue 5min after injection, and then the fluorescence signal intensity gradually weakened and disappeared.

[0195] Example Four, in vitro biological activity of HPV18E6 binding affibody molecules

[0196] 1. CCK-8 method for determining the effect of affibody on HPV cell activity

[0197] A. Cell plating: Good HeLa229, SiHa, C33A cells were plated in 96-well plates at a density of 5000 cells per well, and the medium was supplemented to 100 μL per well, and incubated at 37°C, 5% CO2 overnight;

[0198] B. Incubation of protein: The prepared TAT-Z HPV18E6 :59, TAT-Z HPV18E7 :228 and TAT-Zwt were diluted to 100 μg / mL with basal medium, and TAT-Z HPV18E6 :59, TAT-Z HPV18E7 :228 was diluted to 60 μg / mL, and 100 μL was added to each well after mixing, and incubated at 37°C, 5% CO2 for 72h;

[0199] C. Determination of absorbance: After 12h, 24h, 36h, 48h of incubation, CCK8 detection solution was prepared in the dark at a ratio of 1:9 (10 μL CCK8 solution and 90 μL basal medium), the liquid in the 96-well plate was aspirated, 100 μL CCK8 detection solution was added to each well, and the color was developed in the cell incubator for 2h, and then the absorbance was read at 450nm wavelength using a microplate reader;

[0200] D. Viability calculation: The formula is as follows

[0201] Cell viability (%) = [A drug + B blank] / [C no drug - B blank] x 100%

[0202] A (drug +): The absorbance of the cell well with CCK8 solution and drug; B (blank): The absorbance of the blank well with CCK8 solution and drug; C (no drug): The absorbance of the cell well with only CCK8 solution.

[0203] Previous results in the laboratory showed that TAT-Z HPV18E7 :228 can inhibit the proliferation of HeLa229 cells (HPV18+), so we want to explore whether simultaneous targeting of E6 and E7 can be more effective than targeting E6 or E7 alone in inhibiting the proliferation of HPV18-positive cells. TAT-Z HPV18E6 :59, TAT-Z HPV18E7 :228, TAT-Zwt protein and TAT-Z HPV18E6 :59, TAT-Z HPV18E7 :228 mixture was incubated in 96-well plates containing HeLa229, SiHa (HPV16+), C33A (HPV18-) cells for 0, 12, 24, 36, 48 hours, and cell proliferation was detected by CCK-8 method. The results showed that TAT-ZHPV18E6 :59 and TAT-Z HPV18E7 :228 can reduce the viability of HPV18-positive HeLa229 cells to about 60%, 40%, respectively, and the combination of the two can reduce the viability of HPV18-positive HeLa229 cells to about 30%; the control TAT-Zwt has no significant effect on cell viability Figure 12 A); while TAT-Z HPV18E6 :59, TAT-Z HPV18E7 :228, the combination of the two and TAT-Zwt have less effect on the viability of negative cells SiHa and C33A Figure 12 B, C). When acting on HeLa229 cells for 48h, there is a statistical difference between the experimental group and the control group Figure 12 D), while there is no statistical difference for SiHa and C33A cells Figure 12 E, F) indicating that TAT-Z HPV18E6 / 7 and the combination of the two can specifically inhibit the cell proliferation of HPV18E6-positive HeLa229, and the effect of the combination is more obvious.

[0204] The above only discloses the preferred embodiments of the present application, and of course cannot limit the scope of the rights of the present application, so the equivalent changes made according to the claims of the present application still fall within the scope covered by the present application.

Claims

1. A polypeptide having binding affinity to HPV 18 E6 protein, characterized in that: The polypeptide is a polypeptide with 12-20 amino acid variations based on the amino acid sequence of the Z domain of staphylococcal protein A, and the amino acid sequence of the polypeptide is selected from the group consisting of any one of SEQ ID NO: 2-4.

2. A targeting molecule targeting HPV18 E6 protein, characterized in that, The targeting molecule comprises the polypeptide of claim 1 and a conjugate connected to the polypeptide, wherein the conjugate is a polypeptide tag and / or a detectable label.

3. An isolated polynucleotide, comprising, The polynucleotide is a polynucleotide encoding the polypeptide with binding affinity to HPV18 E6 protein of claim 1.

4. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1 or 2. The polynucleotide is a polynucleotide encoding the targeting molecule targeting HPV18 E6 protein of claim 2, wherein the conjugate is a peptide, and the peptide is a His tag, an HA tag, a Myc tag, or a Flag tag.

5. A recombinant vector, characterized in that, The vector comprises the polynucleotide of claim 3 or 4.

6. A host cell, characterized in that, The host cell comprises the recombinant vector of claim 5, or the genome of the host cell is integrated with the polynucleotide of claim 3 or 4.

7. The polypeptide having binding affinity to HPV 18 E6 protein according to claim 1, characterized in that: The amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO:

3.

8. The targeting molecule targeting HPV18 E6 protein according to claim 2, characterized in that: The amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO:

3.

9. The use of a polypeptide having binding affinity to HPV 18 E6 protein according to claim 7, characterized in that, The polypeptide is a polypeptide with 12-20 amino acid variations based on the amino acid sequence of the Z domain of staphylococcal protein A, and the amino acid sequence of the polypeptide is selected from the group consisting of any one of SEQ ID NO: 2-4.

10. A pharmaceutical composition, characterized by, The polypeptide is a polypeptide with 12-20 amino acid variations based on the amino acid sequence of the Z domain of staphylococcal protein A, and the amino acid sequence of the polypeptide is selected from the group consisting of any one of SEQ ID NO: 2-4.

11. A kit for diagnosing a tumor positive for HPV18 E6 expression, characterized by, The polypeptide is a polypeptide with 12-20 amino acid variations based on the amino acid sequence of the Z domain of staphylococcal protein A, and the amino acid sequence of the polypeptide is selected from the group consisting of any one of SEQ ID NO: 2-4. The polypeptide is a polypeptide with 12-20 amino acid variations based on the amino acid sequence of the Z domain of staphylococcal protein A, and the amino acid sequence of the polypeptide is selected from the group consisting of any one of SEQ ID NO: 2-4.

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