Nano antibody targeting human papilloma virus 18 type E7 protein and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a nanometer antibody targeting human papilloma virus 18 type E7 protein and application of the nanometer antibody. The antibody is any one or a combination of more of 1H10, 4G2 and 4B10, and each of the 1H10, the 4G2 and the 4B10 comprises three antigen complementarity determining regions. Experimental results show that the three nano antibodies provided by the invention all show high affinity binding with the HPV18E7 protein, sandwich method ELISA composed of two pairs of nano antibodies 1H10 and 4G2 as well as 1H10 and 4B10 provided by the invention is used for quantitatively analyzing the level of the HPV18E7 protein, and an auxiliary diagnosis basis can be provided for infection detection of pathological samples by detecting the level of the E7 protein in HPV18 infected cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a nano antibody targeting human papillomavirus type 18 E7 protein and an application thereof. Technical Background

[0002] Cervical cancer is the fourth most common cancer in women worldwide, and 99% of cervical cancers are associated with human papillomavirus (HPV) infection. HPV is a small, non-enveloped, double-stranded circular DNA virus, of which HPV types 16 and 18 account for 80%. Currently, the prevention and treatment methods for cervical cancer are mainly based on screening, mainly TCT cell morphology detection and HPV molecular diagnosis, which are usually used in combination. However, TCT testing has the disadvantages of complicated procedures, long time consumption, and misjudgment and missed judgments caused by human judgment, while HPV molecular diagnosis has the disadvantages of high false positive rate, high instrument configuration requirements, and high cost.

[0003] HPV-infected cells usually highly express viral proteins E6 and E7. Detecting E6 and E7 proteins in HPV-infected cells through a pair of antibodies with different epitopes can reflect whether the cells are infected with HPV. At the same time, detecting the levels of E6 or E7 proteins in cells can semi-quantitatively reflect the severity of HPV infection. Since a pair of antibodies are used for detection, the false positive rate of the test results will be greatly reduced. Currently, there is a cervical cancer test strip on the market for the detection of HPV16E6 and HPV18 E6 proteins, the OncoE6 cervical test. Products and methods for high-sensitivity detection of HPV18 E7 protein are still to be developed. Summary of the invention

[0004] In order to solve the problems in the prior art, one of the objectives of the present invention is to provide a nanobody targeting human papillomavirus type 18 E7 protein.

[0005] The present invention adopts the following technical solutions:

[0006] A nanobody targeting human papillomavirus type 18 E7 protein, the antibody is a combination of any one or more of 1H10, 4G2 and 4B10, wherein 1H10, 4G2 and 4B10 all contain three antigen complementary determining regions CDR1, CDR2 and CDR3, wherein:

[0007] The amino acid sequences of the three antigen complementary determining regions of 1H10 are amino acid sequences having a homology of greater than or equal to 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;

[0008] The amino acid sequences of the three antigen complementary determining regions of 4G2 are amino acid sequences having a homology of greater than or equal to 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;

[0009] The amino acid sequences of the three antigen complementary determining regions of 4B10 are amino acid sequences with a homology greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, and preferably have a homology of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0010] The above homologous sequences also include amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

[0011] Preferably, the three antigen complementary determining region amino acid sequences of the 1H10 are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, the three antigen complementary determining region amino acid sequences of the 4G2 are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and the three antigen complementary determining region amino acid sequences of the 4B10 are shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

[0012] Preferably, the amino acid sequences of 1H10, 4G2, and 4B10 are as follows:

[0013] Amino acid sequence of 1H10:

[0014]

[0015] Amino acid sequence of 4G2:

[0016]

[0017] Amino acid sequence of 4B10:

[0018]

[0019] The amino acid sequences of the three antigen complementary determining regions CDR1, CDR2 and CDR3 of the above-mentioned nanobody are shown in bold lines, namely:

[0020] The amino acid sequence of the complementary determining region of 1H10 is:

[0021] CDR1: GFTFSRNVMN (SEQ ID NO: 1)

[0022] CDR2:GISRGGGTTN (SEQ ID NO:2)

[0023] CDR3:TLIGGVGTK (SEQ ID NO:3)

[0024] The amino acid sequences of the three antigenic complementary determining regions of 4G2 are:

[0025] CDR1: GSTSIMA (SEQ ID NO: 4)

[0026] CDR2: VVRTGLPNTY (SEQ ID NO: 5)

[0027] CDR3:AEASPWRLTQPRAYKYW(SEQ ID NO:6)

[0028] The amino acid sequences of the three complementary determining regions of 4B10 are:

[0029] CDR1:EVTYNNYCMG (SEQ ID NO:7)

[0030] CDR2: GIRPYGGGTY (SEQ ID NO: 8)

[0031] CDR3:ADPIPCLPTIPQLLNFNSR(SEQ ID NO:9)

[0032] The second object of the present invention is to provide an antibody targeting HPV18 oncogenic protein E7, which has any one of the nanobodies 1H10, 4G2 and 4B10 as described above and an Fc domain.

[0033] Preferably, the Fc domain is a human IgG Fc domain, and the amino acid sequence is shown in SEQ ID NO:13.

[0034] The third object of the present invention is to provide a polynucleotide encoding the nanobody as described above, or encoding the antibody with an Fc domain as described above; wherein the nucleotide sequence encoding 1H10 is as shown in SEQ ID NO: 14, the nucleotide sequence encoding 4G2 is as shown in SEQ ID NO: 15, and the nucleotide sequence encoding 4H10 is as shown in SEQ ID NO: 16.

[0035] A fourth object of the present invention is to provide an expression vector comprising the polynucleotide as described above.

[0036] A fifth object of the present invention is to provide a host cell comprising the expression vector as described above; preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, and mammalian cells.

[0037] A sixth object of the present invention is to provide the use of the nanobody or antibody as described above in the preparation of a product for detecting human papillomavirus type 18 E7 protein.

[0038] The present invention provides a detection kit comprising the nanobody or antibody as described above.

[0039] Preferably, the detection product further comprises a second antibody. Further preferably, the second antibody comprises a detectable label, such as a radioactive isotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

[0040] The present invention also provides a use of the nanobody or antibody as described above in preparing a vaccine for preventing human papillomavirus type 18 infection or a drug for treating human papillomavirus type 18 infection.

[0041] The beneficial effects of the present invention are:

[0042] 1. The present invention uses the HPV18 E7 protein expressed by the Escherichia coli prokaryotic expression system Rosetta bacteria to immunize camels four times, then draws blood to separate peripheral blood lymphocytes (PBMCs), extracts total RNA from the separated PBMCs, and then immediately reverse transcribes it into cDNA, which is used as a template to amplify the nanobody sequence, and finally separates and obtains three nanobodies, which are named 1H10, 4G2 and 4B10 respectively.

[0043] The nano antibody (VHH) is derived from natural camel heavy chain antibodies, and has the advantages of 1) simple structure and small molecular weight, which is conducive to expression and use; 2) convenient and efficient large-scale expression in Escherichia coli and various eukaryotic systems; 3) since it has only one binding site, it is a single-domain antibody, and has better permeability, specificity and detection linearity as a diagnostic reagent; 4) it is easy to couple with various fusion proteins or be more easily labeled with various markers; 5) it is easier to prepare bifunctional antibodies, which is more conducive to targeted drug development and directional transport of cell targets; 6) as a drug development, it has the advantages of low immunogenicity to humans and is not easy to produce immune rejection.

[0044] 2. The three nanobodies provided by the present invention have different antigen complementary determining regions. The antibody binding to HPV18E7 protein is expressed and secreted by mammalian cells (HEK 293F), and the antibody is fused with human Fc and cloned into the mammalian expression vector pTT5. The vector is transfected into mammalian cells 293F, and the supernatant is collected after 3 days of culture. The fusion protein in the supernatant is purified using a protein A affinity column, and the yield of the three nanobodies is greater than 50 mg / L. The results of ELISA and SPR show that the three nanobodies have extremely high affinity with HPV18E7, indicating that the nanobodies provided by the present invention are promising for detecting E7 protein in HPV18 infected cells.

[0045] 3. The results of sandwich ELISA showed that nanoantibody 1H10 as capture antibody and 4G2 or 4B10 as detection antibody can quantitatively analyze the protein level of HPV18 E7. The sandwich ELISA composed of two pairs of nanoantibodies 1H10 and 4G2, 1H10 and 4B10 provided by the present invention is used to quantitatively analyze the level of HPV18 E7 protein. By detecting the level of E7 protein in HPV18 infected cells, auxiliary diagnosis can be provided for infection detection of pathological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Figure 1 is the result of antigen purification and SDS-PAGE analysis, where Figure A is the molecular sieve result of antigen HPV18 E7, and Figure B is the SDS-PAGE result of HPV18 E7 protein.

[0047] Figure 2 The ELISA results of the monoclonal phage that specifically binds to the HPV18 E7 protein.

[0048] Figure 3 The purification and SDS-PAGE analysis results of the screened nanoantibodies, wherein Figure A is the elution profile of nanoantibody 1H10-Fc purified using a protein A affinity column, Figure B is the elution profile of nanoantibody 4G2-Fc purified using a protein A affinity column, Figure C is the elution profile of nanoantibody 4B10-Fc purified using a protein A affinity column, and Figure D is the SDS-PAGE results of 1H10-Fc, 4G2-Fc, and 4B10-Fc.

[0049] Figure 4 The results of the ELISA analysis of the binding of nanobodies to HPV18 E7 are shown in Figure 2. The EC values ​​of HPV18 E7 and nanobody 1H10-Fc are shown in Figure 2. 50 At 31.055±2.595 nM (Figure A), the EC of 4G2-Fc 50 At 0.2266±0.314 nM (Fig. B), the EC of 4B10-Fc 50At 0.1874±0.0271nM (Figure C).

[0050] Figure 5 The results of SPR binding analysis of nanoantibodies and HPV18 E7 showed that the KD of HPV18 E7 and nanoantibody 1H10-Fc was 0.0602 nM (Figure A), the KD of 4G2-Fc was 0.0678 nM (Figure B), and the KD of 4B10-Fc was 0.198 nM (Figure C).

[0051] Figure 6 Sandwich ELISA was constructed for two pairs of nanoantibodies to detect HPV18 E7 protein. The analysis results of ELISA are shown in Figure A. Detection signals appeared for both detection antibodies 4G2-Fc and 4B10-Fc, and amplified with the increase of detection antibody concentration, indicating that two pairs of antibodies 1H10 and 4G2-Fc, 1H10 and 4B10-Fc were successfully used for sandwich ELISA to detect HPV18 E7 protein. Figure B is the SDS-PAGE result after the capture antibody 1H10 was removed from Fc.

[0052] Figure 7 The sandwich ELISA was used to quantitatively detect HPV18 E7 protein by pairing antibodies 1H10 and 4B10-Fc. The sensitivity of the sandwich ELISA can reach the hundred nanogram level. DETAILED DESCRIPTION

[0053] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0054] Example 1

[0055] Purification of HPV18 E7 protein for camel immunization and screening

[0056] First, the gene sequence of HPV18 E7 (Genebank: LC509006.1, 1-315 bp) was determined, and the HPV18 E7-pET30a expression plasmid was obtained by gene synthesis from General Biotechnology (Anhui) Co., Ltd. The expression plasmid was transformed into Escherichia coli Rosetta and cultured at 220 rpm and 37 °C for OD 600 At 1:00, 1 mM IPTG was added to induce protein expression for 12 h, and then the bacteria were collected by centrifugation at 3000 rpm for 20 minutes for ultrasonic disruption. The disrupted bacterial solution was centrifuged at 14000 rpm for 30 minutes to collect the supernatant. The HPV18 E7-MBP fusion protein was purified using a Ni affinity column, and the eluted protein was digested with TEV enzyme and purified by a secondary Ni affinity column to remove TEV enzyme and MBP protein. The obtained HPV18 E7 protein was subjected to gel filtration chromatography and then SDS-PAGE electrophoresis analysis, as shown in FIG. Figure 1As shown in Figures A and B, high-purity HPV18 E7 antigen protein was obtained.

[0057] The steps of immunizing camels with the prepared HPV18 E7 antigen protein and isolating the obtained nanoantibodies are as follows:

[0058] 1) Healthy adult camels were selected for subcutaneous injection. The injection solution was an equal volume of HPV18 E7 protein in Freund's complete adjuvant (Tris-HCl, pH 6.8). The first immunization was on day 0, and then immunization was performed every 21 days, for a total of four immunizations (0.5 mg / time).

[0059] 2) After 84 days, blood was drawn from the jugular vein of the camels, and peripheral blood lymphocytes were separated using Ficoll from Solebol.

[0060] 3) Total RNA was extracted using Omega Biotek's RNA extraction kit, and genomic DNA was removed. RNA was reverse transcribed into cDNA using Takara's PrimeScriptTMII first-strand cDNA synthesis kit.

[0061] 4) Construction of nanobody phage display library: Using the above cDNA as a template, the coding sequence of nanobody was obtained by PCR amplification using specific camel VHH primers. The amplified nanobody sequence was inserted into the NcoI and NotI sites of phagemid pR2 by Gibson assembly. The obtained Gibson assembly product is the initial nanobody phage library, and the product is recovered.

[0062] 5) Prepare competent E. coli TG1 (MRC Laboratory of Molecular Biology) cells using the 10% glycerol washing method. The activated TG1 cells were cultured in 300 mL 2×YT (formula 1 L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) medium until the OD 600 About 0.6-0.8, centrifuged at 5000×g for 15 min, washed three times with 250mL, 250mL, and 100mL of pre-cooled 10% glycerol, and finally resuspended with 1mL of 10% glycerol and dispensed into 500μL per tube.

[0063] 6) Use BTX ECM 399 electroporator to transform Escherichia coli TG1 competent cells. Mix the product recovered from Gibson assembly with 500 μL TG1 competent cells and transfer to a 0.1 cm electroporation cuvette, followed by electrotransformation at 2.5 KV. Resuspend the electroporation product in 20 mL 2×YT medium and incubate at 37°C, 220 rpm for 1 h. Take 10 μL of bacterial solution, dilute to 990 μL 2×YT, mix well, then take 40 μL of bacterial solution, dilute to 160 μL 2×YT, plate 100 μL and culture overnight at 37°C. Count and calculate the library size on the second day (library size = number of counts × 10 5 ). The bacteria were spread on five 150 mm 2×YT plates supplemented with 100 μg / ml ampicillin and 2% glucose to amplify the phage library and cultured overnight at 37°C. Next, the transformed colonies were scraped from the plates, vortexed thoroughly with a final concentration of 25% glycerol, snap-frozen in liquid nitrogen in 1 mL aliquots and stored at -80°C, and the size of the phage library was calculated.

[0064] 7) Amplification of the Nanobody Phage Display Library: To amplify the Nanobody phage library, 0.1 ml of the frozen library was thawed on ice, diluted into 100 mL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and cultured at 37°C at 220 rpm. Next, 1×10 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added to the culture and incubated at 37°C (water bath) for 45 minutes. The cell pellet was separated by high-speed centrifugation and resuspended in 100 mL of 2×YT medium to which 0.1% glucose, 50 μg / mL kanamycin and 100 μg / mL ampicillin were added. The cells were incubated at 25°C, 220 rpm for 20 hours to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate phage particles. The precipitated phage particle pellet was dissolved in 1×PBS (formula: 10 mmol / L Na 2 HPO4; 1.75 mmol / L KH2PO4; 137 mmol / L NaCl; 2.65 mmol / L KCl; pH 7.2-7.6) and stored in 1 mL aliquots at -80°C in the presence of 25% glycerol.

[0065] 8) Screening: The purified HPV18 E7 protein was diluted with PBS to a final concentration of 0.1 mg / ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plate), and one well of the ELISA plate was set aside for negative control. After washing three times with 1×PBS, 260 μL of MPBS (1×PBS with 5% skim milk powder dissolved) was added to each well of the ELISA plate and incubated at room temperature for 2 hours to block unbound sites. Next, the plate was washed three times with 1×PBS and 1×10 11 pfu (diluted in 100 μL MPBS) phage library was added to each well, and after one hour of incubation at room temperature, it was washed 15-20 times with PBST (1×PBS containing 0.1% Tween 20). Phages displaying specificity for HPV18 E7 nanobodies were eluted by incubating at room temperature for one hour with trypsin at a final concentration of 0.5 mg / ml. 10 μL of eluted phage was added to 1 mL of E. coli TG1 competent cells and incubated at 37°C (water bath) for 45 minutes for infection, and then the bacterial culture was plated on 2×YT supplemented with 100 μg / mL ampicillin and 2% glucose and incubated overnight at 37°C.

[0066] 9) Preparation of monoclonal phage: After one round of panning, 48 individual colonies were picked into a 96-well round-bottom culture dish containing 100 μL of 2×YT medium supplemented with 100 μg / mL of ampicillin and 2% glucose (w / v). Incubate at 37°C, 220 rpm for 6 hours. Next, 5 μL of the culture solution was inoculated into a new 96-well round-bottom culture dish containing 100 μL / well of 2×YT medium supplemented with 100 μg / mL of ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37°C, 250 rpm for 1.5 hours until the OD 260 is about 0.5. 100 μL contains 4×10 8 pfu KM13 helper phage 2×YT / Amp / 2% glucose liquid medium was added to each well of the 96-well plate and incubated at 37°C for 45 minutes for infection. After infection, centrifuge at 3500×g for 15 minutes, discard the supernatant, resuspend the bacterial pellet in 150μL 2×YT / Amp / Kana / 0.1% glucose (w / v) medium, and incubate overnight at 25°C, 220rpm for about 14-16 hours. The next day, the culture was centrifuged at 3500×g for 30 minutes, and then 150μL of the supernatant was transferred to a new 96-well plate and stored at 4°C for screening of nanobodies.

[0067] 10) Phage ELISA test. Use 1×PBS for dilution, coat 96-well round-bottom immuno-ELISA plates with 100 μL HPV18 E7 at a final concentration of 2 μg / mL, and incubate at 4°C for 16 hours. Next, wash the ELISA plate 3 times with 1×PBS. Block the plate with MPBS (1×PBS with 5% skim milk powder dissolved) at room temperature for 2 hours. After completion, wash the wells 4 times with PBST (PBST). Add 100 μL of 1×10 11 pfu of phage was added to each well and incubated at room temperature for 1 hour. The ELSA plate was then washed 4 times with PBST. HRP-KM13 diluted 1:8000 in MPBS was added to each well at 100 μL and incubated at room temperature for 1 hour. The wells were then washed 4 times with PBST, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well, incubated in the dark for 7 minutes to develop color, and then 50 μL of 1M H 2 SO 4 Terminate the reaction and measure the OD using a microplate reader. 450 nm Value, the result is Figure 2 shown.

[0068] 11) Pick OD 450nm The positive clones with values ​​greater than 1 were sequenced and the sequence results were analyzed. Finally, three positive phage nanobodies specific for HPV18 E7 protein were determined, which were named 1H10 (amino acid sequence as shown in SEQ ID NO: 10), 4B10 (amino acid sequence as shown in SEQ ID NO: 11) and 4G2 (amino acid sequence as shown in SEQ ID NO: 12). These three nanobodies have specific CDR regions that bind to HPV18 E7.

[0069] Example 2

[0070] Expression and purification of nanobody-Fc fusion protein

[0071] The peptide gene sequence that guides secretion is designed and fused to the N-terminus of the nanobody gene to ensure secretion after expression, the human Fc is fused to the C-terminus of the nanobody gene, and the nanobody gene and human Fc are recombined and then cloned into the mammalian expression vector pTT5.

[0072] The constructed vector was transfected into HEK 293F cells (density about 2.5x10 6 cells / ml), in Freestyle TM 293 expression medium (purchased from Yonglian Biotechnology) was used to culture transfected mammalian cells in 5% CO 2, 150 rpm, 37 ° C for 4 days, and then the supernatant of the mammalian cell culture was collected by centrifugation at 3000 rpm for 15 minutes. The nanobody-Fc fusion protein was purified using a Protein A column and eluted with 0.1 M acetic acid. Figure 3 As shown in Figures A, B and C, the prefix a18E7 of the nanobody in the figure refers to the anti-HPV18 E7 protein. The eluted protein was subjected to SDS-PAGE electrophoresis analysis, as shown in Figures A, B and C. Figure 3 As shown in Figure D, high-purity nanobody proteins 1H10-Fc, 4G2-Fc, and 4B10-Fc were obtained.

[0073] Example 3

[0074] Binding affinity analysis of nanobodies to HPV18 E7

[0075] 1) ELISA analysis

[0076] Immuno MaxiSorb plates (Nunc) were coated with 4 μg / mL HPV18 E7 protein and allowed to stand at room temperature for 2 hours. The ELISA plate was washed 3 times with 1×PBS and 260 μL of MPBS (a dilution of 5% skim milk powder dissolved in PBS) was added to each well and allowed to stand at room temperature for 2 hours to block unbound sites. Next, nanoantibody-Fc was gradient diluted with MPBS (starting at 1000 or 4000 nM, 4-fold continuous gradient dilution, 11 gradient dilutions). 100 μL was added to each well and incubated at room temperature for 1 hour. After 1 hour, the ELISA plate was washed 3 times with 1×PBST and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) diluted with MBPS at a dilution factor of 1:6000 was added and incubated at room temperature for 1 hour. The ELISA plate was washed 3 times with 1×PBST and 100 μL of TMB was added to each well and reacted in the dark at room temperature for 8 minutes. Finally, 50 μL of 1M H 2 SO 4 The reaction was stopped and the absorbance at 450 nm was measured.

[0077] The results of ELISA analysis are as follows Figure 4 As shown in Figures A, B, and C, the EC of HPV18 E7 and nanobody 1H10-Fc 50 At 31.055±2.595 nM, the EC of 4G2-Fc 50 At 0.2266±0.314 nM, the EC of 4B10-Fc 50 At 0.1874±0.0271 nM, it was shown that the prepared nanobody-Fc fusion protein had extremely high affinity to HPV18 E7.

[0078] 2) SPR analysis

[0079] The kinetic constants of antibody to antigen HPV18 E7 were detected using a Biacore 8K instrument. HPV18 E7 protein was coated on a CM5 chip, and antibodies with different dilution concentrations (initial concentration: 50 nM for 1H10-Fc, 12.5 nM for 4G2-Fc, 25 nM for 4B10-Fc, two-fold serial dilution) were used to detect binding signals.

[0080] The SPR analysis results are as follows Figure 5 As shown in Figures A, B and C, the KD of HPV18 E7 and nanoantibody 1H10-Fc is 0.0602nM, the KD of 4G2-Fc is 0.0678nM, and the KD of 4B10-Fc is 0.198nM. The prepared nanoantibodies have extremely high affinity for HPV18 E7.

[0081] Example 4

[0082] Detection of HPV18 E7 protein by nanoantibodies

[0083] Using two pairs of nanoantibodies to construct a sandwich ELISA to detect HPV18 E7 protein:

[0084] The nanobody 1H10-Fc was cleaved with TEV enzyme, and the cleaved protein was passed through secondary Ni and protein A affinity columns to remove TEV enzyme and cleaved Fc protein respectively. The results of SDS-PAGE electrophoresis analysis are shown in Figure 6 As shown in Figure B, the purified nanoantibody 1H10 has extremely high purity and meets the requirements of sandwich ELISA capture antibodies.

[0085] Immuno MaxiSorb plates (Nunc) were coated with 5 μg / mL 1H10 nanoantibodies and allowed to stand at room temperature for 2 h. The ELISA plates were washed 3 times with 1×PBS and 260 μL of MPBS was added to each well and allowed to stand at room temperature for 2 h to block unbound sites. Next, HPV18 E7 antigen protein was diluted with MPBS (fixed concentration 100 μg / mL). 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed 3 times with 1×PBS and the detection antibodies 4G2-Fc or 4B10-Fc diluted with MPBS (starting concentration 4000 nM, 4-fold continuous gradient dilution, 11 gradient dilutions) were added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed 3 times with 1×PBST and HRP-anti-humanIgG Fc (Beijing Sino Biological) diluted with MPBS at a dilution factor of 1:6000 was added and incubated at room temperature for 1 h. Wash the ELISA plate three times with 1×PBST and add 100 μL of TMB to each well for 8 min at room temperature in the dark. Finally, add 50 μL of 1M H 2 SO 4 The reaction was stopped and the absorbance at 450 nm was measured.

[0086] The results of ELISA analysis are as follows Figure 6 As shown in Figure A, both detection antibodies 4G2-Fc and 4B10-Fc show detection signals, and the signals are amplified as the concentration of the detection antibodies increases. This indicates that the HPV18 E7 antigen protein is captured by the 1H10 capture antibody and adsorbed to the ELISA plate, and then the detection antibody 4G2-Fc or 4B10-Fc binds to the HPV18 E7 antigen protein, forming a ternary complex of 1H10-HPV18 E7-4G2-Fc or 1H10-HPV18 E7-4B10-Fc in the ELISA plate. Both pairs of antibodies 1H10 and 4G2-Fc, 1H10 and 4B10-Fc were successfully used in sandwich ELISA to detect HPV18 E7 protein.

[0087] Example 5

[0088] Quantitative detection of HPV18 E7 protein by sandwich ELISA with paired antibodies 1H10 and 4B10-Fc

[0089] Immuno MaxiSorb plates (Nunc) were coated with 5 μg / mL 1H10 nanoantibodies and allowed to stand at room temperature for 2 h. The ELISA plates were washed 3 times with 1×PBS and 260 μL of MPBS was added to each well and allowed to stand at room temperature for 2 h to block unbound sites. Next, HPV18 E7 antigen protein was diluted with MPBS (starting concentration 1000 μg / mL, 3-fold gradient serial dilution, 10 gradient dilutions). 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed 3 times with 1×PBS and the detection antibody 4B10-Fc (fixed concentration 1000 nM) diluted with MPBS was added. 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed 3 times with 1×PBST and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) diluted with MPBS at a dilution factor of 1:6000 was added and incubated at room temperature for 1 h. Wash the ELISA plate three times with 1×PBST and add 100 μL of TMB to each well for 8 min at room temperature in the dark. Finally, add 50 μL of 1M H 2 SO 4 The reaction was stopped and the absorbance at 450 nm was measured.

[0090] The results of ELISA analysis are as follows Figure 7 As shown, when the detected HPV18 E7 protein concentration was lower than 1 μg / mL, the experimental group minus the control group still had OD 450nm The readings were close to 0.01, and the error between the two sets of replicate wells was extremely small (small error bar), indicating that the sensitivity of the sandwich ELISA can reach the hundred nanogram level.

[0091] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanobody targeting human papillomavirus type 18 E7 protein, characterized in that: The antibody is a combination of any one or more of 1H10, 4G2 and 4B10, wherein 1H10, 4G2 and 4B10 each comprise three antigen complementary determining regions CDR1, CDR2 and CDR3, wherein: The amino acid sequences of the three antigen complementary determining regions of 1H10 are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; The amino acid sequences of the three antigen complementary determining regions of 4G2 are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively; The three antigen complementary determining region amino acid sequences of 4B10 are amino acid sequences having homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively.

2. A nanobody targeting human papillomavirus type 18 E7 protein according to claim 1, characterized in that: The three antigen complementary determining region amino acid sequences of the 1H10 are shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the three antigen complementary determining region amino acid sequences of the 4G2 are shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and the three antigen complementary determining region amino acid sequences of the 4B10 are shown as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

3. A nanobody targeting human papillomavirus type 18 E7 protein according to claim 1, characterized in that: The amino acid sequence of the 1H10 is shown in SEQ ID NO:10, the amino acid sequence of the 4G2 is shown in SEQ ID NO:11, and the amino acid sequence of the 4B10 is shown in SEQ ID NO:

12.

4. An antibody targeting HPV18 oncoprotein E7, comprising any one of the nanobodies 1H10, 4G2 and 4B10 as claimed in claim 1 and an Fc domain.

5. The antibody according to claim 4, characterized in that The Fc domain is a human IgG Fc domain.

6. A polynucleotide encoding a Nanobody as claimed in any one of claims 1 to 3, or encoding an antibody as claimed in any one of claims 4 to 5. An expression vector comprising the polynucleotide according to claim 6 . A host cell comprising the expression vector according to claim 7 .

9. Use of the nanobody according to any one of claims 1 to 3 or the antibody according to any one of claims 4 to 5 in the preparation of a product for detecting human papillomavirus type 18 E7 protein.

10. The use according to claim 9, wherein the detection is ELISA quantitative detection, the 1H10 is used as a capture antibody, and the 4G2-Fc is used as a detection antibody; or the 1H10 is used as a capture antibody, and the 4B10-Fc is used as a detection antibody.

11. A kit, characterized in that: Comprising the Nanobody according to any one of claims 1 to 3 or the antibody according to any one of claims 4 to 5.

12. Use of the Nanobody according to any one of claims 1 to 3 or the antibody according to any one of claims 4 to 5 in the preparation of a vaccine for preventing human papillomavirus type 18 infection or a medicament for treating human papillomavirus type 18 infection.

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