A nanobody targeting human papillomavirus type 18 E7 protein and its application

Abstract

This invention relates to the field of biopharmaceutical technology, and more particularly to a nanobody targeting the HPV18 E7 protein and its application. The antibody is any one or more combinations of 1H10, 4G2, and 4B10, each of which contains three antigen complementarity-determining regions. Experimental results show that the three nanobodies provided by this invention all exhibit high affinity binding to the HPV18 E7 protein. The sandwich ELISA method composed of two pairs of nanobodies, 1H10 and 4G2, and 1H10 and 4B10, provided by this invention can be used to quantitatively analyze the level of HPV18 E7 protein. Detecting the E7 protein level in HPV18-infected cells can provide auxiliary diagnostic evidence for infection detection in pathological samples.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a nanobody targeting the human papillomavirus type 18 E7 protein and its application. Technical Background

[0002] Cervical cancer is the fourth most common cancer among 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, with HPV types 16 and 18 accounting for 80% of cases. Currently, the prevention and treatment of cervical cancer mainly relies on screening, primarily through TCT cytology testing and HPV molecular diagnostics, which are usually used in combination. However, TCT testing has drawbacks such as a cumbersome process, long processing time, and the possibility of false positives or false negatives due to human error, while HPV molecular diagnostics suffers from a high false positive rate, high equipment requirements, and high costs.

[0003] HPV-infected cells typically highly express viral proteins E6 and E7. Detecting E6 and E7 proteins in HPV-infected cells using a pair of antibodies against different epitopes can reflect whether cells are infected with HPV. Furthermore, detecting the levels of E6 or E7 proteins in cells can semi-quantitatively reflect the severity of HPV infection. Because a pair of antibodies is used, the false positive rate is significantly reduced. Currently, the OncoE6 cervical test strip, which detects HPV16 E6 and HPV18 E6 proteins, is available on the market. Products and methods for highly sensitive detection of HPV18 E7 protein are still under development. 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 that targets the human papillomavirus type 18 E7 protein.

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

[0006] A nanobody targeting the human papillomavirus type 18 E7 protein, wherein the antibody is any one or more combinations of 1H10, 4G2, and 4B10, and each of 1H10, 4G2, and 4B10 contains three antigen complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0007] The amino acid sequences of the three antigen complementarity-determining regions of the 1H10 have a homology of ≥80% with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequences.

[0008] The amino acid sequences of the three antigen complementarity-determining regions of the 4G2 have 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, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequences.

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

[0010] The aforementioned homology sequences also include amino acid sequences that have one or more (preferably 1, 2 or 3) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

[0011] Preferably, the amino acid sequences of the three antigen complementarity-determining regions of 1H10 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the amino acid sequences of the three antigen complementarity-determining regions of 4G2 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the amino acid sequences of the three antigen complementarity-determining regions of 4B10 are shown in 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] The amino acid sequence of 1H10:

[0014]

[0015] The amino acid sequence of 4G2:

[0016]

[0017] The amino acid sequence of 4B10:

[0018]

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

[0020] The amino acid sequence of the antigen complementarity-determining region of 1H10:

[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 antigen complementarity-determining regions of 4G2:

[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 antigen complementarity-determining regions of 4B10:

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

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

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

[0032] The second objective of this invention is to provide an antibody that targets the HPV18 oncogenic protein E7, which has any one of the nanobodies of 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] A third objective of this invention is to provide a polynucleotide that encodes a nanobody as described above, or an antibody having an Fc domain as described above; wherein the nucleotide sequence encoding 1H10 is shown in SEQ ID NO:14, the nucleotide sequence encoding 4G2 is shown in SEQ ID NO:15, and the nucleotide sequence encoding 4H10 is shown in SEQ ID NO:16.

[0035] The fourth objective of this invention is to provide an expression vector comprising the polynucleotides described above.

[0036] A fifth objective 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 exogenous proteins, such as bacteria, yeast, insect cells, or mammalian cells.

[0037] The sixth objective of this invention is to provide the application of the nanobodies or antibodies described above in the preparation of products for detecting human papillomavirus type 18 E7 protein.

[0038] The present invention provides a detection kit comprising nanobodies or antibodies as described above.

[0039] Preferably, the detection product further includes a second antibody; more preferably, the second antibody includes a detectable marker, such as a radioactive isotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

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

[0041] The beneficial effects of this invention are as follows:

[0042] 1. In this invention, camels were immunized four times with HPV18 E7 protein expressed by Rosetta bacteria using the Escherichia coli prokaryotic expression system. Peripheral blood lymphocytes (PBMCs) were then isolated from the blood, and total RNA was extracted from the isolated PBMCs. The RNA was then immediately reverse transcribed into cDNA. The cDNA was used as a template to amplify nanobody sequences, and three nanobodies were finally isolated and named 1H10, 4G2 and 4B10, respectively.

[0043] This nanobody (VHH) is derived from a natural camel heavy chain antibody and has the following advantages: 1) simple structure and small molecular weight, which are conducive to expression and use; 2) convenient for efficient and large-scale expression in E. coli and various eukaryotic systems; 3) due to having only one binding site, it is a single-domain antibody, which has better permeability, specificity and detection linearity as a diagnostic reagent; 4) easy to couple with various fusion proteins or more easily labeled by various markers; 5) easier to prepare bifunctional antibodies, which is more conducive to targeted drug development and cellular target-directed delivery; 6) as a drug development agent, it has the advantages of low immunogenicity in humans and is less likely to cause immune rejection.

[0044] 2. The three nanobodies provided in this invention have different antigen complementarity-determining regions. Antibodies binding to the HPV18 E7 protein were expressed and secreted in mammalian cells (HEK 293F). The antibodies were fused with human Fc cells and cloned into the mammalian expression vector pTT5. The vector was transfected into mammalian 293F cells, and the supernatant was collected after 3 days of culture. The fusion protein in the supernatant was purified using a protein A affinity column. The yields of all three nanobodies were greater than 50 mg / L. ELISA and SPR results showed that all three nanobodies had extremely high affinity for HPV18 E7, indicating that the nanobodies provided in this invention have the potential to be used for detecting the E7 protein in HPV18-infected cells.

[0045] 3. Sandwich ELISA results show that nanobody 1H10, as the capture antibody, and 4G2 or 4B10, as the detection antibody, can quantitatively analyze the protein level of HPV18 E7. The sandwich ELISA method composed of two pairs of nanobodies, 1H10 and 4G2, and 1H10 and 4B10, provided by this invention, is used to quantitatively analyze the level of HPV18 E7 protein. Detecting the E7 protein level in HPV18-infected cells can provide auxiliary diagnostic information for infection detection in pathological samples. Attached Figure Description

[0046] Figure 1 Figure A shows the results of antigen purification and SDS-PAGE analysis. Figure B shows the molecular sieve results of the HPV18 E7 antigen and the SDS-PAGE results of the HPV18 E7 protein.

[0047] Figure 2 ELISA results for monoclonal phages that specifically bind to the HPV18 E7 protein.

[0048] Figure 3 The images show the purification and SDS-PAGE analysis results of the screened nanobodies. Figure A shows the elution image of nanobodies 1H10-Fc purified using a protein A affinity column, Figure B shows the elution image of nanobodies 4G2-Fc purified using a protein A affinity column, Figure C shows the elution image of nanobodies 4B10-Fc purified using a protein A affinity column, and Figure D shows the SDS-PAGE results of 1H10-Fc, 4G2-Fc, and 4B10-Fc.

[0049] Figure 4 The results of ELISA analysis of the binding of nanobodies to HPV18 E7 are shown. The EC50 of HPV18 E7 and nanobodies 1H10-Fc is also described. 50 At 31.055±2.595 nM (Figure A), the EC of 4G2-Fc 50 At 0.2266±0.314 nM (Figure B), EC of 4B10-Fc 50At 0.1874±0.0271 nM (Figure C).

[0050] Figure 5 To illustrate the binding of SPR nanobodies to HPV18 E7, the KD of HPV18 E7 to nanobodies 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 Two pairs of nanobodies were used to construct a sandwich ELISA for detecting HPV18 E7 protein. The ELISA results are shown in Figure A. Both detection antibodies 4G2-Fc and 4B10-Fc showed detection signals, which increased with increasing antibody concentration, indicating that both pairs of antibodies, 1H10 and 4G2-Fc, and 1H10 and 4B10-Fc, were successfully used in the sandwich ELISA for detecting HPV18 E7 protein. Figure B shows the SDS-PAGE results after Fc excision of the capture antibody 1H10.

[0052] Figure 7 For the quantitative detection of HPV18 E7 protein using a sandwich ELISA with paired antibodies 1H10 and 4B10-Fc, the sensitivity of this sandwich ELISA can reach the 100 nanogram level. Detailed Implementation

[0053] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to embodiments:

[0054] Example 1

[0055] Purified HPV18 E7 protein for immunization and screening of camels

[0056] First, the gene sequence of HPV18 E7 was determined (Genebank: LC509006.1, 1-315bp), and the HPV18 E7-pET30a expression plasmid was synthesized by General Biotechnology (Anhui) Co., Ltd. The expression plasmid was transformed into *E. coli* Rosetta and cultured at 220 rpm and 37°C for OD... 600 At 1 hour, 1 mM IPTG was added to induce protein expression for 12 hours. Then, the bacterial cells were collected by centrifugation at 3000 rpm for 20 minutes and subjected to ultrasonic lysis. The lysed bacterial solution was centrifuged at 14000 rpm for 30 minutes, and the supernatant was collected. The HPV18 E7-MBP fusion protein was purified using a Ni affinity column. The eluted protein was digested with TEV enzyme and purified a second time using a Ni affinity column to remove TEV enzyme and MBP protein. The obtained HPV18 E7 protein was identified by gel filtration chromatography and then analyzed by SDS-PAGE electrophoresis. Figure 1As shown in Figures A and B, high-purity HPV18 E7 antigen protein was obtained.

[0057] The nanobodies obtained by immunizing camels with the prepared HPV18 E7 antigen protein and then isolating them are as follows:

[0058] 1) Select healthy adult camels and inject them subcutaneously with an equal volume of HPV18 E7 protein (Tris-HCl, pH 6.8) with Freund's complete adjuvant. The first immunization was on day 0, and then every 21 days thereafter, for a total of four immunizations (0.5 mg / time).

[0059] 2) After day 84, blood was drawn from the jugular vein of the camel and peripheral blood lymphocytes were separated using Solarbio's Ficoll.

[0060] 3) Total RNA was extracted using the Omega Biotek RNA Extraction Kit, and genomic DNA was removed. The RNA was then reverse transcribed into cDNA using the Takara PrimeScript™ II First-Strand cDNA Synthesis Kit.

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

[0062] 5) *E. coli* TG1 (MRC Laboratory of Molecular Biology) competent cells were prepared using the 10% glycerol washing method. Activated TG1 cells were cultured in 300 mL of 2×YT medium (formulation 1L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) until OD500. 600 After centrifuging at 5000×g for 15 min at approximately 0.6-0.8, wash three times with 250 mL, 250 mL, and 100 mL of pre-cooled 10% glycerol. Finally, resuspend in 1 mL of 10% glycerol and dispense into 500 μL tubes.

[0063] 6) Transform *E. coli* TG1 competent cells using a BTX ECM 399 electroporator. The Gibson assembly product was mixed with 500 μL of TG1 competent cells and transferred to a 0.1 cm electroporation cuvette, followed by electroporation at 2.5 kV. The electroporated product was resuspended in 20 mL of 2×YT medium and incubated at 37°C and 220 rpm for 1 h. 10 μL of the bacterial culture was diluted to 990 μL of 2×YT, and then another 40 μL of the bacterial culture was diluted to 160 μL of 2×YT. A 100 μL plate was then plated and incubated overnight at 37°C. The next day, the library size was calculated by counting the cells (library size = count × 10⁻⁶). 5 Bacteria were spread onto five 150 mm 2×YT plates supplemented with 100 μg / ml ampicillin and 2% glucose to amplify the phage library, and incubated overnight at 37°C. Next, transformed colonies were scraped from the plates, vortexed thoroughly with a final concentration of 25% glycerol, flash-frozen in 1 mL aliquots with liquid nitrogen, and stored at -80°C. The size of the phage library was then 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 and diluted in 100 mL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and incubated at 37°C and 220 rpm. Next, 1×10⁻⁶ phage display libraries were added to the library. 12 PFU-modified 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 supplemented with 0.1% glucose, 50 μg / mL kanamycin, and 100 μg / mL ampicillin. The cells were incubated at 25°C and 220 rpm for 20 hours to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate the phage particles. The precipitated phage particles were dissolved in 1×PBS (formulation: 10 mmol / L Na₂HPO₄; 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 ELISA plate (Nunc Maxsorp plate), reserving one well for a negative control. After washing three times with 1×PBS, 260 μL of MPBS (1×PBS dissolved in 5% skim milk powder) was added to each well of the ELISA plate, and the plate was incubated at room temperature for 2 hours to block unbound sites. Next, the plate was washed three times with 1×PBS, and 1×10⁻⁶ ppm of the protein containing the protein targeting HPV18 E7 was added to each well. 11 A PFU (diluted in 100 μL MPBS) phage library was added to each well. After incubation at room temperature for one hour, the cells were washed 15–20 times with PBST (1×PBS containing 0.1% Tween 20). Phages exhibiting specificity against HPV18 E7 nanobodies were eluted by incubation at room temperature for one hour with trypsin at a final concentration of 0.5 mg / mL. 10 μL of the 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. The bacterial culture was then plated on 2×YT supplemented with 100 μg / mL ampicillin and 2% glucose and incubated overnight at 37°C.

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

[0067] 10) Phage ELISA Assay. A 96-well round-bottom immunoassay plate was coated with 100 μL of HPV18 E7 solution (to a final concentration of 2 μg / mL) after dilution with 1×PBS and incubated at 4°C for 16 hours. The plate was then washed three times with 1×PBS. The plate was blocked with MPBS (1×PBS containing 5% skim milk powder) at room temperature for 2 hours. Afterward, the wells were washed four times with PBST. Finally, 100 μL of 1×10⁻⁶ phage ELISA solution was added. 11 PFU phage was added to each well and incubated at room temperature for 1 hour. The ELISA plate was then washed four times with PBST. Next, 100 μL of HRP-KM13 diluted 1:8000 with MPBS was added to each well, and the plate was incubated at room temperature for 1 hour. The wells were then washed four times with PBST, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well. The plate was incubated in the dark for 7 minutes for color development, and then immediately 50 μL of 1M H2SO4 was added to terminate the reaction. The OD was measured using a microplate reader. 450 nm Value, result as Figure 2 As shown.

[0068] 11) Picking OD 450nm Positive clones with a value greater than 1 were sequenced and the sequence results were analyzed. Finally, three positive phage nanobodies specific to HPV18 E7 protein were identified and 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] Design a secretion-guided peptide gene sequence and fuse it to the N-terminus of the nanobody gene to ensure secretion after expression. Fc is fused to the C-terminus of the nanobody gene, and the nanobody gene and human Fc are recombined. Then, it is cloned into the mammalian expression vector pTT5.

[0072] The construct vector was transfected into HEK 293F cells using polyethyleneimine (PEI) at a density of approximately 2.5 x 10⁻⁶ cells / year. 6 (cells / ml), in Freestyle TMTransfected mammalian cells were cultured in 293 expression medium (purchased from Yonglian Biotechnology) at 5% CO2, 150 rpm, and 37°C for 4 days. The supernatant of the mammalian cell culture was then collected by centrifugation at 3000 rpm for 15 minutes. The nanobody-Fc fusion protein was purified using a Protein A column with 0.1 M acetic acid elution. The results are as follows... Figure 3 As shown in Figures A, B, and C, the nanobody prefix a18E7 in the figures refers to the anti-HPV18 E7 protein. The eluted protein was analyzed by SDS-PAGE electrophoresis, as follows... 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 for HPV18 E7

[0075] 1) ELISA analysis

[0076] Immuno MaxiSorb (Nunc) plates were coated with 4 μg / mL HPV18 E7 protein and incubated at room temperature for 2 h. After washing the ELISA plates three times with 1×PBS, 260 μL of MPBS (a PBS-based dilution of 5% skim milk powder) was added to each well and incubated at room temperature for 2 h to block unbound sites. Next, the nanobody-Fc was serially diluted with MPBS (starting at 1000 or 4000 nM, 4-fold serial dilutions, 11 dilutions). 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed three times with 1×PBST, and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) diluted 1:6000 with MBPS was added and incubated at room temperature for 1 h. Finally, the ELISA plates were washed three times with 1×PBST, and 100 μL of TMB was added to each well and reacted at room temperature in the dark for 8 min. Finally, 50 μL of 1M H2SO4 was added to terminate the reaction, and the absorbance at 450 nm was measured.

[0077] The results of the ELISA analysis are as follows: Figure 4 Figures A, B, and C show the EC50 of HPV18 E7 and nanobody 1H10-Fc. 50 EC at 31.055±2.595 nM, 4G2-Fc 50 EC at 0.2266±0.314 nM, 4B10-Fc 50 The concentration at 0.1874 ± 0.0271 nM indicates that the prepared nanobody-Fc fusion protein has extremely high affinity for HPV18 E7.

[0078] 2) SPR analysis

[0079] The kinetic constants of the antibody against the HPV18 E7 antigen were detected using a Biacore 8K instrument. HPV18 E7 protein was coated onto a CM5 chip, and binding signals were detected using antibodies at different dilutions (initial concentrations: 50 nM for 1H10-Fc, 12.5 nM for 4G2-Fc, 25 nM for 4B10-Fc, serially diluted twofold).

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

[0081] Example 4

[0082] Detection of HPV18 E7 protein by nanobodies

[0083] Detection of HPV18 E7 protein using a sandwich ELISA constructed with two pairs of nanobodies:

[0084] The nanobody 1H10-Fc was digested with TEV enzyme. The digested protein was then subjected to two Ni and protein A affinity columns to remove the TEV enzyme and the excised Fc protein, respectively. SDS-PAGE electrophoresis analysis results are shown below. Figure 6 As shown in Figure B, the purified nanobody 1H10 has extremely high purity, meeting the requirements for antibody capture in sandwich ELISA.

[0085] Immuno MaxiSorb (Nunc) plates were coated with 5 μg / mL 1H10 nanobody and incubated at room temperature for 2 h. After washing the ELISA plates three times with 1×PBS, 260 μL of MPBS was added to each well and incubated at room temperature for 2 h to block unbound sites. Next, the 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 three times with 1×PBS, and the detection antibody 4G2-Fc or 4B10-Fc diluted with MPBS (starting concentration 4000 nM, 4-fold serial dilution, 11 dilutions) was added to each well. 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed three times with 1×PBST, and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) diluted 1:6000 with MPBS was added and incubated at room temperature for 1 h. After washing the ELISA plate three times with 1×PBST, add 100 μL of TMB to each well and react at room temperature in the dark for 8 min. Finally, add 50 μL of 1M H2SO4 to terminate the reaction and measure the absorbance at 450 nm.

[0086] The results of the ELISA analysis are as follows: Figure 6 As shown in Figure A, both detection antibodies 4G2-Fc and 4B10-Fc showed detection signals, which increased with increasing antibody concentration. This indicates that the HPV18 E7 antigen protein was captured and adsorbed onto the ELISA plate by the 1H10 capture antibody. Subsequently, the detection antibody 4G2-Fc or 4B10-Fc bound 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, and 1H10 and 4B10-Fc, were successfully used in sandwich ELISA for the detection of HPV18 E7 protein.

[0087] Example 5

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

[0089] Immuno MaxiSorb (Nunc) plates were coated with 5 μg / mL 1H10 nanobody and incubated at room temperature for 2 h. After washing the ELISA plates three times with 1×PBS, 260 μL of MPBS was added to each well and incubated at room temperature for 2 h to block unbound sites. Next, the HPV18 E7 antigen protein was diluted with MPBS (starting concentration 1000 μg / mL, serially diluted 3-fold, 10 dilutions). 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed three times with 1×PBS, and the detection antibody 4B10-Fc diluted with MPBS (fixed concentration 1000 nM) 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 three times with 1×PBST, and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) diluted 1:6000 with MPBS was added and incubated at room temperature for 1 h. After washing the ELISA plate three times with 1×PBST, add 100 μL of TMB to each well and react at room temperature in the dark for 8 min. Finally, add 50 μL of 1M H2SO4 to terminate the reaction and measure the absorbance at 450 nm.

[0090] The results of the ELISA analysis are as follows: Figure 7 As shown, when the detected HPV18 E7 protein concentration was below 1 μg / mL, the experimental group still had OD after subtracting the control group. 450nm The readings are accurate, and the error between the two sets of replicates is extremely small, indicating that the sensitivity of this sandwich ELISA method can reach the level of hundreds of nanograms.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, 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 within the protection scope of the present invention.

Claims

1. A nanobody targeting the human papillomavirus type 18 E7 protein, characterized in that, The antibody is any one of 1H10, 4G2, and 4B10, wherein 1H10, 4G2, and 4B10 each contain three antigen complementarity-determining regions CDR1, CDR2, and CDR3, wherein: The amino acid sequences of the three antigen complementarity-determining regions of the 1H10 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; The amino acid sequences of the three antigen complementarity-determining regions of the 4G2 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; The amino acid sequences of the three antigen complementarity-determining regions of the 4B10 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively.

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

12.

3. A nanobody composition targeting HPV18 oncogenic protein E7, characterized in that, The composition is selected from at least two of 1H10, 4G2 and 4B10 as described in claim 1.

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

5. The antibody as described in claim 4, characterized in that, The Fc domain is the human IgG Fc domain.

6. A polynucleotide encoding a nanobody as described in any one of claims 1-2, or an antibody as described in any one of claims 4-5.

7. An expression vector comprising the polynucleotide as described in claim 6.

8. A host cell comprising the expression vector as described in claim 7.

9. The use of the nanobody according to any one of claims 1-2, the nanobody composition according to claim 3, or the antibody according to any one of claims 4-5 in the preparation of a product for detecting human papillomavirus type 18 E7 protein.

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

11. A reagent kit, characterized in that, It comprises a nanobody as described in any one of claims 1-2, a nanobody composition as described in claim 3, or an antibody as described in any one of claims 4-5.

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