Nano antibody targeting HPV18 E7 protein and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a nano antibody targeting HPV18E7 protein and application of the nano antibody. The nano antibody is named as 1G8, and the 1G8 comprises three antigen complementary determining regions. Experimental results of ELISA and SPR show that the antibody 1G8-Fc obtained after the nano antibody and human IgG1Fc are fused has extremely high affinity with the HPV18E7 protein, and it is indicated that the nano antibody 1G8 provided by the invention is expected to be used for detecting the E7 protein in HPV18 infected cells and can provide an auxiliary diagnosis basis for HPV infection detection of pathological samples.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a nano antibody targeting HPV18 E7 protein and an application thereof. Technical Background

[0002] Cervical cancer is associated with human papilloma virus (HPV) infection. HPV is a small, non-enveloped, double-stranded circular DNA virus, of which HPV16 and 18 account for 80%. E6 and E7 are two critical viral proteins in the HPV infection cycle, of which E7 protein mediates the degradation of retinoblastoma protein pRB, causing the activation of transcription factor E2F and promoting the cell cycle.

[0003] At present, the prevention and treatment methods of cervical cancer are mainly based on screening, mainly TCT cell morphology detection and HPV molecular diagnosis, which are usually used in combination. However, TCT detection has the disadvantages of complicated procedures, long time consumption, misjudgment and omission caused by human judgment, while HPV molecular diagnosis has the disadvantages of high false positive rate, high instrument configuration requirements and high cost.

[0004] 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 level of E6 or E7 protein in the cells can semi-quantitatively reflect the severity of HPV infection. However, since a pair of antibodies is 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 that detects HPV16 E6 and HPV18 E6 proteins, the OncoE6 cervical test. Products and methods for high-sensitivity detection of HPV18E7 protein are still to be developed. Summary of the invention

[0005] In order to solve the problems in the prior art, one of the objectives of the present invention is to provide a nanobody targeting HPV18E7 protein.

[0006] The present invention adopts the following technical solutions:

[0007] A nanobody targeting HPV18 E7 protein, the antibody is named 1G8, and the 1G8 comprises three antigen complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively amino acid sequences with an amino acid sequence homology greater than or equal to 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0008] 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.

[0009] Preferably, the amino acid sequences of the three antigen complementary determining regions CDR1, CDR2 and CDR3 of 1G8 are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0010] Preferably, the amino acid sequences of 1G8 are as follows:

[0011] QLQLVESGGGSVQAGGSLRLSCAPS GSAYSTRPFWMG WFRQVSGKEREGVA CIFVASGRPV YADSVKGRFTISRDNAKTTLFLQMNDLKPEDTAMYYCA AALRVGSTCLRVADVRDW GQGTQVTVSS (SEQ ID NO: 4)

[0012] 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:

[0013] CDR1:GSAYSTRPFWMG(SEQ ID NO:1)

[0014] CDR2: CIFVASGRPV (SEQ ID NO: 2)

[0015] CDR3:AALRVGSTCLRVADVRDW(SEQ ID NO:3)

[0016] A second object of the present invention is to provide an antibody targeting HPV18 E7 protein, which has the 1G8 nanobody and Fc domain as described above.

[0017] Preferably, the Fc domain is a human IgG Fc domain, and the amino acid sequence is shown in SEQ ID NO:5.

[0018] 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 the 1G8 nanobody is shown in SEQ ID NO:6.

[0019] A fourth object of the present invention is to provide an expression vector comprising the polynucleotide as described above.

[0020] 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.

[0021] The 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 HPV18 E7 protein. In particular, the nanobody or antibody can be used as a primary antibody for Western Blot to detect HPV18 E7 protein.

[0022] The present invention provides a detection kit comprising the nanobody or antibody as described above.

[0023] 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.

[0024] 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.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention used the HPV18 E7 protein expressed by the Escherichia coli prokaryotic expression system Rosetta bacteria to immunize camels four times, then drew blood to separate peripheral blood lymphocytes (PBMCs), extracted total RNA from the separated PBMCs, and then immediately reverse transcribed into cDNA, which was used as a template to amplify the nanobody sequence, and finally isolated a nanobody named 1G8.

[0027] 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.

[0028] 2. The nanobody 1G8 provided by the present invention has 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 IgG 1Fc 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 nanobody 1G8 is greater than 50 mg / L.

[0029] The results of ELISA and SPR showed that the antibody 1G8-Fc formed by the fusion of the nanobody and human IgG1 Fc had a very high affinity for the HPV18 E7 protein, indicating that the nanobody 1G8 provided by the present invention has the potential to be used to detect the E7 protein in HPV18-infected cells, providing a basis for HPV diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] Figure 2 The ELISA results of the monoclonal phage that specifically binds to the HPV18 E7 protein.

[0032] Figure 3 Figure 3 is the purification and SDS-PAGE analysis results of the screened nanoantibodies, wherein Figure A is the elution diagram of the nanoantibody 1G8-Fc purified using a protein A affinity column, and Figure B is the SDS-PAGE result of the nanoantibody 1G8-Fc.

[0033] Figure 4 The results of the binding analysis of nanoantibody 1G8-Fc and HPV18 E7 by ELISA and SPR, where Figure A is the EC of antibody 1G8-Fc against HPV18 E7 detected by ELISA 50 Figure 2 is the KD value of antibody 1G8-Fc against HPV18 E7 detected by SPR.

[0034] Figure 5 This is the analysis result of using antibody 1G8-Fc as the primary antibody in Western Blot to detect HPV18 E7. DETAILED DESCRIPTION

[0035] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0036] Example 1

[0037] Purification of HPV18 E7 protein for camel immunization and screening

[0038] 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 1 As shown in Figures A and B, high-purity HPV18 E7 antigen protein was obtained.

[0039] The steps of immunizing camels with the prepared HPV18 E7 antigen protein and isolating the obtained nanoantibodies are as follows:

[0040] 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).

[0041] 2) After 84 days, blood was drawn from the jugular vein of the camels, and peripheral blood lymphocytes were separated using Ficoll from Solebol.

[0042] 3) Total RNA was extracted using Omega Biotek's RNA extraction kit, and genomic DNA was removed using Takara's PrimeScript TM II First Strand cDNA Synthesis Kit, reverse transcribe RNA into cDNA.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 12pfu 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 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.

[0047] 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.

[0048] 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 15min, 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.

[0049] 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 in MPBS 1:8000 was added to the ELISA plate at 100 μL per well 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 H2SO4 was immediately added to terminate the reaction, and the OD was measured by an enzyme reader. 450 nm Value, the result is Figure 2 shown.

[0050] 11) Pick OD 450nmThe positive clones with a value greater than 1 were sequenced and the sequence results were analyzed, and finally a positive phage nanobody specific for HPV18 E7 protein was obtained, named 1G8 (amino acid sequence as shown in SEQ ID NO: 4), which has a specific CDR region that binds to HPV18 E7.

[0051] Example 2

[0052] Expression and purification of nanobody-Fc fusion protein

[0053] 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.

[0054] The constructed vector was transfected into HEK 293F cells (density about 2.5x10 6 cells / ml), in Freestyle TM The transfected mammalian cells were cultured with 293 expression medium (purchased from Yonglian Bio) at 5% CO2, 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. The results are shown in FIG. Figure 3 As shown in Figure A, the prefix a18E7 of the nanobody in the figure refers to the anti-HPV18 E7 protein. The eluted protein was analyzed by SDS-PAGE electrophoresis, as shown in Figure A. Figure 3 As shown in Figure B, a highly pure nanobody protein 1G8-Fc was obtained.

[0055] Example 3

[0056] Binding affinity analysis of nanobodies to HPV18 E7

[0057] 1) ELISA (enzyme-linked immunosorbent assay) analysis

[0058] Immuno MaxiSorb plates (Nunc) were coated with 4 μg / mL HPV18 E7 protein and allowed to stand at room temperature for 2 hours. After washing the ELISA plate 3 times with 1×PBS, 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 was added at a dilution factor of 1:6000 and incubated at room temperature for 1 hour. After washing the ELISA plate 3 times with 1×PBST, 100 μL of TMB was added to each well and reacted at room temperature in the dark for 8 minutes. Finally, 50 μL 1 M H2SO4 was added to terminate the reaction and the absorbance at 450 nm was measured.

[0059] The results of ELISA analysis are as follows Figure 4 As shown in Figure A, the EC of HPV18 E7 and nanobody 1G8-Fc 50 The value was 0.6779±0.0883 nM, indicating that the prepared nanobody-Fc fusion protein had extremely high affinity to HPV18 E7.

[0060] 2) SPR (Surface Plasmon Resonance) Analysis

[0061] 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: 1G8-Fc was 25 nM, two-fold gradient serial dilution) were used to detect binding signals.

[0062] The SPR analysis results are as follows Figure 4 As shown in Figure B, the KD of HPV18 E7 and nanoantibody 1G8-Fc is 2.98nM, and the prepared nanoantibody has extremely high affinity for HPV18 E7.

[0063] Example 4

[0064] Detection of HPV18 E7 protein by nanoantibodies

[0065] As the primary antibody for Western Blot detection of HPV18 E7 protein:

[0066] 293T cells were cultured and transfected with HPV18 E7-pCDNA3.1 plasmid or empty pCDNA3.1 plasmid. 293T cells expressing HPV18 E7 and control 293T cells were collected 48 hours after transfection. Cells were lysed, and cell lysate and purified HPV18 E7 protein were taken for Western Blot. After transfer to PVDF membrane, PBST was used to dissolve 5% skim milk powder to prepare MPBST to block the protein on the membrane for 2 hours.

[0067] Detection of HPV18 E7 protein: The primary antibody was 2 μg / mL 1G8-Fc diluted in PBST, incubated for 1 h, 40 rpm, 25 ° C. The secondary antibody was HRP-anti-human IgG Fc (Sino Biological, Beijing) diluted in MPBST at a dilution of 1:4000, incubated for 1 h, 40 rpm, 25 ° C. The substrate was developed by the chemiluminescent ECL method.

[0068] The results are as follows Figure 5 As shown, the loading order is: lane 1 is 100ng HPV18 E7 protein purified by Rosetta prokaryotic expression system, lane 2 is 293T cells transfected with HPV18 E7-pCDNA3.1 plasmid, lane 3 is 293T cells treated with control, and GAPDH is used as the internal reference of cell loading amount. The results show that 1G8-Fc can be used as a Western Blot primary antibody to specifically detect HPV18 E7 protein expressed by Escherichia coli prokaryotes and HPV18 E7 protein expressed by mammalian cell 293T eukaryotic cells.

[0069] 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 HPV18 E7 protein, It is characterized in that The antibody is named 1G8, and 1G8 comprises three antigen complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 are amino acid sequences with homologies 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.

2. A nanobody targeting HPV18 E7 protein according to claim 1, It is characterized in that The amino acid sequences of the three antigen complementary determining regions CDR1, CDR2 and CDR3 of the 1G8 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

3. A nanobody targeting HPV18 E7 protein according to claim 1, It is characterized in that The amino acid sequence of 1G8 is shown in SEQ ID NO:

4.

4. An antibody targeting HPV18 E7 protein, comprising the 1G8 nanobody as claimed in claim 1 and an Fc domain.

5. The antibody according to claim 4, It is 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 HPV18 E7 protein.

10. A kit, It is 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.

11. 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.

Images