Neutralizing nano antibody for targeting porcine epidemic diarrhea virus RBD and application of neutralizing nano antibody

Abstract

The invention relates to the technical field of biology, in particular to a neutralizing nano antibody targeting a porcine epidemic diarrhea virus RBD and application of the neutralizing nano antibody. The nano antibody is named as C1, and has a specific CDR region combined with PEDV RBD. C1 can be combined with a receptor binding region of the porcine epidemic diarrhea virus with high affinity, and has a good neutralizing effect on the PEDV when the concentration is 6.25 mu g / mL or above. The nano antibody provided by the invention is hopeful to be applied to a porcine epidemic diarrhea virus patent medicine, and has a relatively high application value in prevention, control and treatment of the porcine epidemic diarrhea virus.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a neutralizing nano antibody targeting porcine epidemic diarrhea virus RBD and an application thereof. Technical Background

[0002] Porcine epidemic diarrhea virus (PEDV) has been prevalent in most parts of the world since its emergence in the 1970s. It spreads rapidly by infecting the small intestine and intestinal villus cells, causing infectious intestinal diseases characterized by vomiting, diarrhea and dehydration, resulting in high mortality in newborn piglets with low maternal antibody immunity or loss of natural immunity, causing huge economic losses to the pig industry. Using gene phylogenetic tree analysis, PEDV can be divided into two major gene groups, S INDEL and non-S INDEL, GⅠ S INDEL (GⅠa, GⅠb) and GⅡ (GⅡa, non-S INDEL-GⅡb), according to the insertion or deletion of the S1 subunit of PEDV. Among them, GⅠ type mainly includes some classic strains, such as CV777, DR13 and their cell-adapted strains, and GⅡ type is mainly variant strains isolated since 2010. There is currently no specific treatment for the disease, and vaccines based on classic PEDV strains cannot completely protect pigs from the prevalent PEDV variant strains.

[0003] Nanobodies are derived from alpacas or sharks, and have the advantages of small molecular weight (15kDa), good penetration, high stability, low immunogenicity, etc. They are easy to produce and low in cost. They are also suitable for developing new drug delivery methods other than injections used for traditional antibody drugs. According to its research, PEDV enters cells through the binding of its spike protein (S protein) to host cell receptors.

[0004] Therefore, developing a nanoantibody targeting RBD and screening PEDV therapeutic nanoantibodies with high drugability have great market value for the treatment of porcine epidemic diarrhea and the development of the pig industry. Summary of the invention

[0005] In order to solve the problems in the prior art, one of the objects of the present invention is to provide a neutralizing nanobody targeting porcine epidemic diarrhea virus RBD, which can bind to PEDV RBD with high affinity and provide a broad-spectrum neutralizing effect on porcine epidemic diarrhea virus.

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

[0007] A neutralizing nanobody targeting porcine epidemic diarrhea virus RBD, the nanobody is named C1, and the three antigen complementary determining regions CDR1, CDR2 and CDR3 of C1 are amino acid sequences with a homology 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, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[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 sequence of C1 is as follows:

[0010]

[0011] 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:

[0012] CDR1: GFDFTKYAI (SEQ ID NO: 1)

[0013] CDR2:VVSCISTGDYR(SEQ ID NO:2)

[0014] CDR3:AAFVGLYYPSCPLITEE (SEQ ID NO:3).

[0015] A second object of the present invention is to provide an antibody targeting SARS2 RBD, which has the nanobody C1 and Fc domain as described above.

[0016] Preferably, the Fc domain is a human IgG Fc domain, and the amino acid sequence of the human IgG Fc domain is shown in SEQ ID NO:5.

[0017] Preferably, the Nanobody or antibody further comprises a second antibody, which comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

[0018] The present invention also provides a polynucleotide encoding the nanobody as described above, or encoding the antibody comprising the Fc domain as described above, wherein the nucleotide sequence encoding the C1 is shown in SEQ ID NO:6.

[0019] The present invention also provides an expression vector comprising the polynucleotide as described above, and a host cell comprising the expression vector as described above.

[0020] Preferably, the host cell is a host cell used to express foreign proteins, such as bacteria, yeast, insect cells or mammalian cells.

[0021] The present invention provides the use of the nanobody / antibody as described above in the preparation of a vaccine for preventing porcine epidemic diarrhea or a drug for treating porcine epidemic diarrhea.

[0022] The present invention also provides the use of the nanobody / antibody as described above in the preparation of a kit for diagnosing porcine epidemic diarrhea virus infection.

[0023] Finally, the present invention also provides a pharmaceutical composition comprising a pharmaceutically effective dose of the Nanobody or antibody as described above.

[0024] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more functional adjuvants selected from the group consisting of excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents, such as starch, lipids, wax, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and the like.

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

[0026] 1. The present invention uses PEDV RBD expressed by the mammalian expression system HEK 293F to immunize alpacas 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, uses the cDNA as a template to amplify the nanobody sequence, and finally isolates and obtains a nanobody named C1.

[0027] The nano antibody (VHH) is derived from natural alpaca 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 pigs and is not easy to produce immune rejection.

[0028] 2. The nanobody C1 of the present invention has different antigen complementary determining regions. The ELISA results show that the nanobody C1 shows high affinity binding to PEDV RBD, EC 50 At 0.637 ± 0.134 nM, SPR results showed an affinity of K D =6.37×10 -8 M, virus neutralization test results show that nano antibody C1 has a good neutralization effect at a concentration of 6.25 μg / mL and above. The nano antibody provided by the present invention is expected to be used as a drug for porcine epidemic diarrhea virus, and has a high application value for the prevention, control and treatment of porcine epidemic diarrhea virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A is the result of antigen RBD 24mL molecular sieve, and B is the SDS-PAGE result after RBD Fc removal.

[0030] Figure 2 ELISA results of monoclonal phages that specifically bind to PEDV RBD.

[0031] Figure 3 A is the elution diagram of the nanobody C1-Fc purified by Protein A column, B is the SDS-PAGE result after C1-Fc purification, and C is the SDS-PAGE result of C1 after Fc removal.

[0032] Figure 4 The results of the ELISA analysis of the binding of nanobodies to PEDV RBD; EC 50 =0.637±0.134nM.

[0033] Figure 5 The results of SPR analysis of the binding of nanoantibodies to PEDV RBD; K D =6.37×10 -8 M.

[0034] Figure 6 This is the identification result of the complex formed by 24mL superdex 200 antigen RBD and antibody C1-Fc.

[0035] Figure 7 The results of the neutralization of PEDV using C1-Fc showed that the C1 antibody had a good neutralization effect at concentrations of 6.25 μg / ml and above. Figure A shows the IFA results after the addition of the antibody, and Figure B shows the density of Vero cells infected with PEDV after the addition of the antibody. DETAILED DESCRIPTION

[0036] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0037] Example 1

[0038] Purification of PEDV RBD for immunization of alpacas and screening

[0039] 1) PEDV RBD expression plasmid was constructed according to the gene sequence of PEDV RBD, and the expression plasmid was transfected into HEK 293F cells, cultured at 5% CO2, 150rpm, and 37°C for 4 days, and then the supernatant of the mammalian cell culture was collected by centrifugation at 3000rpm for 10min. The RBD-Fc fusion protein was purified using a Protein A column, eluted with 0.1M acetic acid, and the eluted column was digested with TEV enzyme. The obtained RBD was subjected to gel filtration chromatography and then SDS-PAGE electrophoresis analysis, such as Figure 1 As shown, a highly pure PEDV RBD antigen protein was obtained.

[0040] 2) Alpacas were immunized with highly purified PEDV RBD antigen protein. Healthy adult alpacas were selected for subcutaneous injection. The injection solution was an equal volume of RBD protein in GERBU adjuvant (HEPES, pH 5.3). The first immunization was on day 0, and then immunization was performed every 14 days, for a total of three immunizations (0.2 mg / time), and the third immunization was intramuscular injection; after the 56th day, a booster immunization was performed with an injection of 0.5 mg RBD protein.

[0041] 3) After 70 days, blood was drawn from the jugular vein of the alpacas, and peripheral blood lymphocytes were separated using Ficoll from Solebo.

[0042] 4) Total RNA was extracted using Omega Biotek's RNA extraction kit and genomic DNA was removed. PrimeScript from Takara was used TM II First Strand cDNA Synthesis Kit, reverse transcribe RNA into cDNA.

[0043] 5) 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 alpaca 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] 6) 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 medium (formula 1 L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) 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] 7) 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] 8) 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 / LNa2HPO4; 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] 9) Screening: The purified RBD 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 containing 5% skim milk) 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% Tween20). Phages displaying specific PEDV RBD 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] 10) 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.

[0049] 11) Phage ELISA test. Use 1×PBS for dilution, coat 96-well round-bottom immuno-ELISA plates with 100 μL RBD 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 containing 5% skim milk) 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] 12) OD 450 nm27 positive clones with a value greater than 1 were selected for sequencing and the sequence results were analyzed. Finally, C1 was determined to be a positive phage nanobody specific for PEDV RBD protein, and its amino acid sequence was shown in SEQ ID NO: 1. The nanobody C1 had a specific CDR region that bound to PEDV RBD.

[0051] Example 2

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

[0053] The peptide gene sequence that guides secretion was designed and fused to the N-terminus of the nanobody C1 gene to ensure secretion after expression, the human IgG1 Fc was fused to the C-terminus of the nanobody gene, and the nanobody gene and human IgG1 Fc were recombined and then cloned into the mammalian expression vector pTT5.

[0054] The constructed vector was transfected into HEK 293F cells (density about 2.5×10 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 min. The nanobody-Fc fusion protein was purified using a Protein A column and eluted with 0.1 M acetic acid. The eluted protein was digested with TEV and analyzed by SDS-PAGE electrophoresis. Figure 3 As shown, a highly pure nanobody protein was obtained.

[0055] Example 3

[0056] 1) ELISA analysis of the binding affinity of nanoantibodies to PEDV RBD

[0057] Immuno MaxiSorb plates (Nunc) were coated with 2 μg / mL PEDV RBD 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 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 a diluent containing 5% milk in PBST (starting at 300 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) was diluted with a diluent containing 5% milk in PBST at a ratio of 1:6000 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 at room temperature in the dark for 7 minutes. Finally, 50 μL 1 M H2SO4 was added to terminate the reaction and the absorbance at 450 nm was measured.

[0058] The results of ELISA analysis are as follows Figure 4 As shown, the EC of RBD and nanobody C1-IgG1 Fc 50 At 0.637±0.134nM, it has a higher affinity.

[0059] 2) SPR analysis of the binding affinity of nanoantibodies to PEDV RBD

[0060] The prepared C1-Fc antibody and RBD were subjected to affinity determination using Biacore 8K. Sodium acetate with a pH value of 4.0 was used to dilute to 20 μg / mL, and the RBD was coupled to the activated CM5 chip using the imbolization program. At the same time, a control channel without protein coupling was set up, and RBD was flowed through at a rate of 30 μL / min. When the coupling value reached 200Ru, ethanolamine was used to block. C1-Fc was diluted 3 times from 1000nM for a total of 5 gradients, and the samples were added to a 96-well plate. 50mM NaOH was used as the regeneration solution. The multi-cycle kintics / affinity program was used for kinetic determination, with 120s for sample loading and 600s for dissociation. The results were analyzed using the Biacore evaluation program, as shown in the following figure. Figure 5 The SPR results showed that the affinity of RBD to C1 was 6.37×10 -8 M.

[0061] Example 4

[0062] Analysis of the complex between nanobodies and PEDV RBD

[0063] The PEDV RBD was incubated with the nanoantibody C1-IgG1 Fc on ice for 1 h (molar ratio 1:1.2) to prepare a complex, and superdex 200 molecular sieve was used to identify whether the complex was formed. The results are as follows: Figure 6 As shown. Nanobody-Fc exists in a dimer form (red peak), and the antigen exists in a monomer form (blue peak). Compared with the single peak of C1-Fc or PEDV, the protein absorption peak of the incubation product (PEDV-C1-Fc) is significantly forward-shifted. According to the molecular sieve peak principle, the larger the molecular weight, the more forward the peak position. The appearance of this forward-shifted absorption peak indicates the formation of a substance with a larger molecular weight than C1-Fc or PEDV, that is, the formation of a complex, indicating that the C1-Fc antibody can stably bind to the antigen.

[0064] Example 5

[0065] PEDV virus neutralization assay

[0066] Vero cell line was selected for PEDV virus neutralization test. Vero cells grown in monolayer were stably passaged and diluted to 1.0×10 5 A Vero cell suspension of 100 cells / mL was plated into a 12-well cell culture plate, and 0.1MOI CV777 virus suspension was inoculated 24 hours later. 200μL 4% paraformaldehyde was added to each well 24 hours after inoculation, and the plates were fixed in a 37°C incubator for 15 minutes. After discarding the fixative, 200μL 0.25% TritonX-100 was added to each well and removed after 15 minutes. Wash three times with 1×PBS (K+), add 1% BSA to block for 30 minutes; add C1 antibody diluted with cell maintenance medium (starting from 200μg / mL and diluted to 50μg / mL, 25μg / mL, 12.5μg / mL, 6.25μg / mL, 3.125μg / mL and 1.56μg / mL, 6 dilutions), 2 wells for each dilution, 50μL / well, and set up a positive control of virus inoculation without antibody and a negative control of cells, and incubate at 37°C for 48h; after washing, add fluorescent anti-PEDV S protein antibody and incubate for 1h; take pictures with a fluorescence microscope, observe the number of CPE holes, and record the results.

[0067] like Figure 7 As shown, the results showed that the C1 antibody had a good neutralizing effect on PEDV virus at a concentration of 6.25 μg / ml and above.

[0068] 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 neutralizing nanobody targeting porcine epidemic diarrhea virus RBD, characterized in that: The nanobody is named C1, and the three antigen complementary determining regions CDR1, CDR2 and CDR3 of C1 are amino acid sequences with a homology 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 neutralizing nanobody targeting porcine epidemic diarrhea virus RBD according to claim 1, characterized in that: The amino acid sequences of the three antigen complementary determining regions CDR1, CDR2 and CDR3 of the C1 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

3. A neutralizing nanobody targeting porcine epidemic diarrhea virus RBD according to claim 1, characterized in that: The amino acid sequence of C1 is shown in SEQ ID NO:

4.

4. An antibody targeting porcine epidemic diarrhea virus RBD, comprising the nanobody C1 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, and the amino acid sequence of the human IgG Fc domain is shown in SEQ ID NO:

5.

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 of any one of claims 1 to 3 or the antibody of any one of claims 4 to 5 in the preparation of a vaccine for preventing porcine epidemic diarrhea or in the preparation of a medicament for treating porcine epidemic diarrhea.

10. 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 kit for diagnosing porcine epidemic diarrhea virus infection.

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