CN115850463A - Polypeptide NbM9 capable of recognizing and neutralizing MERS-CoV and application thereof - Google Patents

Abstract

The invention relates to a polypeptide NbM9 capable of identifying and neutralizing MERS-CoV, which comprises 3 complementarity determining regions CDR1-3, and the sequence is shown as SEQ ID NO. 1-3. The invention carries out nano antibody drug development aiming at MERS-CoV, and through preparing MERS-CoV S protein, immunizing a bimodal camel, utilizing a platform technology of phage library display nano monoclonal antibody and the like, nano antibody VHH specifically combined with MERS-CoV is screened, CDR sequence thereof is identified, and humanized VHH-huFc1 is constructed; meanwhile, the curative effect of NbM9 in treating MERS-CoV infection is evaluated by using a pseudovirus neutralization experiment, and a potential detection agent and a treatment drug are provided for preventing and treating MERS-CoV infection.

Description

Polypeptide NbM9 capable of recognizing and neutralizing MERS-CoV and application thereof

Technical Field

The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding MERS-CoV, and also relates to the application of said polypeptide in the preparation of therapeutic medicine and diagnostic agent for MERS-CoV infection.

Background

Middle East Respiratory Syndrome (MERS) was first found in 60 year old saudi arabian male patient who died 6 months old 2012. The latent period of the disease is 2 to 14 days, and the disease is typically manifested by acute respiratory tract infection, acute onset of disease, high fever (39 to 40 ℃), and accompanied symptoms of chills, cough, chest pain, headache, general muscle and joint pain, hypodynamia, anorexia and the like. No vaccine and specific treatment is currently available. Although the disease initially occurs in the middle east region, it gradually spreads to 27 countries of europe, africa, asia, and north america with the development of activities of trade, tourism, religion, and the like. Therefore, the development of safe and effective neutralizing antibodies against MERS pathogens is of great public health importance in the control of such epidemics.

The MERS-causing pathogen is MERS-CoV, belonging to the genus β -coronavirus (β -CoV), which is an enveloped single-stranded positive-strand RNA virus. The single-stranded RNA genome of MERS-CoV is approximately 30kb in size, has 10 Open Reading Frames (ORFs) in total, and encodes 16 nonstructural proteins (nsp 1-16) and four structural proteins: spike protein (S), envelope protein (E), matrix protein (M), and nucleocapsid protein (N). These four structural proteins constitute spherical, crown-like virus particles, of which S and N are strongly immunogenic. The S protein is I type transmembrane glycoprotein in a trimer state, is positioned on the surface of a virus membrane, mediates the fusion of virus attachment host cells and virus-cell membranes, and is a main determinant of cell targeting and pathogenesis. The S protein can induce the body to produce neutralizing antibodies that play a key role in preventing MERS-CoV infection. Therefore, immunization of animals with S protein and screening thereof for antibodies with neutralizing activity would be one of the effective strategies for treating MERS-CoV infection.

In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH 2 and CH 3), one hinge region and one heavy chain Variable region (VHH, i.e. antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of that of conventional antibodies, and with a molecular height and diameter at the nanometer level, is the smallest functional antibody fragment currently available, and is therefore also called Nanobody (Nb). Because of the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80% with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is recently widely used for the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.

Disclosure of Invention

The camel source nanometer monoclonal antibody is obtained by immunizing camel with antigen and is used for detecting and treating MERS-CoV infection. Based on these studies, the present invention provides a polypeptide capable of binding MERS-CoV, comprising 3 complementarity determining regions CDR1-3, the sequences are shown in SEQ ID NO: 1-3.

In a specific embodiment, the polypeptide is a nanobody.

In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, wherein said FR1-4 is sequentially staggered from said CDR 1-3. For example, the FR1-4 sequence may be designed as shown in SEQ ID NOS: 4-7 (sources of alpaca), although the scope of the present invention is not limited thereto. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences have little influence and can be designed according to species, which are well known in the art. FR region sequences of human, murine or camel origin can be designed to link the CDRs, thereby obtaining a nanobody that can bind MERS-CoV.

In a particular embodiment, the polypeptide is a VHH of camelid origin or a humanized VHH.

The invention also provides application of the polypeptide in preparation of a MERS-CoV detection agent or a MERS-CoV S protein detection agent.

The invention also provides application of the polypeptide in preparation of MERS-CoV therapeutic drugs.

The invention also provides nucleic acids encoding the above polypeptides.

The invention also provides application of the nucleic acid in preparation of MERS-CoV therapeutic drugs.

The invention carries out nano antibody drug development aiming at MERS-CoV, and through preparing MERS-CoV S protein, immunizing a bimodal camel, utilizing a platform technology of phage library display nano monoclonal antibody and the like, nano antibody VHH specifically combined with MERS-CoV is screened, CDR sequence thereof is identified, and humanized VHH-huFc1 is constructed; meanwhile, the curative effect of NbM9 in treating MERS-CoV infection is evaluated by using a pseudovirus neutralization experiment, and a potential detection agent and a treatment drug are provided for preventing and treating MERS-CoV infection.

Drawings

FIG. 1 is a curve showing the measurement of antiserum titer of MERS-CoV-S protein after one week of 4 th immunization of alpaca.

FIG. 2 is a graph showing the inhibition of MERS-CoV pseudovirus by antiserum after one week of the 4 th immunization at different dilutions of a gshost cell in vitro, compared with preimmune serum.

FIG. 3 is the panning identification of MERS-VHH phage antibody library, wherein A is the ELISA detection statistical map after phage library panning against MERS-CoV-S protein; b is from the first wheel (1) ^st ) Second wheel (2) ^nd ) And a third wheel (3) ^rd ) Phage antibody library after panning each selected 24,24 and 40 clones for phage ELISA detection statistical map.

FIG. 4 is a statistical chart of ELISA detection of prokaryotically expressed VHH antibodies, each dot representing one clone, with OD450 for S protein/OD 450 of blank on the ordinate, and a positive ratio greater than 5.0 being defined.

Fig. 5 is a statistical chart of SPR detection of NbM9 antibody.

FIG. 6 is a statistical plot of OD450 of different purified concentrations of NbM9 antibody bound to MERS-S protein detected by ELISA.

FIG. 7 is a graph of experiment for NbM9 antibody neutralization of MERS-CoV pseudovirus infection.

Detailed Description

1. Preparation of alpaca immune and antiserum

Priming alpaca with an emulsified mixture of 250 μ g MERS-S protein and 250 μ l Freund 'S complete adjuvant, boosting 3 times with MERS-S protein and 250 μ l Freund' S incomplete adjuvant on days 14, 28 and 42, collecting blood and detecting antiserum titer 1 week after 2 and 3 weeks of immunization; after 1 week of the 4 th immunization, 200ml of blood was collected for the construction of phage antibody library.

Antiserum titers were determined by ELISA using plates coated with MERS-S-his protein at a concentration of 0.5. Mu.g/ml, and 100. Mu.l of either the antiserum or purified antibody was added to each well in a gradient (control for immunization)Pre camel serum), incubated at 37 ℃ for 1.5h, washed 2 times, 1:10000 diluted secondary antibody of horseradish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 μ L TMB substrate is added, incubation is carried out for 10min at 37 ℃,50 μ L of 0.2M H ₂ SO ₄ The reaction was stopped and the OD450nm was measured. ELISA assay serum titers were specified as the highest dilution at OD450 that was more than 2-fold of the blank and greater than 0.2.

As shown in FIG. 1, the antiserum titers of the 4-immunization were 3.28X 10 ⁶ . Therefore, the antigen can induce camel to generate high-titer antiserum specific to MERS-S protein.

To further verify whether this high titer camel antiserum was effective in preventing MERS-CoV virus infection, neutralization experiments for virus infection were performed. Antiserum and preimmune serum with different dilution concentrations are respectively incubated with MERS-CoV pseudoviruses for 60min, then transferred to vero cells, and after 48h, the virus load is detected by a Glo Max chemiluminescence enzyme-linked immunosorbent assay (Promega) instrument and the neutralization effect is calculated. Results of neutralization experiments showed that MERS-S protein-induced antiserum inhibition 90% by volume MERS-CoV infection with ID90 at 1000-fold dilution or more (FIG. 2). In summary, MERS-S protein induces high titer antiserum, and the antiserum has the ability to efficiently inhibit MERS-CoV pseudovirus infection.

Construction and panning of VHH phage library

Collecting 200ml of camel peripheral blood after immunization, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain camel PBMC, extracting RNA according to a TRIzol operation manual, inverting oligo (dT) into cDNA, cloning VHH genes of camels to phagemid plasmids by using techniques such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain a VHH phage library.

In order to further identify whether the MERS-CoV-VHH phage library is successfully constructed, VHH target genes of immune MERS-S protein camels are amplified through PCR, and the target band is 450bp and the size is in accordance with expectation, which indicates that the MERS-CoV-VHH phage antibody library contains VHH genes. 25 clones were selected for sequencing, and 21 clones had the desired fragment inserted, with an insertion rate of about 84%. SequencingThe results showed that these 21 clones had no perfectly identical repeats and the library diversity was 100%. The alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the library capacity of the constructed CD4-VHH phage antibody library is 1.37 multiplied by 10 ⁹ 。

The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The phage antibody library was enriched three times with 50. Mu.g/ml MERS-S-His protein coating. And (3) eluting the enriched phage, converting, coating a plate, selecting a monoclonal antibody to perform binding identification of the phage and CD4 protein ELISA, sequencing the clone with the binding reading value of more than 1.0, cloning to an expression vector pcDNA3.1, and transfecting 293tt cells to express to produce the nano monoclonal antibody.

The panned library was tested for binding to MERS-S protein. The phage ELISA results showed that the read values of the binding between the CD4-VHH phage library and the CD4 protein before enrichment were 0.78, and the read values of the phage library after one, two and three rounds of enrichment were 0.97, 2.59 and 3.34 respectively (FIG. 3A). To further verify the positive phage rate of binding of MERS-CoV-VHH proteins in the enriched library, 24 and 40 clones were selected from each of the 1 st, 2nd and 3rd round enriched libraries for single phage ELISA detection. The results showed that 50% of individual phage clones were positive in the library round 2, 75% of phage clones were positive in the library round 3, and the Target/Blank ratios were all > 5 (FIG. 3B), and the MERS-CoV-VHH phage library with high binding capacity was successfully enriched by MERS-S protein panning.

Construction of VHH prokaryotic expression library and VHH expression

PCR amplification of the enriched 2nd-MERS-CoV-VHH and 3rd-MERS-CoV-VHH phage antibody libraries after the two and three rounds of panning; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; coating a plate with a prokaryotic expression antibody library, culturing overnight, randomly selecting 182 monoclonal colonies the next day, inducing expression of an antibody supernatant by using IPTG, and carrying out ELISA binding detection on the antibody supernatant and S protein.

The results showed that 49 bacterial supernatants bound to S protein while not bound to the blank, with S protein binding/blank reading greater than 5.0 (figure 4). Wherein, the sequence of the antibody NbM9, CDR1-3 is shown as SEQ ID NO. 1-3, and the sequence of FR1-4 is shown as SEQ ID NO. 4-7.

Eukaryotic expression of VHH-huFc

By molecular cloning technology, nbM9 gene is fused with human Fc gene and inserted into pCDNA3.4 eukaryotic expression vector to construct NbM9-huFc-pCDNA3.4 expression plasmid. The constructed NbM9-huFc-pCDNA3.4 was transfected into 293tt cells and expressed to produce NbM9-huFc (4 NB). Cell supernatants were collected for ELISA assay.

Affinity assay for antibody NbM9 and MERS-S protein. Affinity was detected using the Fortebio biomolecular interaction platform. The antibody is solidified on an Anti-human IgG Fc Capture Biosensors (AHC) probe, the solidification time is 400s, the antigen CD4-his protein is combined, the combination time is 180s, the dissociation time is 180s, the combination dissociation condition of the antibody and the antigen is observed, and the instrument is used for fitting a curve to derive data. The results of the affinity assay are shown in Table 1, and the affinity of most antibodies can reach 10 ^-12 (picomole scale), the binding dissociation curve is shown in FIG. 5. From this, we obtained antibodies with high affinity.

TABLE 1NbM9 affinity data

Clone ID	Ka(1/MS)	Kd(1/S)	KD(M)	Response(nm)
					NbM9	3.88E+05	＜1E-07	＜1E-12	0.477

5. Antibody gradient dilution ELISA

Coating the detection plate with 0.5. Mu.g/ml MERS-S protein, 100. Mu.l per well, incubating at 37 ℃ for 2h, washing for 2-4 times, blocking with 4% bovine serum, 250. Mu.l per well, incubating at 37 ℃ for 1h, washing for 2-4 times, adding 100. Mu.l of purified antibody diluted in gradient to each well, incubating at 37 ℃ for 1.5h, washing for 2 times, adding 1:10000 diluted 100 μ l of horseradish peroxidase-labeled anti-human antibody, incubating at 37 deg.C for 1H, washing for 4-6 times, adding 100 μ l of TMB substrate, incubating at 37 deg.C for 10min,50 μ l of 0.2M H ₂ SO ₄ The reaction was stopped and the OD450nm was measured. The results are shown in FIG. 6, when the concentration of antibody NbM9 is as low as 0.00188. Mu.g/ml, the ratio of OD450 bound to MERS-S protein/OD 450 of blank is still greater than 2.

NbM9 neutralizing MERS-CoV pseudovirus

MERS-CoV pseudoviruses were generated by co-transfection of expression vectors expressing firefly luciferase (pNL 43R-E-luciferase) and pcDNA3.1 (Invitrogen) into 293T cells (ATCC). Viral supernatants were collected after 48 h. Viral titers were determined by luciferase activity in relative light units (Bright-Glo luciferase assay vector system, promega Biosciences). The control monoclonal antibody was anti-SFTSV antibody SNB02 (1 mg/ml), and NbM9 was subjected to in vitro neutralization experiments. Antibody was diluted in gradient to different concentrations, along with MERS-CoV pseudovirus, in 5% CO ₂ Incubating at 37 ℃ for 1 hour, adding 1X 10 ⁴ Vero cells, 5% CO ₂ Half maximal inhibitory concentration (IC 50) of monoclonal antibody was evaluated by assay for luciferase activity after incubation in an incubator at 37 ℃ for 48 hours

As a result, as shown in FIG. 7, nbM9 had excellent neutralizing activity, and the inhibition rate reached 90% at an antibody concentration of 0.1376. Mu.g/ml.

From the above experimental results, it can be seen that the antibody NbM9 and its humanized form of the present invention can specifically recognize and bind MERS-CoV and its S protein, and can neutralize MERS-CoV, block its infection, and thus be used to treat MERS.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (8)

1. A polypeptide capable of binding MERS-CoV, which comprises 3 complementarity determining regions CDR1-3, and the sequence is shown in SEQ ID NO 1-3.

2. The polypeptide of claim 1, wherein the polypeptide is a nanobody.

3. The polypeptide of claim 3, further comprising 4 framework regions FR1-4, wherein said FR1-4 is sequentially staggered from said CDR 1-3.

4. The polypeptide of claim 3, wherein the polypeptide is a VHH of camelid or humanized VHH.

5. Use of the polypeptide of any one of claims 1-4 in the preparation of a detector of MERS-CoV or a detector of the S protein of MERS-CoV.

6. Use of a polypeptide according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of MERS-CoV infection.

7. A nucleic acid encoding the polypeptide of any one of claims 1 to 4.

8. Use of the nucleic acid of claim 6 in the preparation of a medicament for the treatment of MERS-CoV infection.

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