CN118359711B

CN118359711B - Anti-poliovirus type 1 antibody, and preparation method and application thereof

Info

Publication number: CN118359711B
Application number: CN202410797193.1A
Authority: CN
Inventors: 刘庆伟; 王晓黎; 阮丽珠; 陈天; 韩文玉; 杨义; 刘艳; 石娜; 李晓霞; 赵胜涛
Original assignee: Chongqing Huazhi Biopharmaceutical Co ltd; Huasong Shanghai Biomedical Technology Co ltd
Current assignee: Chongqing Huazhi Biopharmaceutical Co ltd; Huasong Shanghai Biomedical Technology Co ltd
Priority date: 2024-06-20
Filing date: 2024-06-20
Publication date: 2024-10-15
Anticipated expiration: 2044-06-20
Also published as: CN118359711A

Abstract

The invention relates to the field of antibodies, in particular to an anti-poliovirus type1 antibody, a preparation method and application thereof, wherein the anti-PV 1 antibody has the following technical characteristics: the heavy chain variable region comprises CDR-H1 with an amino acid sequence shown as SEQ ID No.5, CDR-H2 with an amino acid sequence shown as SEQ ID No.6 and CDR-H3 with an amino acid sequence shown as SEQ ID No. 7; the light chain variable region comprises CDR-L1 with an amino acid sequence shown as SEQ ID No.8, CDR-L2 with an amino acid sequence shown as SEQ ID No.9 and CDR-L3 with an amino acid sequence shown as SEQ ID No. 10. The anti-PV 1 antibody can recognize two types of PV1 antigens, and the recognized epitope is a conformational epitope, so that the anti-PV 1 antibody can be used for developing PV1 antiviral drugs, and can also be used as a high-efficiency and sensitive detection reagent in clinical diagnosis of infection of PV1 and vaccine production.

Description

Anti-poliovirus type 1 antibody, and preparation method and application thereof

Technical Field

The invention relates to the field of antibodies, in particular to an anti-poliovirus type 1 antibody, a preparation method and application thereof.

Background

Poliovirus (Poliovirus, PV) is a small RNA virus without a envelope, belonging to the enterovirus genus, is an icosahedral structure without a envelope, whose genome is a single-stranded positive strand RNA, encodes an open reading frame, and contains structural proteins P1 and non-structural proteins P2 and P3. The structural protein P1 can be cleaved by protease 3CD into capsid proteins VP0 (VP 2+VP 4), VP3 and VP1, which can be assembled together with viral RNA into mature viral particles and with VP0 cleavage into VP4 and VP2. Two types of particles exist in PV's natural culture, with infectious mature viral particles, which are well immunogenic (termed D antigen) and empty particles that are not infectious, which are poorly immunogenic (termed C antigen). PV mainly infects children under 5 years old through the faecal route, and can cause symptoms such as fever, fatigue, headache, vomiting, neck stiffness, limb pain and the like, and severe paralysis and even paralysis and death of breathing can be caused. There is no effective treatment for poliomyelitis, and prevention can only be achieved by vaccine. The oral vaccine of attenuated ridge ash (Live-attenuated oral PV VACCINE, OPV) and inactivated vaccine of ridge ash (INACTIVATED-PV VACCINE, IPV) have been used in global immunization programs on a large scale and eventually form the ultimate program for eradicating poliomyelitis. It has been reported that OPV can regain virulence by back-mutations or recombination, causing vaccine-related paralytic poliomyelitis (VAPP) or vaccine-derivative-related poliomyelitis to be prevalent (cVDPV). cVDPV can also be used as a source of infection to cause further transmission. PV can be divided into 3 serotypes, PV1, PV2 and PV3, with wild-type PV2 and PV3 announced to be destroyed in 1999 and 2020, respectively. In recent years, infection with wild-type PV1 has been reported and the popularity of vaccine derivatives of PV1 and PV2 has also increased. Although IPV can avoid this problem, IPV involves the cultivation of large amounts of viruses, with the risk of accidental release. Thus, there is a need to find new alternative vaccines to achieve a global program for eradicating poliomyelitis. Therefore, there is a need to develop new vaccines that are not infectious in order to completely eradicate the infection of PV.

The PV has poor thermal stability, the heat treatment can expand and convert the compact D antigen into the C antigen, and the D antigen content is a key quality control standard of the PV vaccine, so that how to identify two types of antigens is very important in the research and development and production processes of the PV vaccine.

In recent years, the preparation of a passive immune preparation with high efficiency, low cost and small side effect becomes a research hot spot for preventing and treating the viruses. The in vitro antiviral neutralization activity and the in vivo capability of protecting the organism against virus attack of the monoclonal antibody prove that the monoclonal antibody can prevent and treat virus infection, has important significance for promoting clinical research of poliomyelitis, and has extremely important value for further epidemiological investigation and passive treatment.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an anti-poliovirus type 1 antibody, a method for preparing the same and use thereof, for solving the problems of the prior art.

To achieve the above and other related objects, the present invention provides an anti-poliovirus type 1 (PV 1) antibody comprising a heavy chain variable region and a light chain variable region, the anti-PV 1 antibody having the following technical characteristics:

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence as shown in SEQ ID No. 5;

<2> the heavy chain variable region comprises CDR-H2 having the amino acid sequence shown in SEQ ID No. 6;

<3> the heavy chain variable region comprises CDR-H3 having the amino acid sequence shown in SEQ ID No. 7;

<4> the light chain variable region comprises CDR-L1 having the amino acid sequence shown in SEQ ID No. 8;

<5> the light chain variable region comprises CDR-L2 having the amino acid sequence shown in SEQ ID No. 9;

<6> the light chain variable region comprises CDR-L3 having the amino acid sequence shown in SEQ ID No. 10.

In another aspect, the invention provides an isolated polynucleotide encoding a heavy chain variable region or a light chain variable region of the anti-PV 1 antibody.

The invention also provides a nucleic acid construct comprising said isolated polynucleotide.

The invention also provides an engineered cell containing the nucleic acid construct or the polynucleotide integrated with an exogenous source in the genome.

The invention also provides a preparation method of the anti-PV 1 antibody, which comprises the following steps: culturing the engineered cells to express the anti-PV 1 antibody, and purifying and separating the anti-PV 1 antibody.

The invention also provides a kit for detecting PV1 antigen, comprising the anti-PV 1 antibody or immunoconjugate thereof.

The invention also provides the use of said anti-PV 1 antibody, said isolated polynucleotide, said nucleic acid construct, said engineered cell or said kit in drug screening, drug quality control or in the preparation of a diagnostic or therapeutic drug.

The invention also provides the use of said anti-PV 1 antibody, said isolated polynucleotide, said nucleic acid construct, said engineered cell or said kit for non-disease diagnosis in detecting the C-antigen or D-antigen content of PV 1.

As described above, the anti-poliovirus type 1 antibody of the invention, and the preparation method and the use thereof, have the following beneficial effects: the monoclonal antibody 5C6 can recognize two types of PV1 antigens, and the 5C6 monoclonal antibody cannot recognize denatured PV1 sVLP, namely the recognized epitope is a conformational epitope. The pseudovirus neutralization test shows that the pseudovirus has neutralization activity, can be used for developing PV1 antiviral drugs, and can also be used as a high-efficiency and sensitive detection reagent in clinical diagnosis of infection of PV1 and vaccine production.

Drawings

FIG. 1 shows a diagram of the result of SDS-PAGE analysis of monoclonal antibody 5C 6.

FIG. 2 shows the results of a monoclonal antibody 5C6 specific assay.

FIG. 3 shows a graph of the results of Western blot analysis of monoclonal antibody 5C 6.

FIG. 4 shows the detection of binding activity of the PV1 mab miniexpression transfection supernatant.

Detailed Description

The present invention provides an anti-poliovirus type 1 (PV 1) antibody comprising a heavy chain variable region and a light chain variable region, said anti-PV 1 antibody having one or more of the following technical features;

The anti-PV 1 antibodies are capable of neutralizing both D and C antigens of poliovirus type 1.

CDRs (complementarity determining regions, complementarity determining region) generally refer to regions of an antibody that can be spatially complementary to an epitope. Variability in antibodies is typically not evenly distributed throughout the variable regions of the antibody, and the heavy and light chain variable regions of a monoclonal antibody typically each have 3 hypervariable regions (hypervariable region, HVR) which are typically complementary in spatial structure to an epitope, so that the hypervariable regions are also known as complementarity determining regions (complementarity determining region, CDRs), i.e., the heavy chain variable regions typically comprise three complementarity determining regions, i.e., CDR-H1, CDR-H2, and CDR-H3, and the light chain variable regions typically comprise three complementarity determining regions, i.e., CDR-L1, CDR-L2, and CDR-L3.

In certain embodiments of the invention, the complementarity determining regions of the heavy chain variable region of the anti-PV 1 antibody comprise CDR-H1 of the amino acid sequence shown in SEQ ID No.5, CDR-H2 of the amino acid sequence shown in SEQ ID No.6 and CDR-H3 of the amino acid sequence shown in SEQ ID No. 7.

In certain embodiments of the invention, the complementarity determining regions of the light chain variable region of the anti-PV 1 antibody comprise CDR-L1 of the amino acid sequence shown in SEQ ID No.8, CDR-L2 of the amino acid sequence shown in SEQ ID No.9 and CDR-L3 of the amino acid sequence shown in SEQ ID No. 10.

In certain embodiments of the invention, the complementarity determining region of the heavy chain variable region comprises CDR-H1 having the amino acid sequence shown in SEQ ID No.5, CDR-H2 having the amino acid sequence shown in SEQ ID No.6 and CDR-H3 having the amino acid sequence shown in SEQ ID No.7, and the complementarity determining region of the light chain variable region comprises CDR-L1 having the amino acid sequence shown in SEQ ID No.8, CDR-L2 having the amino acid sequence shown in SEQ ID No.9 and CDR-L3 having the amino acid sequence shown in SEQ ID No. 10.

The anti-PV 1 antibody is an antibody fragment, and/or a monoclonal antibody. Further, the monoclonal antibody is an IgG2b antibody.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising an antigen binding or variable region thereof. For example, antibody fragments include single chain antibodies (scFv), fab ', F (ab ') or F (ab ') 2.

The single chain antibody may generally be a polypeptide chain comprising V _H (heavy chain variable region) and V _L (light chain variable region) of the antibody. Typically, the single chain antibody may also include a linker peptide (linker) typically located between V _H and V _L to allow the scFv to form the desired structure for binding to the antigen. For example, the anti-PV 1 antibody may include V _H and V _L,V_H and V _L with a connecting peptide therebetween, the single-chain anti-PV 1 antibody may include V _L, a connecting peptide and V _H in order from the N-terminus to the C-terminus, and the anti-PV 1 single-chain antibody may also include V _H, a connecting peptide and V _L in order from the N-terminus to the C-terminus. The linker peptide may be any of a variety of linker peptides suitable in the art for forming scFv, for example, the linker peptide may be a G4S3 linker, and the selection or design of the G4S3 linker may be referred to in the literature Michel Sadelain etc,Science Translational Medicine,2013;Carl H .June etc, Science Translational Medicine ,2015.

The "Fab" fragment includes the variable and constant regions of the light chain, the variable region of the heavy chain and the first constant region (CH 1). A F (ab') antibody fragment comprises a pair of Fab fragments which are typically covalently linked near the carboxy terminus by a hinge cysteine between them.

In certain embodiments of the invention, the anti-PV 1 antibody is a monoclonal antibody that includes a constant region, which may be a natural sequence constant region (e.g., a human natural sequence constant region) or an amino acid sequence variant thereof.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the various antibodies comprising the population are identical except for possible naturally occurring mutants that are typically present in minute amounts. Monoclonal antibodies are highly specific, i.e., directed against a single epitope on an antigen. Furthermore, unlike polyclonal antibody preparations that include different antibodies directed against different determining regions (epitopes), each monoclonal antibody is directed against a single determining region on the antigen. In addition to their specificity, one advantage of monoclonal antibodies is that they can now be synthesized without contamination by other antibodies. The modifier "monoclonal" refers to the property of the antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring any particular method for producing the antibody.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences derived from a particular species or belonging to a particular antibody type or subtype, and the remainder is identical or homologous to corresponding sequences derived from another particular species or belonging to another particular antibody type or subtype, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

In some embodiments of the invention, the anti-PV 1 antibody is selected from hybridoma cell lines, the heavy chain variable region and the light chain variable region of which have the nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.3, respectively, and the heavy chain variable region and the light chain variable region have the amino acid sequences shown in SEQ ID No.2 and SEQ ID No. 4.

In certain embodiments of the invention, the heavy and light chain variable regions may further comprise framework regions FR, which may be located between or at either end of the complementarity determining regions. In some embodiments of the present invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence of a murine monoclonal antibody variable region is a framework region sequence obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3) amino acids, and the framework region sequence may have homology of 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more with the framework region sequence of the human monoclonal antibody variable region sequence.

In certain embodiments of the invention, the isolated polynucleotide comprises nucleotides as set forth in nucleotide sequences SEQ ID NO. 11-16.

In certain embodiments of the invention, the nucleotide sequence encoding the heavy chain variable region of the anti-PV 1 antibody is set forth in SEQ ID No. 1.

In certain embodiments of the invention, the nucleotide sequence encoding the light chain variable region of the anti-PV 1 antibody is set forth in SEQ ID No. 3.

The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue, and can be a variety of expression vectors, including vector backbones, i.e., empty vectors, and expression frameworks. The term "expression cassette" refers to a sequence having the potential to encode a protein.

The kind of the expression vector is not particularly limited. Expression vector refers to a nucleic acid molecule that allows insertion of exogenous nucleotides without disrupting the ability of the vector to replicate and/or integrate in a host cell. Expression vectors may include nucleic acid sequences that allow for replication in a host cell, such as an origin of replication. Expression vectors may also include one or more selectable marker genes and other genetic factors. An expression vector is a vector that contains the necessary regulatory sequences to transcribe and translate an inserted gene or genes. The expression vector is selected from eukaryotic expression vector or prokaryotic expression vector.

The prokaryotic expression vector is selected from an escherichia coli expression vector, a bacillus subtilis expression vector or a streptomyces expression vector. In a preferred embodiment, the prokaryotic expression vector is selected from the group consisting of E.coli expression vectors.

The eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector or a mammalian expression vector. The mammalian expression vector is a non-viral expression vector or a viral expression vector. The viral expression vector is selected from the group consisting of a retrovirus expression vector, a lentivirus expression vector, an adenovirus expression vector, and an adeno-associated virus expression vector.

The host cell is selected from eukaryotic host cells or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeasts, insects, birds, plants, caenorhabditis elegans (c.elegans) or nematodes (nematode) or mammalian host cells. A non-limiting example of an insect cell is a spodoptera frugiperda (Sf) cell. Examples of yeast host cells are Saccharomyces cerevisiae (S.cerevisiae), kluyveromyces lactis (Kluyveromyceslactis, K.lactis) or yarrowia lipolytica (yarrowia lipolytica). Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, chinese Hamster Ovary (CHO) cells, human Embryonic Kidney (HEK) cells, african green monkey cells, CV1 cells, vero or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as E.coli, streptomyces, bacillus subtilis, salmonella typhi (Salmonellatyphi) or Mycobacterium (mycobacteria).

The person skilled in the art can transfect the expression vector into a host cell according to methods well known in the art to obtain a cell comprising a gene encoding an antibody of the invention. For example, the introduction of the expression vector into eukaryotic cells can be performed by lipofection.

The kit typically detects the PV1 antigen as a target. The kit may also include a label for the anti-PV 1 antibody, which may generally be used to label the anti-PV 1 antibody, optionally in a variety of labels including, but not limited to, fluorescent labels, radioactive labels, enzyme-labeled labels, chemiluminescent labels, and the like. The kit may also typically comprise one or more reagents required for detection, depending on the detection principle of the kit. In addition, the kit may further include, as required: containers, controls (negative or positive controls), buffers, adjuvants, etc., which can be selected by the skilled artisan according to the particular circumstances.

In one embodiment, the kit is an enzyme-linked immunosorbent assay kit.

In one embodiment, the kit is a double antibody sandwich enzyme-linked immunosorbent assay kit.

The medicament may be a medicament targeting the PV1 antigen, binding to or acting on the PV1 antigen, thereby treating and/or preventing an indication.

In certain embodiments of the invention, the drug in the drug screening, drug quality control may be a poliomyelitis treatment drug, a poliomyelitis diagnostic drug, or a poliomyelitis prevention drug. The diagnostic, therapeutic or prophylactic agent may be an agent that targets an antigen on the functional surface of the PV1 virus surface, binds to or acts on the antigen, thereby diagnosing, treating and/or preventing poliomyelitis.

In certain embodiments of the invention, the poliomyelitis preventing agent is a vaccine.

The drug screening is in vitro screening.

The drug quality control is, for example, in vitro potency assay of the drug.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

A method for preparing a PV1 neutralizing monoclonal antibody using recombinant PV1 virus-like particles (sVLP) as immunogens is described below. Through screening, purification and identification analysis, monoclonal antibody 5C6 can recognize two types of PV1 antigens. Western blots showed that the 5C6 mab was unable to recognize denatured PV1 sVLP, indicating that the epitope recognized by the mab was a conformational epitope. The pseudovirus neutralization assay showed neutralization activity. In conclusion, the 5C6 monoclonal antibody can be used for developing PV1 antiviral drugs, and can also be used as a high-efficiency and sensitive detection reagent in clinical diagnosis of infection of PV1 and vaccine production.

Examples

1. Materials and methods

1.1 Antigen preparation and mouse immunization

The structural proteins P1 (VP 0+ VP3+ VP 1) of the PV1 Mahoney strain, the PV2 MEF-1 strain and the PV3 Saukett strain are used for preparing and expressing wtVLP ⁰³¹ (hereinafter wtVLP, C antigen) by using a Pichia expression system, and a mutation site is introduced in a reference (PMID: 28103317) to obtain sVLP ⁰³¹ (hereinafter sVLP, D antigen), and a specific preparation method can be referred to in patent CN115707778B.

5 Μg of PV1 sVLP was mixed with 60 μg of aluminum adjuvant, and 100 μl of the mixed formulation was immunized on 6 week old female Balb/c mice intraperitoneally 3 times, with a 2 week interval between two immunizations. 2 weeks after the last immunization, 7 μg of PV1 sVLP was injected into the tail vein for booster immunization.

1.2 Preparation and selection of hybridoma cell lines

3 Days after mouse tail vein booster immunization, mouse spleen cells were fused with myeloma cells SP2/0 using PEG1450 to prepare hybridoma cells. After 9 days of hybridoma cell culture, antibodies specifically secreted against PV1 sVLP were screened using ELISA and pseudovirus neutralization experiments. 50 μl of the monoclonal hybridoma cell supernatant was taken and subjected to antibody subtype identification according to the subtype identification kit instructions.

1.3 Ascites production and antibody purification

Female Balb/c mice were intraperitoneally injected with 500. Mu.l liquid paraffin oil, and after two weeks each mice were intraperitoneally injected with 80 ten thousand hybridoma cells. After 1-2 weeks, collecting ascites with needle, centrifuging at 4,000rpm for 10min, removing upper layer oil and lower layer precipitate, and purifying clarified ascites with antibody. According to the instructions, the ascites was purified using iProtein G Purose 4 Fast Flow affinity column (thousand pure organisms) to obtain antibodies.

1.4 Indirect ELISA

Coating 96-well elisa plate (200 ng/well) with PV1, PV2 and PV3 sVLP or wtvlps, incubating overnight at 4 ℃; adding 5% of skimmed milk PBST, and sealing at 37 ℃ for 1-2 hours; adding an antibody sample to be detected, and incubating for 2 hours at 37 ℃; HRP-labeled secondary antibody was then added and incubated for 1 hour, and finally absorbance OD450nm was read.

1.5 Polyacrylamide gel electrophoresis and western blot analysis

The protein samples were mixed with a polypropylene gel electrophoresis (SDS-PAGE) loading buffer, boiled for 5min, and the protein samples were separated by a 12% polyacrylamide gel. Protein bands were visualized by coomassie blue staining or proteins were transferred to PVDF membranes for western blot analysis. Monoclonal antibody concentration 5. Mu.g/mL, rabbit anti-VP 1 and VP3 polyclonal antibodies 1:5000 were diluted for use, followed by incubation with HPR-labeled secondary antibodies, and finally recorded with a luminescent image analyzer.

1.6 Neutralization test

Packaging the pseudoviruses: the P1 sequence in the virus PV1 Maloney strain (Gene Bank ID: V01149.1) is replaced by GFP and a T7 promoter sequence is added in front of the 5' UTR, and the mixture is synthesized and then connected to pUC57simple to obtain replicon plasmid pUC57-PV1M-eGFP (gold Style); the Sabin 1 virus strain P1 sequence (Gene Bank ID: AY 184219) and dsRed Gene sequence are synthesized in Kirsrui, the Sabin 1 P1 Gene is connected into pcDNA3.3 (thermo), and then dsRed is connected to the P1N end to obtain pcDNA3.3-dsRed-Sabin 1 P1; the T7 polymerase (Gene Bank ID: M38308) was synthesized and then ligated into pcDNA3.3 to obtain pcDNA3.3-T7. 3 μg pcDNA3.3-dsRed-Sabin 1 P1 and lipo 3000 (Thermo) were mixed and added to a 6-well plate containing 50-60% 293T cells; 1. Mu.g of pcDNA3.3-T7 and 2. Mu.g of pUC57-PV1M-eGFP in combination with lipo 3000 (Thermo) were added after 1 day; after 4 days, the transfectants were harvested, repeatedly frozen and thawed 3 times at-80℃and the supernatant was centrifuged. The packaged virus was used to infect 293T cells, and after 19 hours the GFP count was counted to calculate the pseudovirus titer.

Neutralization titer determination: diluting a sample to be tested by using DMEM (DMEM) containing 2% FBS (FBS) in a ratio of 2 times, mixing 50 μl of diluted serum with an equal volume of diluent containing 220 PV1 pseudoviruses, and arranging multiple holes; after incubation at 37℃for 2h, 100ul of 1.5X10 ⁵/mL 293T cells were added to each well and incubated at 37℃for 20h with 5% CO ₂, the GFP count was observed; the reciprocal of the highest dilution at which the sample could neutralize 50% of pseudovirus was defined as the neutralization titer.

1.7 Gene isolation and sequencing of the variable region of monoclonal antibody

After the monoclonal antibody hybridoma cells are centrifugally collected, RNA is extracted by a Trizol method, after reverse transcription, a mouse antibody gene modulation system is utilized to amplify variable region genes, and the correct amplified products are sent to a sequencing company for sequencing.

1.8 Recombinant expression identification of monoclonal antibody genes

The primers were designed and the chimeric antibody expression plasmids (containing the variable region and the hFc) were constructed by inserting the antibody heavy chain variable region and the light chain variable region into pcDNA3.4-mIgG2b-3C-hFc and pcDNA3.4-mIg kappa vectors, respectively, using the overlap method.

The expression of the antibodies in the culture supernatants was determined by indirect ELISA by cotransfecting heavy and light chain chimeric antibody expression plasmids (2. Mu.g each) into 293T cells using liposome methods, and harvesting the culture supernatants after 48h for analysis.

Plates (100 ng/well) were covered with PV1 sVLP overnight at 4 degrees. Cell transfection supernatant was diluted 2-fold from stock solution in a total of 6 gradients; transfection of empty supernatant as negative control; incubating the primary antibody for 2 hours at room temperature; secondary antibody G-alpha-H-HRP (1:8000) or G-alpha-M-HRP (1:10000), and incubating for 1H at room temperature; after 5min of development, the reaction was terminated.

2. Results

2.1 Screening of hybridoma cells secreting PV 1-specific antibodies

Hybridoma cells were prepared by fusing spleen cells from PV1 sVLP immunized mice with SP2/0 cells. Hybridoma cell supernatants were screened by ELISA to obtain hybridoma cell lines capable of secreting cells with PV1 sVLP binding capacity. Finally 5C6 was selected and the identification information is shown in Table 1.

TABLE 1 identification of monoclonal antibody secreting hybridoma cell lines

Hybridoma cell strain	Heavy chain	Light chain	PV1 sVLP binding Capacity	PV1 wtVLP binding capability	Pseudo virus IC50 (μg/mL)
						5C6	IgG2b	kappa	+++	+++	4.133

Note that: -: OD450<0.15; +: OD450 is more than 0.3 and is more than or equal to 0.15; ++: OD450 is more than 0.5 and is more than or equal to 0.3; +++: OD450 is more than or equal to 0.5.

2.2 Specificity analysis of anti-PV 1 monoclonal antibody

The purity and integrity of the purified PV1 mab from ascites was identified using a polypropylene gel electrophoresis (SDS-PAGE) method. As shown in fig. 1, mab 5C6 shows two bands, about 50kDa and 25kDa in size, corresponding to the heavy and light chains, respectively. The specificity of monoclonal antibodies to recognize PV1 sVLP (D antigen) and wtVLP (C antigen) was examined by ELISA, as shown in table 1, both 5C6 antigens were recognized. At the same time, the specificity of the reaction of the antibodies with PV1, PV2 and PV3 sVLP was examined, as shown in fig. 2, 5C6 could specifically recognize PV1 sVLP, but not PV2 and PV3 sVLP. Finally, the binding of the monoclonal antibodies to PV1, PV2 and PV3 sVLP was analyzed by Western blot, as shown in fig. 3, 5C6 monoclonal antibodies were unable to recognize denatured PV1, PV2 and PV3 sVLP, suggesting that the epitopes recognized by the antibodies are conformational epitopes.

2.3 Neutralizing Activity of monoclonal antibodies

The neutralization activity of 5C6 mab was examined by a pseudo-virus neutralization assay, and the result is shown in Table 1, wherein the neutralization concentration of 5C6 was 4.133. Mu.g/mL, and the neutralizing agent was found to be a detection reagent in the development of PV1 antiviral drugs and in the production of vaccines.

2.4 Gene sequence analysis of 5C6 monoclonal antibody

The sequences of the heavy chain variable region and the light chain variable region of the 5C6 mab obtained by the 5' RACE technique are as follows:

5C6 monoclonal antibody heavy chain variable region nucleotide sequence:

CAAGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTATTATAACCCATCCCTGAAGAGCCAACTCACAATCTCCAAGGATACATCCAGAAACCAGGTATTCCTCAAAATCACCAGTGTGGGCACTGCAGATACTGCCACTTACTACTGTGCTCGAAGTGAAGGTAACTCCCTCTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA（SEQ ID NO.1）

5C6 monoclonal antibody heavy chain variable region amino acid sequence:

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVGTADTATYYCARSEGNSLYYAMDYWGQGTSVTVSS（SEQ ID NO.2）

5C6 mab light chain variable region nucleotide sequence:

AGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTTGATTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATATTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGCTATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAGGACCTGGCAGTTTATTTCTGTCACCACCATTATAGCTCTCCGTTCACGTTCGGTGGGGGGACCAAGCTGGAAATG（SEQ ID NO.3）

5C6 mab light chain variable region amino acid sequence:

SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVDWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCHHHYSSPFTFGGGTKLEM（SEQ ID NO.4）

The heavy chain variable region belongs to the IGHV8 family and the light chain variable region belongs to the IGKV6 family.

The amino acid sequence and nucleotide sequence of each CDR region are summarized as follows:

Heavy chain CDR1:

GFSLSTSGMG（SEQ ID NO.5）

GGGTTTTCACTGAGCACTTCTGGTATGGGT（SEQ ID NO.11）

heavy chain CDR2:

IWWDDDK（SEQ ID NO.6）

ATTTGGTGGGATGATGATAAG（SEQ ID NO.12）

Heavy chain CDR3:

ARSEGNSLYYAMDY（SEQ ID NO.7）GCTCGAAGTGAAGGTAACTCCCTCTACTATGCTATGGACTAC（SEQ ID NO.13）

light chain CDR1:

QSVSND（SEQ ID NO.8）

CAGAGTGTGAGTAATGAT（SEQ ID NO.14）

Light chain CDR2:

YAS（SEQ ID NO.9）

TATGCATCC（SEQ ID NO.15）

light chain CDR3:

HHHYSSPFT（SEQ ID NO.10）

CACCACCATTATAGCTCTCCGTTCACG（SEQ ID NO.16）

2.5 recombinant expression verification of mab 5C6

To verify that the antibody variable region genes were correct, the 5C6 heavy and light chain plasmids were cotransfected 293T, empty plasmid transfection was used as a negative control and supernatants were collected for detection of binding to PV1 sVLP. As shown in FIG. 4, the recombinantly expressed 5C6 antibody specifically recognizes PV1 sVLP, indicating that the amplified 5C6 sequence is correct.

The invention successfully screens and obtains the PV1 specific monoclonal antibody 5C6, and the identified epitope is a conformational epitope. In conclusion, the 5C6 obtained by the research can sensitively detect the PV 1D antigen, and can be used for clinical diagnosis and quality control of vaccine development and production.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. An anti-poliovirus type 1 antibody, characterized in that the anti-poliovirus type 1 antibody comprises a heavy chain variable region and a light chain variable region, said anti-poliovirus type 1 antibody having the following technical characteristics:

2. The anti-poliovirus type 1 antibody according to claim 1, characterized in that the anti-poliovirus type 1 antibody is a monoclonal antibody.

3. The anti-poliovirus type 1 antibody according to claim 1, characterized in that the heavy chain variable region amino acid sequence of the anti-poliovirus type 1 antibody is shown as SEQ ID No.2 and/or the light chain variable region amino acid sequence is shown as SEQ ID No. 4.

4. An isolated polynucleotide encoding the anti-poliovirus type 1 antibody of any one of claims 1-3.

5. The isolated polynucleotide of claim 4, wherein the isolated polynucleotide comprises nucleotides having a nucleotide sequence set forth in SEQ ID No. 11-16.

6. The isolated polynucleotide of claim 4, wherein the isolated polynucleotide comprises a nucleotide sequence as set forth in SEQ ID No.1 and a nucleotide sequence as set forth in SEQ ID No. 3.

7. A nucleic acid construct comprising the isolated polynucleotide of any one of claims 4 to 6.

8. An engineered cell comprising the nucleic acid construct of claim 7 or the polynucleotide of any one of claims 4-6 integrated into the genome.

9. A method for preparing an anti-poliovirus type 1 antibody according to any one of claims 1 to 3, comprising the steps of: culturing the engineered cell of claim 8 to express the anti-poliovirus type 1 antibody, and purifying and separating to obtain the anti-poliovirus type 1 antibody.

10. A kit for detecting poliovirus type 1 antigens, characterized by comprising an anti-poliovirus type 1 antibody or an immunoconjugate thereof according to any one of claims 1 to 3.

11. Use of an anti-poliovirus type 1 antibody of any one of claims 1 to 3, an isolated polynucleotide of any one of claims 4 to 6, a nucleic acid construct of claim 7, an engineered cell of claim 8 or a kit of claim 10 for the preparation of a reagent for detecting the C-or D-antigen content of poliovirus type 1.

12. Use of an anti-poliovirus type 1 antibody of any one of claims 1 to 3, an isolated polynucleotide of any one of claims 4 to 6, a nucleic acid construct of claim 7, an engineered cell of claim 8 or a kit of claim 10 for the preparation of a medicament for diagnosing or treating polio.