CN106608907B

CN106608907B - Preparation and application of anti-ERPV-ATIp recombinant truncated protein monoclonal antibody

Info

Publication number: CN106608907B
Application number: CN201510710538.6A
Authority: CN
Inventors: 蓝柯; 姜福伟; 张弛宇; 何志祥
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Shanghai Institute Of Immunology And Infection Chinese Academy Of Sciences
Priority date: 2015-10-27
Filing date: 2015-10-27
Publication date: 2021-08-03
Anticipated expiration: 2035-10-27
Also published as: CN106608907A

Abstract

The invention provides a monoclonal antibody of an ERPV-ATIp recombinant truncated protein, and preparation and application thereof. Specifically, a drosophila S2 cell expression system is utilized to introduce a specific ERPV-ATIp truncated protein coding gene to obtain a high-purity recombinant ERPV-ATI truncated protein, and an anti-ERPV-ATIp truncated protein monoclonal antibody is obtained by taking the high-purity recombinant ERPV-ATIp truncated protein as an antigen. The monoclonal antibody can specifically recognize ERPV-ATIp truncated protein and has high affinity to ERPV-ATIp full-length protein.

Description

Preparation and application of anti-ERPV-ATIp recombinant truncated protein monoclonal antibody

Technical Field

The invention relates to the field of biological medicines, in particular to preparation and application of an ERPV-ATIp recombinant truncated protein resisting monoclonal antibody.

Background

Erythromelalgia-related poxvirus (ERPV), a member of the orthopoxvirus genus, was named ZHENG-ZHANG Virus and was isolated from pharyngeal swabs of patients with erythromelalgia. The epidemic erythromatous limb pain is mostly caused in female middle school students of 15 to 17 years old; the disease is acute and usually occurs suddenly at night; initially the patient has toe pain, followed by forefoot pain; patients often can not fall asleep and the cold compress can relieve pain; edema and congestion appear on the feet and legs of some patients, and simultaneously the patients feel numb; walking difficulty for severe people; in some patients, the foot is diseased, and the fingers are similarly diseased.

EPPV viruses develop A-type inclusion bodies (ATIs) during the growth cycle, which are assembled from ATIp expressed by A25L; the inclusion body is virus-free (ATI V-); IgG specific to ERPV A type inclusion body appears in the serum of some patients with epidemic erythromatous acrodynia. Currently, there are no commercially available antibodies against ERPV A-type inclusion bodies.

Therefore, there is an urgent need in the art to develop anti-ERPV virus drugs and detection antibodies with good clinical application prospects.

Disclosure of Invention

The invention aims to provide preparation and application of an ERPV-ATIp recombinant truncated protein resisting monoclonal antibody.

In the first aspect of the invention, an antigenic polypeptide is provided, and the sequence of the antigenic polypeptide is shown as 497-919 site in SEQ ID NO. 2.

In another preferred embodiment, the antigenic polypeptide is recombinantly expressed in a eukaryotic host cell.

In another preferred embodiment, the eukaryotic host cell comprises a drosophila S2 cell.

In a second aspect of the invention, there is provided an antibody specific for an antigenic polypeptide according to the first aspect of the invention.

In another preferred embodiment, the antibody is a polyclonal antibody or an antiserum.

In another preferred embodiment, the antibody is a monoclonal antibody.

In a third aspect of the invention, there is provided a test kit comprising an antigenic polypeptide according to the first aspect of the invention and/or an antibody according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided a use of the antigenic polypeptide of the first aspect of the invention and/or the antibody of the second aspect of the invention in the preparation of a test reagent or kit for detecting erythromelalgia-associated poxvirus (ERPV).

In another preferred embodiment, the detection reagent comprises a lateral flow strip.

In a fifth aspect of the invention, there is provided a test reagent for testing a poxvirus associated with erythromelalgia, said test reagent comprising an antigenic polypeptide according to the first aspect of the invention and/or an antibody according to the second aspect of the invention.

In another preferred embodiment, the detection reagent comprises a test strip.

In another preferred example, the test strip includes:

the device comprises a sample pad, a combination pad, a reaction membrane, an absorption pad and a back lining, wherein the reaction membrane is provided with a detection area and a quality control area; and

wherein the detection zone is immobilised with a polypeptide according to the first aspect of the invention and the conjugate pad is immobilised with an antibody according to the second aspect of the invention.

In a sixth aspect of the present invention, there is provided a heavy chain variable region of an antibody, comprising the following three complementarity determining regions CDRs:

XH1 CDR1,

XH2 and CDR2 of SEQ ID NO

CDR3 as shown in SEQ ID NO XH 3;

preferably, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: XHV.

In a seventh aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region and a heavy chain constant region according to the sixth aspect of the invention.

In another preferred embodiment, the heavy chain amino acid sequence of the antibody is as set forth in SEQ ID No. XH.

In an eighth aspect of the present invention, there is provided a light chain variable region of an antibody, the light chain variable region having complementarity determining regions CDRs selected from the group consisting of:

CDR 1' as shown in SEQ ID NO: XL1,

CDR 2' as shown in SEQ ID NO: XL2, and

CDR 3' as shown in SEQ ID NO XL 3;

preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO XLV.

In a ninth aspect of the invention there is provided a light chain of an antibody, said light chain having a light chain variable region and a light chain constant region as described in the eighth aspect of the invention.

In another preferred embodiment, the light chain amino acid sequence of the antibody is as set forth in SEQ ID No. XL.

In a tenth aspect of the present invention, there is provided an antibody having:

(1) a heavy chain variable region according to the sixth aspect of the invention; and/or

(2) A light chain variable region according to the eighth aspect of the invention;

alternatively, the antibody has:

a heavy chain according to the seventh aspect of the invention; and/or a light chain according to the ninth aspect of the invention.

In another preferred embodiment, the antibody is of the subtype IgG 1.

In another preferred embodiment, the monoclonal antibody is produced by a hybridoma cell line selected from the group consisting of: 5E9, 6G12, and/or 11B 2.

In an eleventh aspect of the present invention, there is provided a recombinant protein having:

(i) a heavy chain variable region according to the sixth aspect of the invention, a heavy chain according to the seventh aspect of the invention, a light chain variable region according to the eighth aspect of the invention, a light chain according to the ninth aspect of the invention, or an antibody according to the tenth aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In a twelfth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the sixth aspect of the invention, a heavy chain according to the seventh aspect of the invention, a light chain variable region according to the eighth aspect of the invention, a light chain according to the ninth aspect of the invention, or an antibody according to the tenth aspect of the invention; or

(2) A recombinant protein according to the eleventh aspect of the invention.

In another preferred embodiment, the sequence of the polynucleotide has a polynucleotide sequence as shown in SEQ ID No. XHDNA and/or SEQ ID No. XLDDNA.

In a thirteenth aspect of the invention, there is provided a vector comprising a polynucleotide according to the twelfth aspect of the invention.

In a fourteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the thirteenth aspect of the invention into which has been integrated a polynucleotide according to the twelfth aspect of the invention.

In a fifteenth aspect of the present invention, there is provided a kit comprising:

an antibody according to the tenth aspect of the present invention.

In another preferred embodiment, the kit is an enzyme-linked immunoassay kit.

In a sixteenth aspect of the invention, there is provided an immunoconjugate comprising:

(a) an antibody according to the tenth aspect of the invention or a recombinant protein according to the eleventh aspect of the invention; and

(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In a seventeenth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody according to the tenth aspect of the invention, a recombinant protein according to the eleventh aspect of the invention, or an immunoconjugate according to the sixteenth aspect of the invention; and

a pharmaceutically acceptable carrier.

In an eighteenth aspect of the present invention, there is provided a method of producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the tenth aspect of the invention or a recombinant protein according to the eleventh aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of electrophoresis of fragments of the ERPV-ATIp coding sequence amplified by PCR.

FIG. 2 shows an SDS-PAGE pattern of the purification of ERPV-ATIp recombinant truncated proteins using affinity chromatography.

FIG. 3 shows the purification of ERPV-ATIp recombinant truncated protein immunoblots (WBs) using affinity chromatography methods.

FIG. 4 shows a graph of serum antibody titers after a third immunization of Balb/c mice.

FIG. 5 shows SDS-PAGE analysis of antibody purification in ascites using a ProteinG affinity chromatography column.

FIG. 6 shows the results of immunoblot analysis of the monoclonal antibody of the invention and His-Tag (2A8) Mouse mAb from Abmart corporation.

FIG. 7 shows the results of immunoprecipitation analysis of the monoclonal antibody of the invention and His-Tag (2A8) Mouse mAb from Abmart corporation.

FIG. 8 shows the results of immunofluorescence experiments performed with the Monoclonal Antibody of the present invention against Anti-Flag Tag Mouse Antibody (1B10) (control), available from Abbkine.

FIG. 9 shows the detection of anti-ERPV-ATIp antibodies in serum by the indirect enzyme-linked immunoassay provided herein.

FIG. 10 shows the results of detection of recombinant proteins in cell lysates after 4 days of infection of Sf9 cells with recombinant baculovirus P1 virus.

FIG. 11 shows WB detection map of ERPV-ATIp full-length protein expressed by Drosophila S2 cells.

FIG. 12 shows a map of WB expression for different lengths of truncated proteins.

FIG. 13 shows the examination of the purification effect of recombinant ERPV-ATIp protein expression of different lengths. Wherein FIG. 13-A is SDS-PAGE detection; FIG. 13-B shows WB detection.

Detailed Description

The inventor obtains an anti-ERPV-ATIp truncated protein monoclonal antibody through extensive and intensive research. Experiments show that by utilizing a drosophila S2 cell expression system and introducing a specific ERPV-ATIp truncated protein coding gene, high-purity recombinant ERPV-ATI truncated protein can be obtained, and then an anti-ERPV-ATIp truncated protein monoclonal antibody can be obtained. The monoclonal antibody can specifically recognize ERPV-ATIp truncated protein and has high affinity to ERPV-ATIp full-length protein. On the basis of this, the present invention has been completed.

Term(s) for

ERPV virus

ERPV virus, an erythromelalgia-related poxvirus (erythromelalgi-related poxvirus), was isolated from pharyngeal swabs of patients with epidemic erythromelalgia. Serological identification shows that ERPV is an orthopoxvirus intraformational member, but has a neutralizing epitope difference with the orthopoxvirus intraformational member vaccinia virus and vaccinia virus. The serum of the patients with the epidemic erythromelalgia contains neutralizing antibodies and anti-ERPV inclusion body IgG antibodies of the ERPV, and the detection rate of the serum is obviously higher than that of local unforeseen patients and American residents.

EPPV-ATI is an A-type inclusion body which appears in the virus in the growth cycle and is formed by the assembly of ATIp expressed by A25L. The nucleotide sequence of the full-length ATIp protein is shown in SEQ ID NO. 1, and the amino acid sequence of the full-length ATIp protein is shown in SEQ ID NO. 2.

The inventor obtains a section of specific ERPV-ATIp truncated protein through multiple screening, and the truncated protein can be stably expressed by utilizing a drosophila S2 cell expression system and can be separated and purified. The truncated protein is taken as an antigen, and a high-affinity monoclonal antibody which can specifically identify the ERPV-ATIp recombinant truncated protein and the ERPV-ATIp full-length protein can be obtained. In a preferred embodiment of the invention, the amino acid sequence of the ERPV-ATIp truncated protein is as shown in SEQ ID NO. 2 at position 497-919.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The invention also comprises a monoclonal antibody with the corresponding amino acid sequence of the anti-ERPV-ATIp truncated protein monoclonal antibody, a monoclonal antibody with the variable region chain of the anti-ERPV-ATIp truncated protein monoclonal antibody, and other proteins or protein conjugates with the chains and fusion expression products. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the anti-ERPV-ATIp truncated protein monoclonal antibody or fragment thereof. The invention also comprises a cell surface marker or antigen combined with the anti-ERPV-ATIp truncated protein monoclonal antibody or the fragment thereof.

The present invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; an antibody heavy chain; the light chain of the antibody.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the following three complementarity determining regions CDRs:

CDR1, the amino acid sequence of which is shown as SEQ ID NO: XH1, and the coding nucleotide sequence of which is SEQ ID NO: XH1 DNA;

CDR2, the amino acid sequence of which is shown in SEQ ID NO. XH2, and the coding nucleotide sequence of which is shown in SEQ ID NO. XH2 DNA;

CDR3, the amino acid sequence of which is shown in SEQ ID NO. XH3, and the coding nucleotide sequence of which is shown in SEQ ID NO. XH3 DNA.

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and a heavy chain constant region, which may be of murine or human origin.

As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:

CDR 1', the amino acid sequence of which is shown as SEQ ID NO: XL1, and the coding nucleotide sequence of which is SEQ ID NO: XL1 DNA;

CDR 2', the amino acid sequence of which is shown in SEQ ID NO. XL2, and the coding nucleotide sequence of which is shown in SEQ ID NO. XL2 DNA;

CDR 3', the amino acid sequence of which is shown in SEQ ID NO. XL3, and the coding nucleotide sequence of which is SEQ ID NO. XL3 DNA.

In a preferred embodiment of the invention, the light chain of the antibody comprises the light chain variable region and a light chain constant region, which may be murine or human.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and all refer to an antibody that specifically binds to anti-ERPV virus, e.g., a protein or polypeptide having a heavy and/or light chain. They may or may not contain the initial methionine.

In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody against ERPV virus, which heavy chain constant region and/or light chain constant region may be humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the invention refers to a polypeptide which has the binding activity of anti-ERPV virus A-type inclusion body and is specifically combined with the ERPV-ATIp truncated protein. The term also includes variants of the polypeptides that specifically bind to the ERPV-ATIp truncated protein having the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID NO. XVHDNA, or XVLDNA, or may be a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differs from the coding region sequence shown in SEQ ID No. XVH, XVL.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Composition comprising a metal oxide and a metal oxide

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the invention can be directly used for binding ERPV virus, thus being used for preventing and treating the ERPV virus which causes epidemic erythromelalgia. In addition, other scientific studies and clinical diagnostics may be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Hybridoma cell strain

The invention also provides a hybridoma cell strain capable of producing the anti-ERPV-ATIp truncated protein monoclonal antibody; preferably, the invention provides a hybridoma cell strain with high titer aiming at the anti-ERPV-ATIp truncated protein monoclonal antibody.

After obtaining the hybridoma producing the anti-ERPV-ATIp truncated protein monoclonal antibody of the invention, the skilled person can conveniently prepare the antibody by using the hybridoma cell strain. In addition, the structure of the antibody of the present invention (e.g., the heavy chain variable region and the light chain variable region of the antibody) can be easily known by those skilled in the art, and then the monoclonal antibody of the present invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the antigens of the invention can be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, they can be prepared using hybridoma technology or by recombinant DNA methods.

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to medium (HAT medium matrix), including myeloma Cell lines, such as murine myeloma Cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, rockwell, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies.

The medium in which the hybridoma cells are grown is assayed to detect the production of monoclonal antibodies of the desired specificity, e.g., by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA). The location of the antibody-expressing cells can be detected by FACS. The hybridoma clones can then be subcloned (subcloned) by limiting dilution procedures and grown by standard methods. Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow in animals as ascites tumors.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention provides a monoclonal antibody aiming at ERPV virus. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. Taking supernatant fluid of hybridoma cell culture, carrying out saturated ammonium sulfate precipitation to obtain IgG, and purifying the antibody obtained by crude extraction through an affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells are inoculated into the abdominal cavity of the sensitized mouse, and the abdomen is obviously swelled after about 10 days. Ascites was extracted, the supernatant was centrifuged, diluted with the loading buffer, and the diluted solution was purified by an affinity column (HiTrap Ptoeing G HP column (1 mL)).

Labeled immunoglobulins (antibodies)

In a preferred embodiment of the invention, the immunoglobulin is provided with a detectable label. More preferably, the marker is selected from the group consisting of: colloidal gold labels, horseradish peroxidase labels, colored labels or fluorescent labels.

The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the invention, the monoclonal antibody against the truncated ERPV-ATIp protein is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.

The ERPV-ATIp resisting truncated protein monoclonal antibody has high specificity and high titer.

Test plate and material therefor

The detection plate can be made of detection plate materials commonly used in the field by adopting a conventional detection plate preparation method.

The invention relates to an immunoassay plate for detecting ERPV virus, which comprises a test strip and a supporting plate for supporting the test strip, for example, a PVC polyester rubber plate and the like can be adopted; the test strip is formed by sequentially overlapping sample filtering paper, chromatographic materials, a nitrocellulose membrane and absorbent paper, and the overlapped part can be fixedly connected by adopting a conventional method, such as an adhesive tape and the like; wherein: the chromatography material is pre-coated with a colloidal gold-labeled or colored-labeled anti-ERPV-ATIp truncated protein monoclonal antibody, preferably a colloidal gold-labeled anti-ERPV-ATIp truncated protein monoclonal antibody, and an adsorption detection line and a quality control line are arranged on a nitrocellulose membrane;

in a preferred embodiment: the colloidal gold-labeled anti-ERPV-ATIp truncated protein monoclonal antibody pre-coated on the chromatographic material is dissolved by the colloidal gold-labeled anti-ERPV-ATIp truncated protein monoclonal antibody with the concentration of 0.5-1.5mg/mLThe solution is pre-coated with a coating amount of 50. mu.L/cm²(ii) a The preferred concentration is 0.5 or 1.5mg/mL, 50. mu.L/cm²。

Detection method and result judgment

And (4) flatly placing the detection plate, dropping the sample on the sample filtering paper, and observing the chromatography result within 3-5 min. And judging the result according to the position of the appearing stripe.

Negative: the quality control area and the detection area both have obvious colored bands and are shown as negative;

positive: a clear color band appears only in the quality control area, and no color band appears in the detection area, which is shown as positive;

and (4) invalidation: the quality control area and the detection area have no color band or no color band appears in the quality control area and a color band appears in the detection area, which indicates that the detection method is wrong or the detection plate is deteriorated or invalid, and the detection plate is required to be replaced for detection.

Method and sample

The present invention relates to methods for detecting ERPV virus in a sample lysed with cells and/or tissue. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of ERPV virus in the lysed sample. The sample used in the method of the present invention may be any sample comprising cells present in a cell preservation solution, as used in liquid-based cytology methods.

Reagent kit

The present invention also provides a kit comprising an antibody (or fragment thereof) of the present invention or an assay plate of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention further designs a detection kit for detecting ERPV virus level, which comprises an antibody for identifying anti-ERPV-ATIp truncated protein, a lysis medium for dissolving a sample, and general reagents and buffers required by detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.

The invention further contemplates the development of a kit for diagnostic assessment of conditions associated with ERPV virus infection from a sample of solution which can detect ERPV virus present in the sample solution, wherein the cell preservation fluid used to preserve the sample can be, for example, a cell preservation fluid used in a liquid-based cytology procedure.

The ERPV-ATIp resistant truncated protein monoclonal antibody has the advantages of high affinity, high specificity and the like, can be widely applied to the detection field of preparing ERPV viruses, such as the preparation field of detection reagents or detection equipment and the like, and has obvious advantages in the aspects of specificity, sensitivity, detection rate and the like compared with the traditional detection method or detection reagent.

The main advantages of the invention include:

(a) the monoclonal antibody can be conveniently and rapidly obtained, can be used for detecting ATIp, and provides a sequence basis for the development of a humanized recombinant antibody.

(b) Can be used for the research and development of ERPV diagnostic kits and can be used for preparing enzyme linked immunosorbent assay kits and colloidal gold rapid diagnostic kits.

(c) Because the recombinant protein expressed by the drosophila S2 cell is used, the obtained recombinant protein is close to a natural conformation, the immunogenicity is good, and the affinity and the specificity of the obtained monoclonal antibody are good.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1

Acquisition of ERPV-ATI truncated protein coding Gene

According to the basic information of ERPV-ATIp gene In Virus Pathologen Resource database, by using pFLAG-CMV5.1-ATI (pFLAG-CMV5.1 Vector is given by tumor Virus research group of Shanghai Pasteur institute In China as a gift In laboratory, and inserted ATI full-length sequence is synthesized by Shanghai Burkhstan biology company) as a template, using an In-Fusion kit, using Vector NTI Advance 11 software to design specific primers, carrying out PCR amplification to obtain a coding sequence of ERPV-ATIp truncated protein, and introducing enzyme cutting sites at two ends of a PCR product respectively: kpn I and ApaL I. Wherein, the primer sequences used for PCR amplification are as follows:

primer 1(SEQ ID No.: 3):

5’-TCTCCATGGCCCGGGGTACCTAGAGAATCTCTTGATAGGGAACGGGA-3’

primer 2(SEQ ID No.: 4):

5’-GGCTTACCTTCGAAGGGCCCAGAATAGTCTTTCTGAAGACGATCGAATTCTA-3’

FIG. 1 is an agarose gel electrophoresis of a PCR amplified fragment of the ERPV-ATIp coding sequence, molecular weight markers 1Kb DNA Ladder RTU (DM010) from GeneDirex, in which the lane samples are, in order: 1:1Kb DNA Ladder RTU (Genedirex),2: ERPV-ATIp protein coding sequence fragment, 3: pMT-BIP-V5/His-A,4: pMT-BIP-V5/His-A after double enzyme digestion, and 5: recombinant pMT-BIP-V5/His connected with ERPV-ATIp protein coding sequence fragment.

As a result, as shown in FIG. 1, a 1.2kb fragment (truncated) was obtained by PCR amplification. The plasmid is naturally present in a supercoiled state. The supercoiled plasmid pMT-BIP-V5/His-A is approximately 2kb, and the size after double digestion is about 5 kb. The constructed supercoiled recombinant plasmid is about 2.7 kb.

Example 2

Construction of recombinant protein expression vectors

The PCR product of example 1 was digested with KpnI and ApaL I, cut into a gel, recovered, ligated with pMT/BIP/V5-His-A vector similarly digested with KpnI and ApaL I, and then introduced into DH5 a. Plates were coated overnight and single spots were picked for sequencing validation by Boshang Biotech Ltd. Wherein, the vector pMT/BIP/V5-His-A site is an Expression vector used by Drosophila S2 Expression System.

The sequencing result shows that the nucleotide sequence of the inserted fragment in the vector is completely consistent with the nucleotide sequence of the target fragment, and the recombinant protein expression vector is constructed correctly.

Example 3

Construction of Drosophila S2 stable transfer cell strain for stably expressing recombinant protein

The recombinant protein expression vector and the pcolblast plasmid constructed in example 2 were transformed into DH5a competent cells, respectively, individual clones were selected, amplified, plasmids were extracted, and plasmid concentration was determined using NanoDrop. Transfection reagents were prepared as provided with reference to the Drosophila S2 expression system.

One day before transfection, well-prepared Drosophila S2 cells were passaged into six-well plates at 1.5X 10 per well⁶Cells cultured at 28 ℃ for 12-18h to reach 4 multiplied by 10⁶Individual cells can be used for transfection.

On the day of transfection, transfection mixtures were prepared according to the following recipe:

solution A: 150 μ L

Solution a was slowly added dropwise to solution B on the cell bench while mixing continuously with Vortex. The whole process is about 1 min. The mixture was then placed on a cell bench for 30min until a precipitate of more uniform size was formed. The mixture was added dropwise to a 6-well plate plated with Drosophila S2 cells, 150. mu.L of the mixture per well. The cells were incubated at 28 ℃ for 18 h.

Cells were changed 18h after transfection, centrifuged to remove calcium phosphate solution, washed twice with complete medium, resuspended transfected cells in 2mL complete medium and passaged to the original 6-well plate.

Adding screening antibiotic Blasticidin HCl into the cell culture solution 3 days after transfection to the final concentration of 25 mug/mL, replacing the culture solution containing the selection antibiotic at intervals of 4-5 days, and screening for about 2 weeks to obtain the stably transfected cells.

100 stably transfected cells obtained were diluted by the limiting dilution method into 10mL of a previously prepared normal cell having a cell density of 105, mixed well, and plated in a 96-well plate in an amount of 100. mu.L per empty. Adding 100 mu L culture solution with 50 mu g/mL concentration of screening antibiotic Blasticidin into 96-well plate after 24h, and supplementing 96-well plate with the culture solution every 4-5 days50 mu.L of culture solution containing 25 mu g/mL of screening antibiotic Blasticidin HCl, selecting single cell cloning wells after 10 days, subculturing to 48-well plates, and gradually expanding and culturing. The cells are subcultured to a 24-well plate for 48h, serum-free culture solution is replaced, and an inducer CdCl with the final concentration of 5 mu M is added₂And after 72h, cell culture fluid and cells are collected for WB analysis. WB assay used antibodies: a first antibody: His-Tag (2A8) Mouse mAb (available from Abmart, M20001S); secondary antibody: Anti-Mouse IgG (H + L), AP Conjugate (available from Promega, S3721).

Example 4

Large-scale induced expression and purification of ERPV-ATIp truncated recombinant protein

The stably transfected cell line obtained in example 3 was expanded and cultured, and then inoculated into 3L of Spinner flash, 1L of serum-free culture medium was added to each Flask, and the cell density of the inoculum was 0.5-1X 10⁶cell/mL. Culturing at 28 deg.C for 24-48h, and adding inducer CdCl into the culture solution₂The final concentration was set to 5. mu.M, and the cell culture broth was collected 4 days after the addition of the inducer. The operation is as follows:

centrifuging the cell culture solution at 4 deg.C and 6000rpm/min for 10min to collect cell culture supernatant, filtering with 0.45 μm filter membrane, and adding protease inhibitor PMSF into the culture supernatant to obtain final concentration of 1 mM. The culture solution was applied to QuixStand, GE^TMbenchmark systems performed 10-fold concentration and buffer exchange. The concentrated sample can be stored at-20 ℃.

The human recombinant ERPV-ATI truncated protein was purified by affinity chromatography using His GraviTrap Column from GE. The purification results were analyzed by SDS-PAGE and WB. The primary antibody used in WB assay was His-Tag (2A8) Mouse mAb (Abmart) and the secondary antibody used was Goat Anti-Mouse Ig capture antibody (SBA).

FIG. 2 shows an SDS-PAGE pattern of the purification of ERPV-ATIp recombinant truncated proteins using affinity chromatography. Wherein, the samples of each lane are sequentially Marker, PageRuler Prestated Protein Ladder (10-170 kDa); lane1-6 His

Eluent for purification treatment of affinity chromatography column (1ml sample collected in each tube)

FIG. 3 shows the results of immunoblot (WB) analysis of recombinant truncated proteins purified from ERPV-ATIp using affinity chromatography. The sample loading sequence for each lane is the same as that of FIG. 2.

The results are shown in FIGS. 2 and 3. As can be seen from a comparison of FIGS. 2 and 3, the truncated protein with higher purity was obtained, and the size of the obtained monomeric protein (which was confirmed to exist as a dimer in example 9) was about 60kDa, wherein the purity of eluate 4 was better and the expression level was higher.

Example 5

Animal immunization

Balb/c mice (8 weeks, female) were immunized 4 times with 1 week intervals, using the ERPV-ATIp recombinant truncated protein purified in example 4 as an immunogen. The concrete method comprises the following steps:

primary immunization: antigen 50 μ g, plus Freund's complete adjuvant subcutaneous multiple injection;

↓ 1 week later

And (3) second immunization: the dosage is reduced to 20 mu g, and subcutaneous multi-point injection is carried out by adding Freund incomplete adjuvant;

↓ 1 week later

And (3) third immunization: the dosage is the same as above, subcutaneous multi-point injection is carried out by adding Freund's incomplete adjuvant, and serum is collected 7 days later to measure the antibody titer;

↓ 1 week later

The fourth immunization dose is the same as the above, and subcutaneous multi-point injection of Freund incomplete adjuvant is added;

↓ 1 week later

Boosting immunity, adding 10 mu g of antigen, adding no adjuvant, and performing subcutaneous multipoint injection;

↓3days later

Taking spleen cells for fusion.

The results are shown in FIG. 4, where the serum antibody titers of 2 mice after the third immunization exceeded 1: 1000000.

Example 6

Hybridoma preparation and screening

Myeloma cells were prepared from the conventional cell line SP2/0 prepared from a monoclonal antibody. The cells before fusion are treated with a culture medium containing 8-azaguanine to kill cells that are not resistant to HATSensitive progenitor cells. Selecting cells with good cell morphology and activity and growing in logarithmic phase during fusion, and adjusting cell density to about 2 × 10 with fresh culture solution one day before fusion⁵cells/mL, the next day cells were used for the fusion experiment.

On the third day after the booster immunization, splenocytes from immunized mice were taken and subjected to cell fusion with myeloma cells under 50% PEG-mediated conditions, followed by termination of the fusion with the culture medium. The ratio of splenocytes to myeloma cells at fusion was 10: 1. After completion of the fusion, the cells were added to a 96-well plate plated one day in advance with feeder cells.

Positive clones were screened using indirect ELISA. The antigen selected was the ERPV-ATIp recombinant truncated protein purified in example 4, and the secondary antibody was HRP-labeled Goat Anti-Mouse IgG1 (SBA). The confirmed positive clones are subcloned by using a limiting dilution method, and cell clones which are positive after three times of cloning are monoclonal strains which can be subjected to enlarged culture and frozen storage.

Example 7

Ascites production and antibody purification

Balb/c mice were selected for ascites preparation for 8 weeks. Injecting liquid paraffin 0.5mL into abdominal cavity of each mouse, injecting hybridoma cell strain 7 days later, and injecting cell amount 1-3 × 10 into each mouse⁶cells. After 1-2 weeks, the abdomen of the mouse is obviously enlarged, ascites is extracted by a syringe, and a large amount of antibody can be obtained by purification from the ascites.

Centrifuging ascites fluid (4 deg.C, 2000rpm for 5min), removing uppermost layer adipose tissue by aspiration, removing cell components and other precipitate, collecting supernatant, diluting with cold binding buffer 20 times, centrifuging at 12000rpm for 15min, filtering supernatant with 0.45 μm filter membrane, and standing until no bubbles are formed. According to the manual of the Bio-Rad apparatus, methanol and ddH were used₂And O, cleaning the instrument pipeline. HiTrap protein G HP column (1mL) was attached, and the flow rate and UV absorbance were set. Then, the purification is carried out according to the following steps:

20mL ddH₂o wash → 20mL binding buffer equilibration → load → 10mL binding buffer wash → 20mL elution buffer (where absorbance reaches a high value)First, the column was collected by a separate collection tube, and once the absorbance appeared, the column was immediately collected in multiple tubes, 1mL was collected in each tube, 100. mu.L of neutralization buffer was added to each 1mL of eluate) → 20mL of pure water → 20mL of 20% ethanol washing, and the column was removed and stored at 4 ℃. Samples were taken from each run, loading buffer was added, boiling water bath was carried out for 10min, and samples were analyzed for antibody purity by SDS-PAGE. The remaining eluate was collected in a pre-treated dialysis bag and dialyzed in PBS solution at 4 ℃ for 24h, during which time the dialysate was changed 4 times. Collecting dialyzed sample, adding glycerol to final concentration of 50%, determining antibody concentration with Bio-Rad Bradford reagent, subpackaging purified antibody, and storing in-80 deg.C refrigerator.

FIG. 5 shows an SDS-PAGE analysis of the antibodies in ascites. Wherein, the lane samples are sequentially as follows: lane1: diluting the treated ascites by Binding Buffer; lane 2: penetrating fluid treated by HiTrap protein G HP affinity chromatographic column; lane 3-7: eluent after Elution buffer treatment.

As shown in FIG. 5, two bands, 55kDa and 20kDa, were detected in the eluate (Lane3-7), which are the heavy chain and light chain of the antibody, respectively, and no impurity band, and the purification effect was good. .

Example 8

Monoclonal antibody immunoblotting (Western Blot) application

Taking the drosophila S2 cell stable transfer strain obtained by screening in the example 3, changing the complete culture medium to a serum-free culture medium 48h after the normal passage to T75 flash, and adding an inducer CdCl with the final concentration of 5 mu M after 24h₂And inducing the expression of ERPV-ATIp recombinant truncated protein. Cell culture supernatants were collected 72h after addition of inducer, 1600 μ L of each supernatant was added to a 1.5mL EP tube, 240 μ L of 100% TCA was added, and ice-cooled for 20 min. The mixture was centrifuged at 13000rpm at 4 ℃ for 30min, and the supernatant was discarded. Adding 4800 μ L acetone, ice-cooling for 20min, centrifuging at 13000rpm at 4 deg.C for 30min, discarding supernatant, and opening cover to allow acetone to evaporate. Adding 50 μ L2 xSDS-PAGE sample buffer, treating at 100 deg.C for 10min, loading (30 μ L for each 15-well PAGE gel, 1 μ g for ERPV-ATIp recombinant truncated protein), and performing 10% SDS-PAGE electrophoresis (90V, 30min, 120V, 40min) to transfer membrane (90V, 60min under constant pressure). PVDF film after transfer printing is at room temperatureSealing in 5% skimmed milk for 60 min. Membranes were cut into small strips according to lane position, each strip was incubated with 1mL of a final concentration of 1. mu.g/mL monoclonal antibody (monoclonal antibody from the antibody purified in example 5 and His-Tag (2A8) Mouse mAb from Abmart corporation) overnight at 4 ℃ and washed 4 times with TBST solution, all membrane strips were incubated with HRP-labeled Goat Anti-Mouse Ig capture antibody (SBA) antibody solution diluted 1:1000 for 1h, and scanned with Las2000 after washing 4 times with TBST.

As shown in FIG. 6, the three monoclonal antibodies can be combined with the monomeric protein of 60 kDa; the monomeric protein bound by the control His antibody was also 60kDa, indicating that the three monoclonal antibodies correctly recognized the truncated protein.

Example 9

Monoclonal antibody Immunoprecipitation (IP) applications

Taking the drosophila S2 cell stable transfer strain obtained by screening in the example 3, carrying out normal passage for 72h after T75 flash, and adding an inducer CdCl with the final concentration of 5 mu M₂And inducing the expression of ERPV-ATIp recombinant truncated protein. Collecting cell culture supernatant 72 hours after adding inducer, adding 1mL of supernatant to 1.5mL of a tube, adding 20. mu.L of Recombinant Protein G (rProtein G) Agarose (purchased from Invitrogen: 15920-010) to the supernatant, incubating the supernatant at 4 ℃ for 1 hour in a rotary shaker of an EP tube, centrifuging at 10000rpm for 5min, collecting the centrifuged supernatant, adding 50. mu.L of Recombinant Protein G (rProtein G) Agarose and 1. mu.g of each of the antibodies 5E9, 6G7 and 11B2 purified in example 7 to each of the EP tube, incubating the EP tube at 4 ℃ overnight, centrifuging at 10000rpm for 5min, collecting the centrifuged precipitate, washing 3 times with PBS, adding 100. mu.L of 2 xSDS-supernatant, collecting the supernatant, centrifuging at 4000rpm for 5min, collecting the supernatant, performing SDS-PAGE, performing a SDS-PAGE using a running gel (30. mu.g-PAGE), performing a single-PAGE to obtain a supernatant, and performing single-PAGE, performing single-PAGE on the supernatant, performing single-PAGE on the single-PAGE, washing, incubating with a secondary antibody Polyclonal Rabbit Anti-Goat Immoglobulins/HRP (abcam), and imaging with Las2000 mini after washing.

As shown in FIG. 7, all the antibodies can bind to free ERPV-ATIp recombinant truncated protein in the culture solution; the truncated protein exists as a dimer, and the addition of high concentrations of DTT dissociates the dimer.

Example 10

Monoclonal antibody Immunofluorescence (IF) assay

According to the method in lipofectamine 2000, 2ug of plasmid pFLAG-CMV5.1-ATI is transferred into 293T growing on an inner cover glass sheet of a 24-well plate, the liquid is changed after 6h, and the sample is collected after 48 h. The medium was blotted dry, washed twice with PBS and fixed with 500. mu.L of 2% paraformaldehyde in PBS for 20 min. The membrane was washed twice with PBS, and 500. mu.L of 0.1% Triton X-100PBS was added and soaked at room temperature for 15min to perforate the cell membrane. The coverslips were then transplanted to a new 24-well plate and washed twice with PBS. Add 500. mu.L Blocking Buffer (PBS with 5% FBS) and block for 20min at room temperature. The Blocking Buffer was blotted dry and 40. mu.L of primary Antibody (3 antibodies purified in example 7 and Anti-Flag Tag Mouse Monoclonal Antibody (1B10, 1: 100) from Abbkine) was added overnight. PBS wash 3 times, 500. mu.L blocking buffer for 5 min. Add 50. mu.L of goat Anti-mouse IgG H diluted 1:1000&L(Alexa

555) (abcam) incubation for 60 min. PBS was washed 1 time, 300 μ L1: DAPI at 5000-fold dilution was blocked for 10min and washed 2 times with PBS. In ddH₂Soaking in O once, and sucking to remove water. Drop a drop of nail polish on the slide, flip the cover slip over the nail polish, drive off the steam pocket, and close the cover slip with nail polish. Detection was performed using an immunoelectron microscope, Lecia DM 2500.

As a result, as shown in FIG. 8, all three monoclonal antibodies were able to bind to the full-length ERPV-ATIp, and the monoclonal antibody secreted from monoclonal strain 5E9 was very strong in binding ability. In the figure, the blue part is the nuclei after DAPI staining and the red part is the ERPV-ATIp full-length protein distributed in the cytoplasm.

Example 9

Indirect ELISA detection of anti-ERPV-ATIp antibodies

The purified ERPV-ATIp recombinant truncated protein was diluted to 2. mu.g/mL with coating buffer (Na2CO 3: 1.59g, NaHCO 3: 2.93g dissolved in 800mL water, pH adjusted to 7.4, volume adjusted to 1000mL), coated with blank ELISA plate (Nunc: 468667), 100. mu.L/well, overnight at 4 ℃, washed andblocking was performed by adding blocking solution (1% BSA, PBST, pH7.4) for 2h at 37 ℃. And adding the liquid to be detected after fully washing. Mouse positive sera (serum-mouse-pos) were purchased from the tumor virus research group of the shanghai pasteur institute of the Chinese academy of sciences. The serum was obtained after immunization with full-length ERPV-ATIp. Mouse sera were obtained from 1:1000 starts with 10-fold dilution. After incubation at 37 ℃ for 40min, after 4 washes with PBST, add 1: the Goat Anti-Mouse Ig capture antibody (SBA) was diluted 10000 times. After incubation at 37 ℃ for 30min, the cells were washed 4 times with PBST, 0.5mL of TMB buffer (10mg/5mL absolute ethanol), 10mL of substrate buffer (0.2M Na2HPO4(25.7 mL. times.0.1M citric acid 24.3mL plus distilled water 50mL), 0.75% H₂O₂32 μ L) was incubated at 37 ℃ for 20min, 50 μ L of stop buffer (2M H) was added₂SO₄) The absorbance of each well at a wavelength of 450nm was recorded by a Molecular Device SpectraMax 190 plate reader, and the measurement results were analyzed by Prizm 6.

The results are shown in FIG. 9, with a positive mouse serum dilution of 10⁶Fold was still detectable, while mouse negative sera had essentially no readings. The results show that the anti-ERPV-ATIp antibody of the invention has better specificity.

Comparative example 1

Expression of ERPV-ATIp full-length protein by baculovirus expression system

Designing a recombinant primer by taking pFlag-CMV5.1-ERPV-ATIp as a template and referring to an In-Fusion Kit application instruction, carrying out PCR amplification to obtain an ERPV-ATIp gene, connecting a PCR product with a baculovirus expression vector pFast-bactobab-HTB, transforming DH5 alpha E. Wherein, the primers used for PCR amplification are as follows:

primer 3(SEQ ID No.: 5):

5’-TTTCAGGGCGCCATGGTTGAGGTCACGAACCTTATTGAAAAATGTAC-3’

primer 4(SEQ ID No.: 6):

5’-TTCTCGACAAGCTTGGTACCAGTAGATATTGGTAGAAGATATGCACTAACA-3’

coli competent cells were transformed with the above recombinant plasmid DH10Bac e. Screening is carried out by using a 50 mu g/ml Kan, 7 mu g/ml Tet and 10 mu g/ml Gen triple-resistance solid agarose plate, colonies obtained by screening are inoculated into a three-resistance liquid culture medium, and plasmids are detected and extracted by PCR. Namely the constructed shuttle vector (Bacmid).

The incomplete medium, pre-warmed at 27 ℃ adjusted the density of Sf9 cells cultured in suspension in Spinner flash to 4X 10⁵cell/ml. 0.5ml (2X 10) was added to each well of a 24-well plate 1h before transfection⁵cells) the above incomplete medium Sf-9 cell dilution solution, and culturing in an insect cell incubator at 27 ℃.

On the day of transfection mixtures of transfection were prepared according to the recipe provided in the instructions:

solution B was slowly added drop-wise to solution a in a biosafety cabinet. After the mixture was vortexed and mixed by Vortex, the AB mixture was allowed to stand for 15 min. Then, 160. mu.L of incomplete medium was slowly dropped to dilute the medium. After removing the incomplete medium from the 24-well plate, a dilution of the AB mixture was added to ensure that the dilution covered the entire well bottom. Subsequently, the 24-well plate was placed in a wet box and incubated in an insect cell incubator at 27 ℃ for 8 hours, and then a dilution of the AB mixture solution in the plate was removed and 200. mu.L of complete medium was added. After culturing at 27 ℃ for 8 days in an insect cell incubator, the medium containing the first generation virus (P1 virus) was collected while observing the presence or absence and morphology of CPE. Sf9 cells were then infected with varying amounts of P1virus and harvested 4 days later, during which time CPE appearance and morphology was observed.

Immunoblotting (WB) was performed to examine protein expression according to the method of example 3.

FIG. 10 shows the WB detection results of recombinant proteins in cell lysates after 4 days of infection of Sf9 cells with recombinant baculovirus P1virus, wherein Lane1 is untransfected cells; lane2-6 WB assay of cell lysates 4 days after infection of Sf9 cells with varying amounts of P1virus, Lane2-6: the amounts of P1 viruses were: 50 μ L of P1 virus-containing medium, 100 μ L of P1 virus-containing medium, 200 μ L of P1 virus-containing medium, 400 μ L of P1 virus-containing medium, and 800 μ L of P1 virus-containing medium. The red arrow indicates the detection band for protein WB expressed by Sf9 cells.

As shown in FIG. 10, the recombinant baculovirus expressed the recombinant protein with a size significantly lower than 130kDa, which is very different from the estimated 130kDa molecular weight of ERPV-ATIp protein. Since the proteins expressed by the insect baculovirus expression system undergo post-translational modification, this suggests that ERPV-ATIp may be cleaved.

The results indicate that the full-length protein expressed by the baculovirus expression system is not suitable as an immunogen for ERPV-ATIp antibodies.

Comparative example 2

Expression of ERPV-ATIp full-length protein by using drosophila S2 cells

According to the method in the embodiment 1-4, ERPV-ATI full-length protein is obtained, recombinant protein expression vector is constructed, drosophila S2 stable transfer cell strain which stably expresses recombinant protein is constructed, the ERPV-ATIp full-length protein is subjected to large-scale induction expression and purification, and SDS-PAGE and WB are used for testing the expression condition of the protein.

Wherein, the primers used for PCR amplification are as follows:

primer 5(SEQ ID No.: 7):

5’-TCTCCATGGCCCGGGGTACCTATGGAGGTCACGAACCTTATTGAAAA-3’

primer 6(SEQ ID No.: 8):

5’-GGCTTACCTTCGAAGGGCCCAGTAGATATTGGTAGAAGATATGCACTAACATCATATG-3’

as a result, as shown in FIG. 11, WB showed a band of about 130kDa, but SDS-PAGE showed no corresponding band, indicating a lower expression level. Therefore, the ERPV-ATIp full-length protein expressed by Drosophila S2 cells is not suitable for immunizing mice as an antigen.

Example 10

Screening of truncated-Gamma protein

The truncated protein in example 1 was truncated-gamma, which was screened in this example to obtain an expression level unexpectedly higher than other analogous ATIp proteins (full-length or fragment) and was effective in purifying the polypeptide.

The method is the same as the method in the embodiment 1-4, namely, a plurality of different ERPV-ATI truncated protein genes are obtained by PCR respectively, the expression is carried out by utilizing a drosophila S2 expression system, and the expression condition of the protein is tested by WB.

The positions of the truncated proteins in this example in the gene encoding the ERPV-ATI full-length protein and the primers used are shown in the following table:

truncated proteins	Start and stop position	Forward primer	Reverse primer
				truncated-1	1-1404	f-1	r-1404
truncated-2	1-1554	f-1	r-1554
				truncated-3	1-1667	f-1	r-1667
truncated-4	1-1797	f-1	r-1797
				truncated-5	1-1929	f-1	r-1929
truncated-6	1-2064	f-1	r-2064
				truncated-7	1-2193	f-1	r-2193
truncated-8	1-2325	f-1	r-2325
				truncated-α	1-1269	f-1	R-α
truncated-β	445-1659	F-β	R-β
				truncated-γ	1489-2757	F-γ	R-γ
truncated-δ	1801-3339	F-δ	R-δ

The partial primer sequences used are shown in the following table:

FIG. 12 shows a map of WB expression for different lengths of truncated proteins. Sampling and detecting for different induction time, 3 days and 4 days. The number of lanes contained in the horizontal line on the graph is actually 2, and WB detection is performed on day 3 and day 4, respectively. Lanes: 1-12, Drosophila S2 cell induction medium supernatant encoding truncated-delta, truncated-alpha, truncated-beta, truncated-gamma, truncated-1, truncated-8, truncated-4, truncated-5, truncated-3, truncated-2, truncated-7, and truncated-6 fragment protein plasmids.

As a result, as shown in FIG. 12, the 12 truncated proteins were not expressed, and the expression level of each protein fragment varied with the increase in induction time. In order to obtain cell lines with high expression of truncated proteins, three higher cell lines (truncated-gamma, truncated-1 and truncated-8) were selected for mass culture and purification.

Three stable transgenic cell lines (truncated-gamma, truncated-1 and truncated-8) with higher expression levels were expanded and induced to express and purify the three truncated proteins according to the method of example 4. SDS-PAGE and WB were examined for protein expression.

FIG. 13 shows the examination of the purification effect of recombinant ERPV-ATIp protein expression of different lengths. Wherein FIG. 13-A is SDS-PAGE detection; FIG. 13-B shows WB detection. The order of SDS-PAGE is identical to that of WB, and the eluents are found in truncated-1, truncated-8 and truncated- γ, respectively.

As shown in FIG. 13, the truncated protein truncated-gamma was purified much more effectively than the other two proteins. The estimated molecular weight of truncated-gamma protein is about 47kDa, and SDS-PAGE and WB show a protein molecular weight greater than 47kDa, indicating the presence of post-translational modification of truncated-gamma. The eluate E3 of the truncated protein truncated-gamma has a high protein purity.

Furthermore, as a result of comparing SDS-PAGE and WB, it was found that the expression levels of truncated-1 (estimated 52kDa) and truncated-8 (estimated 85kDa) were very low, and that SDS-PAGE could not detect protein expression but WB could detect protein expression.

The above results show that the truncated protein truncated-gamma is an unexpectedly particularly suitable truncated protein for expression using Drosophila S2 cells, and retains a conformation similar to that of the original ATIp protein.

The above experiments of the present invention have shown that monoclonal antibodies or polyclonal antibodies (e.g., antisera) prepared by immunizing mice with the truncated protein as an immunogen can effectively bind ATIp.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. An antigenic polypeptide, characterized in that the sequence of the antigenic polypeptide is shown as 497-919 site in SEQ ID NO. 2.

2. The antigenic polypeptide of claim 1 which is recombinantly expressed in a eukaryotic host cell.

3. The antigenic polypeptide of claim 2 where the eukaryotic host cell is a Drosophila S2 cell.

4. A test kit comprising the antigenic polypeptide of claim 1.

5. The test kit of claim 4, further comprising an antibody specific for the antigenic polypeptide of claim 1.

6. The test kit of claim 4, wherein the test kit further comprises a test strip.

7. The test kit of claim 6, wherein the test strip comprises:

the device comprises a sample pad, a combination pad, a reaction membrane, an absorption pad and a back lining, wherein the reaction membrane is provided with a detection area and a quality control area;

the detection region is immobilized with the antigen polypeptide of claim 1, and the conjugate pad is immobilized with an antibody against the antigen polypeptide of claim 1.