CN106188281B - The preparation and application of anti-norovirus GII.4 type source of mouse monoclonal antibody - Google Patents

Abstract

The invention discloses the preparations and application of a kind of anti-norovirus GII.4 type source of mouse monoclonal antibody.The experimental results showed that the monoclonal antibody has the high neutralization activity to norovirus GII.4, and the cross reaction with GI.1 virus-like particle is not present in the antibody, being capable of specific recognition GII.4 virus-like particle.

Description

Preparation and application of anti-Norovirus GII.4 murine monoclonal antibody

technical field

The invention belongs to the field of biomedicine, in particular, the invention relates to the preparation and application of an anti-Norovirus GII.4 mouse monoclonal antibody.

Background technique

Noroviruses (NoVs) are one of the major pathogens causing sporadic cases and large outbreaks of acute gastroenteritis, and Noroviruses can infect people of all ages. Although the symptoms caused by norovirus infection are generally mild and self-limiting, and the course of the disease lasts for about 1-3 days, it can still cause more serious symptoms and even death in children, the elderly, and people with immunocompromised . According to the amino acid sequence of VP1 capsid protein, Norovirus can be divided into 6 genomes (G1-GVI), but only GI, GII and GIV can infect humans. Human norovirus infection is mainly caused by norovirus GII, and large outbreaks are mostly caused by norovirus GII.4. Norovirus infection caused by variants of the GII.4 virus strain accounts for about 55%-85% of gastroenteritis cases worldwide, and those cases that are more serious and require hospitalization or even death are mostly caused by Norovirus. Caused by virus GII.4.

Norovirus is prevalent in both developed and developing countries, bringing serious economic losses to all countries, and posing a great threat to the health of children and the elderly. So far, there are no preventive vaccines and specific therapeutic drugs on the market. Norovirus lacks a cell culture model, and there is no small animal model, which has brought great obstacles to the research of vaccines and antiviral drugs.

Therefore, those skilled in the art are committed to developing anti-norovirus drugs with good clinical application prospects.

Contents of the invention

The purpose of the present invention is to provide the preparation and application of an anti-Norovirus GII.4 mouse monoclonal antibody.

The first aspect of the present invention provides a heavy chain variable region of an antibody, and the heavy chain variable region includes the following three complementarity determining regions (CDRs):

CDR1 shown in SEQ ID NO:8,

CDR2 shown in SEQ ID NO: 9, and

CDR3 shown in SEQ ID NO: 10;

Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:6.

The second aspect of the present invention provides a heavy chain of an antibody, the heavy chain having the heavy chain variable region and the heavy chain constant region as described in the first aspect of the present invention.

In another preferred example, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO.:3.

In the third aspect of the present invention, there is provided a light chain variable region of an antibody, the light chain variable region has a complementarity determining region CDR selected from the group consisting of:

CDR1' shown in SEQ ID NO: 14,

CDR2' shown in SEQ ID NO: 15, and

CDR3' shown in SEQ ID NO: 16;

Preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO:7.

The fourth aspect of the present invention provides an antibody light chain, which has the light chain variable region and the light chain constant region as described in the third aspect of the present invention.

In another preferred example, the light chain amino acid sequence of the antibody is shown in SEQ ID NO.:5.

In the fifth aspect of the present invention, an antibody is provided, the antibody has:

(1) The heavy chain variable region according to the first aspect of the present invention; and/or

(2) The light chain variable region as described in the third aspect of the present invention;

Alternatively, the antibody has:

The heavy chain according to the second aspect of the present invention; and/or the light chain according to the fourth aspect of the present invention.

The sixth aspect of the present invention provides a recombinant protein, the recombinant protein has:

(i) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the fourth aspect of the present invention A light chain according to the above aspect, or an antibody according to the fifth aspect of the present invention; and

(ii) Optional tag sequences to aid in expression and/or purification.

The seventh aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the fourth aspect of the present invention A light chain according to the above aspect, or an antibody according to the fifth aspect of the present invention; or

(2) The recombinant protein as described in the sixth aspect of the present invention.

In another preferred example, the polynucleotide sequence has a polynucleotide sequence as shown in SEQ ID NO.:2 and/or SEQ ID NO.:4.

The eighth aspect of the present invention provides a vector containing the polynucleotide described in the seventh aspect of the present invention.

The ninth aspect of the present invention provides a genetically engineered host cell, which is characterized in that it contains the vector described in the eighth aspect of the present invention or the polynucleotide described in the seventh aspect of the present invention is integrated in the genome.

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

The antibody of the fifth aspect of the present invention.

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

In the eleventh aspect of the present invention, an immunoconjugate is provided, which comprises:

(a) the antibody described in the fifth aspect of the present invention or the recombinant protein described in the sixth aspect of the present invention; and

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

The twelfth aspect of the present invention provides a pharmaceutical composition, said composition comprising the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, or the recombinant protein described in the eleventh aspect of the present invention. the immunoconjugates described above; and

pharmaceutically acceptable carrier.

The thirteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

(a) cultivating the host cell described in the ninth aspect of the present invention under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, and the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the polyacrylamide gel electrophoresis analysis of the purified anti-GII.4 mAb. The five purified antibodies were treated with loading buffer containing a reducing agent and loaded onto a 12% polyacrylamide gel for electrophoresis, and the protein bands were displayed by Coomassie brilliant blue staining. M, protein molecular weight standard; 1, D11 monoclonal antibody; 2, G9 monoclonal antibody; 3, 2D8 monoclonal antibody; 4, 7D8 monoclonal antibody; 5, 8E1 monoclonal antibody

Figure 2 shows the binding ability of monoclonal antibodies to different antigens identified by enzyme-linked immunosorbent assay (Elisa). Each well of the Elisa plate was coated with 100ngGII.4(A) or GI.1(B) virus-like particles, each well was added with different concentrations of purified monoclonal antibody and incubated at 37°C for 2 hours, and then HRP-labeled anti- Mouse secondary antibody was incubated. Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an irrelevant control. Each point in the figure shows the mean and standard deviation of OD450nm measured in triplicate samples.

Figure 3 shows Western blot analysis. After the GII.4 virus-like particles were treated, they were electrophoresed in a 12% polyacrylamide gel, then transferred to PVDF membrane, and hybridized with purified monoclonal antibodies. M, protein molecular weight standard; 1, D11 monoclonal antibody; 2, G9 monoclonal antibody; 3, 2D8 monoclonal antibody; 4, 7D8 monoclonal antibody; 5, 8E1 monoclonal antibody; 6, mouse anti-GII.4 virus-like particle clone antibody.

Figure 4 shows the detection of GI.1 and GII.4 VLPs by sandwich Elisa. Each well of the Elisa plate was coated with 50ul 1:5000 diluted rabbit anti-GII.4 (A) or rabbit anti-GI.1 (B), and each well was added with different concentrations of GII.4 virus-like particles (A) and GI.1 virus-like particles (B) were incubated at 37°C for 2 hours, then 10 ng of purified monoclonal antibody was added to each well, and finally incubated with HRP-labeled anti-mouse secondary antibody. Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an irrelevant control.

Figure 5 shows the activity of the purified monoclonal antibody to inhibit the interaction between GII.4 virus-like particles and PGMIII detected by alternative neutralization assay. Coat 50ul 10ug/ml PGMII per well on the Elisa plate, incubate different concentrations of monoclonal antibodies and 0.5ug/ml GII.4 virus-like particles at room temperature for 1 hour, add to the Elisa plate, then add rabbit anti-GII.4, Finally, incubation was performed with HRP-labeled anti-rabbit secondary antibody. Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an irrelevant control.

Figure 6 shows the identification of recombinantly expressed monoclonal antibodies. Each well of the Elisa plate was coated with 100ngGII.4 virus-like particles, each well was added with different concentrations of purified monoclonal antibody and incubated at 37°C for 2 hours, followed by incubation with HRP-labeled anti-mouse secondary antibody. The culture supernatant of cells not transfected with plasmid was used as blank control. The histogram in the figure shows the mean and standard deviation of OD450nm measured in three replicate samples.

Detailed ways

The inventor obtained a mouse-derived monoclonal antibody against Norovirus GII.4 through extensive and in-depth research. Experimental results show that the monoclonal antibody has extremely high neutralizing activity against Norovirus GII.4 , and the antibody has no cross-reaction with GI.1 virus-like particles, and can specifically recognize GII.4 virus. The present invention also provides the application of the above-mentioned monoclonal antibody.

Specifically, this study used GII.4 virus-like particles as an immunogen to prepare a monoclonal antibody 8E1 that can specifically recognize GII.4. Methods such as ELISA and surrogate neutralization experiments show that the antibody can be used to detect and analyze GII.4 sensitively, and more importantly, it also has strong neutralizing activity.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, the scope of the present invention being limited only by the appended claims.

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. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are exemplified herein.

Norovirus GII.4

Norovirus GII.4 belongs to the Norovirus genus GII type and is the main pathogen causing norovirus outbreaks. Norovirus is prevalent in both developed and developing countries, bringing serious economic losses to all countries, and posing a great threat to the health of children and the elderly. So far, there are no preventive vaccines and specific therapeutic drugs on the market.

The present invention uses recombinant GII.4 virus-like particles as immunogen to prepare GII.4 monoclonal antibody. The antibody prepared by the present invention can not only be used as a tool for laboratory detection, but also be a reliable candidate for the preparation of therapeutic humanized monoclonal antibody and a useful reagent for the development of diagnostic methods.

In the present invention, norovirus GII.4 VP1 is used to prepare virus-like particles, and its amino acid sequence is:

MASSDANPSDGSAANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIVVDVRQLEPVLIPLPDVRNNFYHYNQSNDPTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVESRTKPFSVPVLTVEEMTNSRFPIPLEKLFTGPSSAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHITGSRNYTMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGVLTQTTRTDGSTRGHKATVYTGSADFAPKLGRVQFETDTDHDFEANQNTKFTPVGVIQDGSTTHRNEPQQWVLPSYSGRNTPNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMDLDCLLPQEWVQYFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRAL(SEQ ID NO.:1，GenBank ID:KC631827.1)，309位丝氨酸(Ser)突变为天冬酰胺(Asn)。

In the present invention, norovirus GI.1VP1 is used to prepare virus-like particles, and its amino acid sequence is:

MASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGVLSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR(SEQ ID NO.:20，NP_056821.2)

Antibody

As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions. Each light chain has a variable region (VL) at one end and a constant region 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 . Specific amino acid residues form the interface between the variable domains of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. 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 domains are called the framework regions (FR). The variable domains of native heavy and light chains each contain four FR regions that are roughly in a β-sheet configuration connected by three CDRs that form connecting loops, in some cases forming partial β-sheet structures. The CDRs in each chain are brought into close proximity by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.

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. Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, 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, ie, the individual antibodies comprised within the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the identity of the antibody, which is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring any particular method for producing the antibody.

The present invention also includes the monoclonal antibody with the corresponding amino acid sequence of the anti-GII.4 virus monoclonal antibody, the monoclonal antibody with the variable region chain of the anti-GII.4 virus monoclonal antibody, and the monoclonal antibody with these chains Other proteins or protein conjugates and fusion expression products. Specifically, the present invention includes any protein or protein conjugates and fusion expression products (ie immunoconjugates and fusion expression products) having light chains and heavy chains containing hypervariable regions (complementarity determining regions, CDRs), as long as the 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 (cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules and the anti-GII.4 virus monoclonal Conjugates formed by combining antibodies or fragments thereof. The present invention also includes cell surface markers or antigens combined with the anti-GII.4 virus monoclonal antibody or fragments thereof.

The present invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') ₂ fragments; antibody heavy chains; antibody light chains.

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

As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes the following three CDRs:

CDR1, its amino acid sequence is GYSFTDYYMH (SEQ ID NO: 8), its coding nucleotide sequence is, GGTTACTCATTCACTGACTACTACATGCAC (SEQ ID NO.: 11);

CDR2, its amino acid sequence is YIDPFNGDTGYNQKFKV (SEQ ID NO.: 9), its coding nucleotide sequence is TATATTGATCCTTTCAATGGTGATACTGGCTACAACCAGAAATTCAAGGTC (SEQ ID NO.: 12);

The amino acid sequence of CDR3 is DDWGSPFSY (SEQ ID NO.: 10), and the coding nucleotide sequence is GATGACTGGGGATCCCCGTTTTTCTTAC (SEQ ID NO.: 13).

In another preferred example, the amino acid sequence of the heavy chain variable region is:

EIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIGYIDPFNGDTGYNQKFKVRATLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQGTLVTVSA (SEQ ID NO.: 6).

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

In another preferred example, the heavy chain amino acid sequence of the antibody is:

MGWSWIFLFLLSGTAGVHSEIQLQQSGPELMKPGASVKISCKASGYSFTDYYMHWVKQSHGKSLEWIGYIDPFNGDTGYNQKFKVRATLTVDESSTTAYMHLSSLTSDDSAVYYCARDDWGSPFSYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENCKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO.:3)

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

In a preferred embodiment of the present invention, the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the following group:

CDR1', its amino acid sequence is RASHDIGSNLD (SEQ ID NO: 14), its coding nucleotide sequence is, CGGGCAAGTCATGACATTGGTAGTAACTTAGAC (SEQ ID NO.: 17);

CDR2', its amino acid sequence is ATSTLDS (SEQ ID NO: 15), its coding nucleotide sequence is, GCCACATCCACTTTAGATTCT (SEQ ID NO.: 18)

CDR3', its amino acid sequence is LQYASSPRT (SEQ ID NO: 16), its coding nucleotide sequence is, CTACAATATGCTAGTTCCTCGGACG (SEQ ID NO.: 19)

In another preferred example, the amino acid sequence of the light chain variable region is:

DIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLIYATSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIK (SEQ ID NO.: 7).

In a preferred embodiment of the present invention, the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.

In another preferred example, the light chain amino acid sequence of the antibody is:

MRAAAQIFGFLLLLFPGTRCDIQMTQSPSSLSASLGESVSLTCRASHDIGSNLDWLQQEPDGTIKRLIYATSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFVEYYCLQYASSPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO.:5)

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably, and all refer to antibodies specifically binding to anti-GII.4 virus, for example having a heavy chain (such as SEQ ID NO.:3 amino acid sequence) and/or light chain (such as the amino acid sequence of SEQ ID NO.:5) protein or polypeptide. They may or may not contain starting methionine.

In another preferred example, the antibody is an anti-GII.4 virus mouse or human-mouse chimeric monoclonal antibody, and its heavy chain constant region and/or light chain constant region can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2 or the like.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. Specifically, the present invention includes any protein or protein conjugates and fusion expression products (ie immunoconjugates and fusion expression products) having heavy chains and light chains containing variable regions, as long as the variable regions are compatible with the antibody of the present invention The variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous.

In general, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs), which are separated into four framework regions (FRs), four The amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a ring structure, and the β sheets formed by the FRs in between are close to each other in the spatial structure. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute FR or CDR regions can be determined 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 because at least some of them are involved in binding antigen. Accordingly, the present invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs, as long as the CDRs are more than 90% (preferably more than 95%, most preferably 98%) identical to the CDRs identified herein. Homology above).

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

As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It 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 mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

The antibody of the present invention refers to a polypeptide that has anti-GII.4 virus-binding activity and includes the above-mentioned CDR region. The term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change 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, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to substantially full-length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, 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 of an antibody of the invention.

In the present invention, "conservative variants of the antibody of the present invention" refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention. An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A

The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 2 or 4 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but with the same amino acid sequence as SEQ ID NO.: 3, 5, 6, 7, 8, 9, 10, Nucleic acid sequences that differ from the coding region sequences shown in 14, 15, and 16.

A polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .

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

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with Denaturing agent, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.:12 and/or SEQ ID NO.:22.

The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) involved in the present invention include biomolecules in an isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.

Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or products capable of producing detectable enzymes.

The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention can be directly used for binding GII.4 virus-like particles, and thus can be used for preventing and treating noroviruses (NoVs) that cause acute gastroenteritis. In addition, other therapeutic agents may also be used concomitantly.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and pharmaceutical acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually 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 about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

hybridoma cell line

The present invention also provides a hybridoma cell strain capable of producing the anti-GII.4 virus monoclonal antibody of the present invention; preferably, the present invention provides a high-titer hybridoma cell strain directed against the anti-GII.4 virus monoclonal antibody.

After obtaining the hybridoma producing the anti-GII.4 virus monoclonal antibody of the present invention, those skilled in the art can conveniently use the hybridoma cell line to prepare antibodies. In addition, those skilled in the art can easily know the structure of the antibody of the present invention (such as the heavy chain variable region and light chain variable region of the antibody), and then prepare the monoclonal antibody of the present invention by recombinant methods.

Preparation of monoclonal antibodies

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture 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, Rockville, MD, USA). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications (Monoclonal Antibodies Production Techniques and Applications), pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

The culture medium in which the hybridoma cells were grown is assayed for the production of monoclonal antibodies with the desired specificity, eg, by an in vitro binding assay, eg, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of antibody-expressing cells can be detected by FACS. The hybridoma clones can then be subcloned by a limiting dilution step and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59-103 Page). Suitable media for use for this purpose include, for example, DMEM or RPMI-1640 media. In addition, hybridoma cells can grow in animals as ascites tumors.

Monoclonal antibodies secreted by subclones are suitably isolated from culture medium, ascitic fluid or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxyphospho Limestone chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention provides a monoclonal antibody against GII.4 virus. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant of the hybridoma cell culture was taken, and the IgG was crudely extracted by saturated ammonium sulfate precipitation, and then the crudely extracted antibody was purified by an affinity chromatography column (Protein G-Sephrose).

In a preferred solution of the present invention, the monoclonal antibody is prepared by a method for producing monoclonal antibody from ascites of Balb/C mice. About 10 hybridoma cells were inoculated into the peritoneal cavity of the sensitized mice, and the abdomen was obviously swollen about 10 days later. Ascitic fluid was extracted, and after crude extraction by saturated ammonium sulfate precipitation, the crude antibody was purified by affinity chromatography (Protein G-Sephrose).

Labeled immunoglobulin (antibody)

In a preferred example of the present invention, the immunoglobulin has a detectable label. More preferably, the label is selected from the group consisting of colloidal gold label, horseradish peroxidase label, colored label or fluorescent label.

Colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody against GII.4 virus is labeled with colloidal gold to obtain the monoclonal antibody labeled with colloidal gold.

The anti-GII.4 virus monoclonal antibody of the invention has good specificity and high titer.

Test board and its material

The detection board of the present invention can be made of commonly used detection board materials in the field, and is made by a conventional detection board preparation method.

The present invention detects the immunoassay board of GII.4 virus, comprises test strip and the supporting plate of supporting test strip, as can adopt PVC polyester glue board etc.; Described test strip is made of filter sample paper, chromatographic material, nitrocellulose Membrane and absorbent paper are overlapped sequentially, and the overlapping parts can be fixedly connected by conventional methods, such as adhesive tape; wherein: the chromatography material is pre-coated with colloidal gold-labeled or colored-labeled anti-GII.4 virus monoclonal antibody or polyclonal Antibody, preferably colloidal gold-labeled anti-GII.4 virus monoclonal antibody, adsorption detection line and quality control line on nitrocellulose membrane;

In a preferred scheme: the colloidal gold-labeled anti-GII.4 virus monoclonal antibody pre-coated on the chromatographic material is carried out by using a concentration of 0.5-1.5mg/ml colloidal gold-labeled anti-GII.4 virus monoclonal antibody solution Pre-coated, the coating amount is 50 μl/cm ² ; the preferred concentration is 0.5 or 1.5 mg/ml, 50 μl/cm ² ;

Detection method and result judgment

Lay the detection plate flat, drop the sample on the filter paper, the sample is about 120 μl, and observe the chromatographic results within 3 to 5 minutes. The result is judged by the location of the streaks that appear.

Negative: There are obvious color bands in both the quality control area and the detection area, indicating negative;

Positive: Only obvious color bands appear in the quality control area, but no color bands in the detection area, which is positive;

Invalid: There is no color band in the quality control area and detection area, or there is no color band in the quality control area but a color band appears in the detection area, indicating that the detection method is wrong or the detection board is deteriorated or invalid, and the detection board should be replaced for testing.

Methods and Samples

The present invention relates to a method for the detection of norovirus in samples lysed with cells and/or tissues. The steps of the method are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; detect the level of GII.4 virus in the dissolved sample. The sample used in the method of the present invention may be any sample including cells present in a cell preservation solution, as used in liquid-based cell assays.

Reagent test kit

The present invention also provides a kit containing the antibody of the present invention (or a fragment thereof) or the detection plate of the present invention. In a preferred example of the present invention, the kit further includes a container, an instruction manual, a buffer agent etc.

The present invention further designs a detection kit for detecting the level of GII.4 virus, which includes an antibody that recognizes anti-GII.4 virus, a lysis medium for dissolving samples, common reagents and buffers required for detection, such as various buffer, detection label, detection substrate, etc. The test kit may be an in vitro diagnostic device.

The present invention further designs and develops a test kit for the diagnosis and evaluation of GII.4 virus infection-related conditions from solution samples, and the test kit can detect GII.4 viruses present in the sample solution, wherein the cell preservation solution for preserving samples can be Such as cell preservation solution in liquid-based cell assay.

The anti-GII.4 virus monoclonal antibody of the present invention has the advantages of high affinity, high specificity, etc., and can be widely used in the detection field of preparing GII.4 virus, such as the preparation field of detection reagents or detection equipment, etc., in specificity, sensitivity Compared with traditional detection methods or detection reagents, it has significant advantages in terms of detection rate and detection rate.

The main advantages of the present invention are:

(1) The monoclonal antibody 8E1 of the present invention can specifically recognize norovirus-like particles;

(2) The monoclonal antibody 8E1 of the present invention can specifically bind Norovirus GII.4, and has no cross-reaction with Norovirus GI.1, thereby realizing the anti-norovirus GI.1 and Norovirus GII.4 Identification.

(3) The monoclonal antibody 8E1 of the present invention has strong neutralizing activity against Norovirus.

Below in conjunction with specific embodiment, further elaborate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate detailed condition in the following examples, generally according to conventional conditions such as people such as Sambrook, molecular cloning: the condition described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

Materials and methods

1 Antigen preparation and mouse immunization

Using the baculovirus-insect expression system, virus-like particles were prepared by expressing Norovirus GII.4 VP1 ^[1] . After mixing 10 ug of virus-like particles (50 ul volume) with an equal volume of aluminum adjuvant (500 ug), the female Balb/c mice were immunized intraperitoneally for 6 weeks, and immunized once at 0 weeks, 2 weeks, and 4 weeks. At week 6, mouse serum was taken to detect the neutralization titer. At week 7, a mouse with the highest neutralizing titer was boosted with 15ugGII.4 VLP through the tail vein. After 3 days, the mouse spleen was taken for the preparation of hybridoma cells.

2 Preparation and screening of hybridoma cell lines

Three days after the tail vein booster immunization of the mice, the spleen cells of the mice were fused with myeloma cells SP2/0 through PEG1500 to prepare hybridoma cells. Nine days later, antibodies secreted specifically against GII.4 virus-like particles were screened by enzyme-linked immunosorbent assay. In short, GII.4 virus-like particles were coated on a 96-well plate with 100ng per well, overnight at 4°C, blocked with PBST containing 5% skimmed milk, and incubated at 37°C for 2 hours by adding 50ul of hybridoma culture medium to each well , then incubated with HRP-labeled secondary antibody (sigma) for 1 hour, and finally carried out color reaction, and read the absorbance value of OD450.

3 Ascites preparation and antibody purification

Female Balb/c mice were intraperitoneally injected with 500ul of liquid paraffin oil, and two weeks later, each mouse was intraperitoneally injected with 300,000 hybridoma cells. After 7 days, the ascites was collected with a 12-gauge needle, centrifuged at 10,000 rpm for 10 minutes, the upper layer of oil and the lower layer of sediment were removed, and the clarified ascites was taken for antibody purification. According to the instructions, ascitic fluid was purified using HiTrap HiTrapTM Protein G affinity column (GEhealth care) to obtain antibodies.

4 ELISA identification of monoclonal antibodies

100 ng GI.1 or GII.4 virus-like particles per well were used to coat 96-well Elisa plates at 4°C overnight to identify the binding ability of monoclonal antibodies. After the Elisa plate was blocked by PBST containing 5% skimmed milk at 37°C for 1 hour, different concentrations (5ug/ml, 2.5ug/ml, 1.25ug/ml and 0.625ug/ml) were added to the monoclonal antibody 37 at 50ul per well. Incubate at ℃ for 2 hours, then incubate with HRP-labeled anti-mouse secondary antibody, and finally read the absorbance value OD450.

5 Polyacrylamide gel electrophoresis and western blot analysis

After the protein sample was mixed with SDS-PAG loading buffer, it was boiled for 10 min, and the protein sample was separated by 12% polyacrylamide gel. The protein bands were displayed by Coomassie brilliant blue staining or the protein was transferred to PVDF membrane for western blot analysis. The monoclonal antibody was diluted into PBST containing 1% skim milk at a final concentration of 1 ug/ml. The mouse anti-GII.4 polyclonal antibody was diluted 1:1000, then incubated with HPR-labeled mouse secondary antibody (sigma), and finally recorded with LAS-400 luminescence image analyzer.

6 Preparation of rabbit polyclonal antibody against GI.1 virus-like particle or GII.4 virus-like particle

Mix GI.1 virus-like particles or GII.4 virus-like particles with Freund's complete adjuvant in equal volumes to fully emulsify, and subcutaneously inject 150ug/rat into healthy rabbits. After 3 weeks and 6 weeks, 150ug of GI.1 virus-like particles or GII.4 virus-like particles were mixed with an equal amount of Freund's incomplete adjuvant, fully emulsified and boosted. Serum was collected 2 weeks after the last immunization, and stored at -80°C after aliquoting.

7 sandwich Elisa detection of GI.1 and GII.4 virus-like particles

Use rabbit polyantiserum against GI.1 virus-like particles (preparation method as above) and rabbit polyantiserum against GII.4 virus-like particles (preparation method as above) 1:5000 dilution (50ul/well) overnight at 4°C Coat a 96-well Elisa plate, block the Elisa plate with PBST containing 5% skimmed milk at 37°C for 2 hours, then add the virus-like particles to the Elisa plate, starting with 40ng/50ul/well, 2-fold ratio dilution to 12 concentrations , incubate at 37°C for 2 hours, then incubate VLP-specific monoclonal antibody 10ng/50ul/well at 37°C for 1 hour, then incubate with HPR-labeled mouse secondary antibody, and finally read the absorbance value OD450.

8 In vitro alternative neutralization experiments

Coat 96-well Elisa plate with 10ug/ml porcine gastric mucin III (PGM) (Shanghai Yuanmu Biotechnology Co., Ltd.) (50ul/well) at room temperature, and block the Elisa plate overnight at 4°C with PBST containing 5% skimmed milk Backup. Start with GII.4 virus-like particle-specific monoclonal antibody 8ug/ml, dilute it 2 times, and incubate with an equal volume of 0.5ug/ml GII.4 virus-like particles at room temperature for 1 hour, then add to the PGM-coated 96 Well Elisa plate, incubate at room temperature for 1 hour, then add rabbit anti-GII.4 virus-like particle polyclonal antibody (preparation method as above) 1:1000 dilution solution and incubate at 37°C for 1 hour, then use HPR-labeled rabbit secondary antibody (sigma ) for incubation, and finally read the absorbance value OD450.

9 Gene sequence amplification of monoclonal antibody and construction of expression vector

First, the cells of the hybridoma cell line were extracted with Trizol reagent for total RNA, and then the heavy chain and light chain full-length genes were amplified according to the instructions of the 5'RACE kit. Use PCR amplification method to introduce HindIII and EcoRI restriction sites at the 5' end and 3' end of the heavy chain and light chain, respectively, and clone the amplified heavy chain and light chain genes into pGEM-T In (Promage), the positive clones were screened for sequencing, and then the clones with the correct sequence were digested with HindIII and EcoRI, and after the target fragment was purified by agarose gel electrophoresis, they were used with the isodigested plasmid pcDNA3.1 (Promage) T4DNA ligase was connected to construct eukaryotic expression vectors pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L).

Identification of recombinant expression of 10 monoclonal antibody genes

Using liposome method to co-transfect pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L) into CHO cells, collect the culture supernatant after 72 hours for analysis, and use ELISA to determine the expression of the antibody in the culture supernatant: Cover the plate with GI.1 virus-like particles, block with PBST containing 5% milk at 37°C for 1 hour, add different dilutions of the culture supernatant to be tested and incubate at 37°C for 2 hours, then use HRP-labeled anti-mouse IgG secondary antibody Incubate, and finally read the absorbance value OD450.

Example 1 Screening of hybridoma cells secreting GII.4 specific antibody

Spleen cells from mice immunized with GII.4 virus-like particles were used to prepare hybridoma cells. The hybridoma cell supernatant was screened by Elisa test, so as to obtain a hybridoma cell line capable of secreting virus-binding ability. Finally, five monoclonal antibodies with excellent binding ability were screened out, all of them could bind GII.4 VLP. Subtype identification showed that G9, D11, 7D8 and 8E1 belonged to IgG1, and 2D8 belonged to IgG3.

Table 1. Identification of hybridoma cell lines secreting monoclonal antibody

hybridoma cell line heavy chain light chain Binding ability to GII.4VLP* G9 IgG1 kappa +++ D11 IgG1 kappa +++ 2D8 IgG3 kappa +++ 7D8 IgG1 kappa +++ 8E1 IgG1 kappa +++

All samples used for analysis were 50ul hybridoma cultured cells.

*, +: OD450nm > 0.15; ++: OD450nm > 0.3; +++: OD450nm > 0.5.

Example 2 Specific Analysis of Anti-GII.4 Monoclonal Antibody

The purity and integrity of GII.4 mAb purified from ascitic fluid were first identified by SDS-PAGE. Figure 1 shows that the heavy chain and light chain of five mAbs are about 50KD and 25KD respectively. Next, the reactivity of the monoclonal antibody with different antigens, including GI.1 virus-like particles and GII.4 virus-like particles, was detected by Elisa method. Figure 2 shows that G9, D11, 2D8, and 8E1 can specifically recognize GII.4 virus-like particles, and there is no cross-reaction with GI.1 virus-like particles, while some monoclonal antibodies (eg, 7D8), simultaneously with GI.1 virus The combination of GII.4 virus-like particles and GII.4 virus-like particles cannot specifically recognize GII.4 viruses.

Finally, the binding of monoclonal antibody to GII.4 was analyzed by Western blot. Figure 3 shows that among the five antibodies: D11, G9 and 8E1 can recognize the natural structure of GII.4 virus-like particles, but cannot recognize the denatured GII. 4 VLPs, suggesting that the epitopes recognized by D11, G9 and 8E1 may be conformational epitopes.

Example 3 Monoclonal antibody-based sandwich Elisa can specifically and sensitively detect GI.1 and GII.4 virus-like particles

The minimum detection limit of the monoclonal antibody to virus-like particles was determined by sandwich Elisa (when OD450nm>0.15, it was judged as positive). Figure 4 shows that G9, D11, 7D8, and 8E1 monoclonal antibodies can all detect GII.4 virus-like particles with specificity and sensitivity, and the minimum detection limits are: 0.3125ng, 0.3125ng, 0.3125ng, and 0.625ng, respectively.

Example 4 Potential neutralizing activity of monoclonal antibodies

Histoblood group antigen (HBGA) is a carbohydrate present on mucosal tissues and red blood cells and is a receptor required for norovirus infection. The binding inhibition assay for HBGA is widely used as a surrogate assay for antibody-mediated norovirus neutralization. Porcine gastric mucin Ⅲ (PGM) contains HBGA, which has been verified as an alternative neutralization test ^[2] . The potential neutralizing activities of five mAbs, G9, D11, 2D8, 7D8 and 8E1, were detected by alternative neutralization assays. Figure 5 shows that D11, G9, and 8E1 have good neutralizing activity against GII.4, and their EC50s for preventing virus-like particles from binding to PGM are: 0.0979ug/ml, 0.6948ug/ml, and 0.04357ug/ml, respectively. The above results show that the neutralizing activity of 8E1 against GII.4 is far superior to other antibody strains. The neutralizing activity of 8E1 monoclonal antibody against GII.4 is more than twice that of the D11 antibody strain and about 16 times that of the G9 antibody strain.

Example 5 Gene Sequence Analysis of Monoclonal Antibody

The heavy chain and light chain sequences of the cloned 8E1 monoclonal antibody are as follows (the underlined part is the signal peptide sequence, the italicized part is the variable region sequence, is the constant region sequence):

8E1 monoclonal antibody heavy chain nucleotide sequence:

ATGGGATGGAGCTGGATCTTTCTCTTCCTTCTGTCAGGAACTGCAGGTGTCCACTCT (SEQ ID NO.:2)

8E1 monoclonal antibody heavy chain amino acid sequence:

MGWSWIFLFLLSGTAGVHS (SEQ ID NO.: 3)

8E1 monoclonal antibody light chain nucleotide sequence:

ATGAGGGCTGCTGCACAGATTTTTGGCTTCTTGTTGCTCTTGTTTCCAGGTACCAGATGT (SEQ ID NO.:4)

8E1 monoclonal antibody light chain amino acid sequence:

MRAAAQIFGFLLLLFPGTRC (SEQ ID NO.:5)

Further analysis of the 8E1 monoclonal antibody heavy chain variable region and light chain variable region sequences, the amino acids of the 8E1 monoclonal antibody heavy chain variable region are as follows (the heavy chain CDR region is underlined):

EIQLQQSGPELMKPGASVKISCKAS GYSFTDYYMH WVKQSHGKSLEWIG YIDPFNGDTGYNQKFKV RATLTVDESSTTAYMHLSSLTSDDSAVYYCAR DDWGSPFSY WGQGTLVTVSA (SEQ ID NO.: 6)

The heavy chain variable region described above belongs to the IGHV1 subgroup.

The amino acids of the light chain variable region of 8E1 monoclonal antibody are as follows (underlined is the heavy chain CDR region):

DIQMTQSPSSLSASLGESVSLTC RASHDIGSNLD WLQQEPDGTIKRLIY ATSTLDS GVPKRFSGSRSGSDYSLTISSLESEDFVEYYC LQYASSPRT FGGGTKLEIK (SEQ ID NO.: 7)

The light chain variable region described above belongs to the IGKV9 subgroup.

The amino acid sequence and nucleotide sequence of each CDR region are summarized in Table 2.

Table 2

Example 6 Recombinant expression and identification of monoclonal antibody genes

In order to verify whether the cloned 8E1 monoclonal antibody gene is correct, the coding sequences of the heavy chain and light chain were inserted into pcDNA3.1 to construct expression vectors pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L), Then CHO cells were co-transfected, and whether there was an antibody specifically binding to GII.4 virus-like particles in the cell supernatant was detected by ELISA. Figure 6 shows that the supernatant of cells expressing the 8E1 monoclonal antibody sequence has a high binding signal, and it is related to the dilution factor of the supernatant; while the supernatant of the control cells that have not been transfected with the relevant plasmid has no binding signal no matter whether it is diluted or not. This result shows that the 8E1 monoclonal antibody of the present invention can be successfully expressed in host cells.

discuss

The present invention obtains the monoclonal antibody 8E1 capable of specifically binding to GII.4 virus particles, which has strong potential neutralizing activity, and can be used as a therapeutic monoclonal antibody drug or as a detection reagent after humanization. Through sandwich ELISA detection, the minimum detection limit of monoclonal antibody 8E1 to GII.4 virus-like particles is 0.625ng, which provides a favorable theoretical basis for the development of these monoclonal antibodies into norovirus detection kits. However, the EC50 of the monoclonal antibody 8E1 against GII.4 obtained in the present invention for the interaction between GII.4 and PGM is 0.04357ug/ml, which is far superior to other monoclonal antibodies. Moreover, Western analysis showed that the epitope recognized by the 8E1 monoclonal antibody is a conformational epitope, which makes 8E1 only recognize virus particles with the correct spatial conformation, but not depolymerized and denatured virus particles, which can be used for reliable virus particle quantification.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

references

1. Huhti, L., et al., A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles. Arch Virol, 2010.155(11): p.1855-8

2. Lindesmith, L.C., et al., Immunogenetic mechanisms driving norovirus GII.4 antigenic variation. PLoS Pathog, 2012.8(5): p.e1002705.

Claims (13)

1. An antibody, characterized in that the antibody has a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region includes the following three CDRs: CDR1 shown in SEQ ID NO:8, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 10; And, the light chain variable region includes the following three CDRs: CDR1' shown in SEQ ID NO: 14, CDR2' shown in SEQ ID NO: 15, and CDR3' shown in SEQ ID NO:16. 2. The antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:6. 3. The antibody of claim 1, wherein the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:3. 4. The antibody of claim 1, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:7. 5. The antibody of claim 1, wherein the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:5. 6. A recombinant protein, characterized in that, said recombinant protein has: (i) the antibody of any one of claims 1-5; and (ii) Optional tag sequences to aid in expression and/or purification. 7. A polynucleotide, characterized in that it encodes a polypeptide selected from the group consisting of: (1) the antibody according to any one of claims 1-5; or (2) recombinant protein as claimed in claim 6. 8. A vector comprising the polynucleotide according to claim 7. 9. A genetically engineered host cell, characterized in that it contains the vector according to claim 8 or the polynucleotide according to claim 7 is integrated in its genome. 10. A kit, characterized in that, the kit includes: The antibody of any one of claims 1-5. 11. An immunoconjugate, characterized in that the immunoconjugate contains: (a) the antibody according to any one of claims 1-5 or the recombinant protein according to claim 6; and (b) A conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme. 12. A pharmaceutical composition, characterized in that the composition comprises the antibody according to any one of claims 1-5, the recombinant protein according to claim 6, or the immunoconjugate according to claim 11 ;as well as pharmaceutically acceptable carrier. 13. A method for preparing a recombinant polypeptide, characterized in that the method comprises: (a) under conditions suitable for expression, culturing the host cell according to claim 9; (b) isolating the recombinant polypeptide from the culture, the recombinant polypeptide is the antibody according to any one of claims 1-5 or the recombinant protein according to claim 6.