阅读说明：本技术 抗金黄色葡萄球菌锰离子结合蛋白c的抗体及其应用 (Antibody for resisting staphylococcus aureus manganese ion binding protein C and application thereof ) 是由 曾浩 葛爽 宋振 邹全明 刘圆圆 敬海明 赵莉群 张怡 于 2019-11-29 设计创作，主要内容包括：本发明公开了抗金黄色葡萄球菌锰离子结合蛋白C的抗体及其应用,所述抗金黄色葡萄球菌锰离子结合蛋白C的抗体识别的表位肽至少包括MntC<Sub>126-128aa</Sub>,氨基酸序列为LDN；该抗体通过培养表达载体的宿主细胞来生成单克隆抗体,生成的单克隆抗体被分泌到上清液中,通过层析技术将其纯化。本发明的抗体可以治疗由MRSA感染引起的脓毒血症,在预防和治疗金黄色葡萄球菌感染方面有重要意义。(The invention discloses an antibody for resisting staphylococcus aureus manganese ion binding protein C and application thereof, wherein epitope peptide recognized by the antibody for resisting staphylococcus aureus manganese ion binding protein C at least comprises MntC 126‑128aa The amino acid sequence is LDN; the antibody is produced by culturing host cells expressing the vector to produce a monoclonal antibody, which is secreted into the supernatant and purified by chromatographic techniques. The antibody can treat sepsis caused by MRSA infection, and has important significance in preventing and treating staphylococcus aureus infection.)

1. An antibody against staphylococcus aureus manganese ion binding protein C, comprising: the epitope peptide recognized by the antibody against staphylococcus aureus manganese ion binding protein C at least comprises MntC_126-128aaAnd the amino acid sequence is LDN.

2. The antibody against staphylococcus aureus manganese ion-binding protein C of claim 1, wherein: the amino acid sequence of the epitope peptide identified by the antibody of the anti-staphylococcus aureus manganese ion binding protein C is shown in SEQ ID NO. 12-SEQ ID NO. 15.

3. The antibody against staphylococcus aureus manganese ion-binding protein C of claim 1, wherein: the antibody comprises a heavy chain and a light chain, wherein the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 or consist of CDR variants with equivalent functions; the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11 or consist of CDR variants with equivalent functions.

4. The antibody against staphylococcus aureus manganese ion-binding protein C of claim 3, wherein: the constant region of the antibody comprises any one of a human IgM, IgA or IgA constant region.

5. The antibody against staphylococcus aureus manganese ion-binding protein C of claim 3, wherein: the amino acid sequence of the heavy chain is shown as SEQ ID NO.4 or a sequence which has at least 85 percent of homology with SEQ ID NO.4 and is specifically combined with the full-length amino acid or partial amino acid of the staphylococcus aureus manganese ion binding protein C; the light chain amino acid sequence is shown as SEQ ID NO.8 or a sequence which has at least 85% homology with SEQ ID NO.8 and is specifically combined with the full-length amino acid or partial amino acid of the staphylococcus aureus manganese ion binding protein C.

6. The antibody against staphylococcus aureus manganese ion-binding protein C of claim 3, wherein: the full-length amino acid of the staphylococcus aureus manganese ion binding protein C is shown in SEQ ID No. 1.

7. The antibody against Staphylococcus aureus manganese ion-binding protein C according to any one of claims 1 to 6, wherein: the equilibrium dissociation constant of the antibody combined with the staphylococcus aureus manganese ion binding protein C is not higher than 1 x 10^-8。

8. A nucleotide sequence encoding the antibody of any one of claims 1 to 7.

9. Use of an antibody according to any one of claims 1 to 7 in the preparation of a reagent which specifically binds to manganese ion binding protein C of Staphylococcus aureus.

10. Use of an antibody according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment or prevention of s.

Technical Field

The invention relates to the field of medicine and immunology, in particular to an antibody for resisting staphylococcus aureus manganese ion binding protein C, and also relates to application of the antibody for resisting staphylococcus aureus manganese ion binding protein C.

Background

Staphylococcus aureus (staphylococcus aureus for short) is a gram-positive bacterium, is a main human pathogenic bacterium which is widely popularized and causes hospital and community related infection, and can cause various diseases of human. The infection is mainly acute and purulent. The systemic infection can cause serious infection and complications such as osteomyelitis, septic arthritis, endocarditis, pneumonia, sepsis and the like, and the fatality rate can reach 20%. The problem of bacterial resistance is increasingly exacerbated and highlighted by long-term antibiotic abuse. Methicillin-resistant Staphylococcus aureus (MRSA) was discovered for the first time in 1961 and is called as 'super bacteria' to develop multiple drug resistance, and the MRSA has become a gram-positive bacterium with the highest infection rate in ICU wards, postoperative infection, burns and the like, becomes a difficult problem for clinical treatment and seriously threatens human health. With the successive emergence of vancomycin-resistant staphylococcus aureus (VRSA) in 2002, the last line of defense for vancomycin treatment of MRSA has been broken through, and clinical infection at present faces a severe situation of "no drug rescue". Therefore, the search for anti-MRSA drugs has become an important direction in the research field of treating MRSA in recent years.

Staphylococcus aureus manganese ion binding protein C (MntC) is a highly conserved cell surface protein and also an important virulence factor of staphylococcus aureus systemic infection, and plays an important role in Staphylococcus aureus infection by promoting the acquisition of manganese ions from the host environment. Loss or reduction of manganese ion transporter activity sensitizes staphylococcus aureus to oxidative stress, rendering the bacteria vulnerable to attack by immune cells and death, while MntC has adhesin activity, a feature that may be characteristic of the pathogenesis of staphylococcus aureus. In recent years, staphylococcus aureus vaccine is subjected to a plurality of stages of capsular polysaccharide vaccine, DNA vaccine and inactivated vaccine, but the staphylococcus aureus vaccine is not successfully obtained, and passive immunization based on antibody medicine is used for treating MRSA infection, so that on one hand, the staphylococcus aureus vaccine can quickly establish specific immune protection for infected people at the first time and quickly and efficiently eliminate pathogens, and on the other hand, the staphylococcus aureus vaccine can help MRSA susceptible people with low immunity and even immunodeficiency to obtain instant and effective immune protection.

Therefore, the present invention is based on the above background, and it is necessary to develop an antibody against manganese ion binding protein C of Staphylococcus aureus.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an antibody against staphylococcus aureus manganese ion binding protein C; the second object of the present invention is to provide a nucleotide sequence encoding said antibody; the invention also aims to provide the application of the antibody in preparing a reagent for specifically binding the manganese ion binding protein C of the staphylococcus aureus; the fourth purpose of the invention is to provide the application of the antibody in preparing a medicament for treating or preventing staphylococcus aureus infection.

In order to achieve the purpose, the invention provides the following technical scheme:

1. antibody against staphylococcus aureus manganese ion-binding protein C, which recognizes epitope peptide comprising at least MntC_126-128aaAnd the amino acid sequence is LDN.

Preferably, the amino acid sequence of the epitope peptide identified by the antibody of the anti-staphylococcus aureus manganese ion binding protein C is shown in SEQ ID NO. 12-SEQ ID NO. 15.

Preferably, the antibody comprises a heavy chain and a light chain, wherein the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 or CDR variant compositions with equivalent functions; the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11 or consist of CDR variants with equivalent functions.

The CDR regions of the heavy chains are positioned as follows:

CDR1 region amino acids 26 to 33 within the VH exon;

CDR2 region amino acids 51 to 58 within the VH exon;

CDR3 region amino acids 97 to 108 within the VH exon.

The CDR regions of the light chain are positioned as follows:

amino acids 27 to 32 of the CDR1 region within the V λ exon;

amino acids 50 to 52 of the CDR2 region within the V λ exon;

CDR3 region amino acids 89 to 97 within the V λ exon.

Preferably, the constant region of the antibody comprises any one of a human IgM, IgA or IgA constant region.

Preferably, the amino acid sequence of the heavy chain is shown as SEQ ID NO.4 or a sequence which has at least 85% homology with SEQ ID NO.4 and specifically binds to the full-length amino acid or partial amino acid of the manganese ion binding protein C of staphylococcus aureus; the light chain amino acid sequence is shown as SEQ ID NO.8 or a sequence which has at least 85% homology with SEQ ID NO.8 and is specifically combined with the full-length amino acid or partial amino acid of the staphylococcus aureus manganese ion binding protein C.

Preferably, the full-length amino acid of the staphylococcus aureus manganese ion binding protein C is shown as SEQ ID No. 1.

Preferably, the equilibrium dissociation constant of the antibody combined with the manganese ion binding protein C of staphylococcus aureus is not higher than 1 x 10^-8(ii) a Such as: 1X 10^-9M、1×10^-10M、1×10^-11M or less is dissociated from S.aureus manganese ion-binding protein C. The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, and refers to the degree of dissociation of an antibody and antigen when in equilibrium. Smaller KD indicates less dissociation, which represents greater affinity between the antibody and antigen.

The antibody of the invention is to sort single plasma cells from peripheral mononuclear cells of volunteers with high MntC antibody titer, and amplify the heavy chain and light chain variable region genes of the antibody by using mRNA of the single plasma cells as a template. The heavy chain and light chain variable region genes of the antibody are respectively connected with a promoter, a human constant region and a poly-A tail by overlapping extension PCR to construct a linear expression vector of the monoclonal antibody. Host cells containing recombinant expression vectors for the nucleic acid molecules are then constructed. Prokaryotic and eukaryotic expression systems are common, prokaryotic host cells such as bacterial cells; eukaryotic host cells such as plant cells and mammalian cells. Particularly preferred are human producer cell lines.

A nucleotide sequence encoding said antibody.

Because the nucleotide sequence and the amino acid sequence are in corresponding relation, the specific amino acid sequence of the antibody determines the nucleotide sequence of the nucleic acid molecule, and under the condition of determining the amino acid sequence of the antibody, a corresponding, proper and reasonable nucleotide sequence can be obtained according to the amino acid sequence. Preferably, the heavy chain nucleotide sequence is shown as SEQ ID NO.16, and the light chain nucleotide sequence is shown as SEQ ID NO. 17.

Preferably, the antibody is used for preparing a reagent which specifically binds to the manganese ion binding protein C of the staphylococcus aureus.

Preferably, the antibody is used for preparing a medicament for treating or preventing staphylococcus aureus infection.

The term "variant" as used herein refers to a sequence resulting from modification of such a sequence by insertion, deletion or substitution of one or more amino acids, or by chemical derivatization of one or more amino acid residues in the amino acid sequence, or nucleotides in the nucleotide sequence, or one or both distal ends of the sequence, and also includes natural allelic mutations, wherein the modification does not affect (and in particular does not lose) the activity of such a sequence.

In the present invention, the term "CDR region" means a complementarity determining region of an antibody, i.e., a region that determines the specificity of an antibody for a specific antigen. The CDRs 1 to 3 on both the heavy and light chains collectively make up the antigen binding site of the antibody.

The invention has the beneficial effects that: the invention provides an antibody against staphylococcus aureus manganese ion binding protein C, which is prepared by culturing a host cell of an expression vector to generate a monoclonal antibody, secreting the generated monoclonal antibody into a supernatant and purifying the monoclonal antibody by a chromatography technology.

The antibody can treat sepsis caused by MRSA infection, and has important significance in preventing and treating staphylococcus aureus infection.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a diagram of single plasma cell sorting in the present invention.

FIG. 2 shows the SDS-PAGE result of MntC monoclonal antibody M0686 of the present invention.

FIG. 3 shows the binding activity of MntC monoclonal antibody M0686 to MntC measured in the present invention.

FIG. 4 shows the result of analyzing the interaction between MntC and M0686 by using the biomembrane interference technique according to the present invention.

FIG. 5 is a diagram showing the results of the MntC denatured glue WB of the present invention.

FIG. 6 is a graph showing the results of the inhibition of cytokine production by MntC by M0686 in the present invention (A: IL-5; B: IL-17A; C: IFN-. gamma.).

FIG. 7 is a graph showing the results of M0686 in the present invention on the evaluation of MRSA sepsis animal model.

FIG. 8 is a histological analysis in the present invention to evaluate the effectiveness of antibodies.

FIG. 9 is a graph showing the results of the MntC Mapping polypeptide Elisa of the present invention (A: the result of binding of the polypeptide to the M0686 monoclonal antibody; B: MntC_129-143aa、MntC_132-143aaAnd MntC_135-143aaBinding to M0686 results).

Detailed Description

The technical solution of the present invention is clearly and completely described below. Technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention pertains, and experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.