阅读说明：本技术 流感病毒基质蛋白m1的单克隆抗体及其应用 (Monoclonal antibody of influenza virus matrix protein M1 and application thereof ) 是由 蒲娟 佟琪 宗亚楠 王明暘 曲润康 刘金华 于 2019-09-25 设计创作，主要内容包括：本发明涉及生物技术领域,具体涉及流感病毒基质蛋白M1的单克隆抗体及其应用。本发明提供流感病毒基质蛋白M1的单克隆抗体,所述单克隆抗体包含重链可变区和轻链可变区,所述重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1-3所示；所述轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.4-6所示。该单克隆抗体能够特异识别并结合多种亚型的流感病毒基质蛋白M1、在流感病毒亚型间具有广谱适用性,同时具有较高的特异性和敏感性,可以很好地应用于免疫印迹实验、间接免疫荧光实验以及免疫沉淀等技术,具有较高的应用价值。(The invention relates to the technical field of biology, in particular to a monoclonal antibody of influenza virus matrix protein M1 and application thereof. The invention provides a monoclonal antibody of influenza virus matrix protein M1, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6. The monoclonal antibody can specifically recognize and combine influenza virus matrix protein M1 of various subtypes, has broad-spectrum applicability among influenza virus subtypes, has high specificity and sensitivity, can be well applied to technologies such as immunoblotting experiments, indirect immunofluorescence experiments, immunoprecipitation and the like, and has high application value.)

1. The monoclonal antibody of the influenza virus matrix protein M1, which comprises a heavy chain variable region and a light chain variable region, and is capable of specifically binding to the influenza virus matrix protein M1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is any one of:

(1) as shown in SEQ ID NO. 7;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 7;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.7 and has the same function; preferably, the homology is at least 85%; more preferably at least 95%;

the amino acid sequence of the light chain variable region is any one of the following:

(1) as shown in SEQ ID NO. 8;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 8;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.8 and has the same function; preferably, the homology is at least 85%; more preferably at least 95%.

3. The monoclonal antibody of claim 1 or 2, characterized in that the amino acid sequence of the heavy chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 9;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 9;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.9 and has the same function; preferably, the homology is at least 85%; more preferably at least 95%;

the amino acid sequence of the light chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 10;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 10;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.10 and has the same function; preferably, the homology is at least 85%; more preferably at least 95%.

4. A hybridoma cell producing the monoclonal antibody of any one of claims 1 to 3.

5. A nucleic acid encoding the monoclonal antibody of any one of claims 1 to 3;

preferably, the nucleic acid sequence encoding the heavy chain of the monoclonal antibody is shown as SEQ ID No.11 or is the complementary sequence thereof, and the nucleic acid sequence encoding the light chain of the monoclonal antibody is shown as SEQ ID No.12 or is the complementary sequence thereof.

6. A labeled complex obtained by biochemically labeling the monoclonal antibody according to any one of claims 1 to 3;

preferably, the biochemical label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a radioactive label.

7. Use of the monoclonal antibody of any one of claims 1 to 3 or the hybridoma cell of claim 4 or the labeled complex of claim 6 for the preparation of a kit for detecting influenza virus, influenza matrix protein M1 or an influenza virus antibody.

8. Use of the monoclonal antibody according to any one of claims 1 to 3 or the hybridoma cell according to claim 4 or the marker complex according to claim 6 for the preparation of a medicament for the prophylaxis or treatment of influenza virus or for quality control of an influenza virus vaccine.

9. A medicament comprising a monoclonal antibody according to any one of claims 1 to 3.

10. An influenza virus detection kit comprising the monoclonal antibody according to any one of claims 1 to 3 or the labeled complex according to claim 6.

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody of an influenza virus matrix protein M1, a hybridoma cell secreting the monoclonal antibody of the influenza virus matrix protein M1 and application thereof.

Background

Antibody technology is largely divided into monoclonal antibody technology and polyclonal antibody technology. An antigen is determined by a plurality of antigenic determinants, one antigenic determinant stimulates the body, and a B lymphocyte receives an antibody produced by the antigen, which is called a monoclonal antibody. Stimulation of the body by multiple epitopes correspondingly produces a wide variety of monoclonal antibodies, which when mixed together are polyclonal. Polyclonal antibodies may exist in multiple subtypes, resulting in poor specificity compared to monoclonal antibodies. When the polyclonal antibody is used for an immunity experiment, a background value is generated, and the experiment result is influenced to different degrees. The characteristics of high specificity and sensitivity of the monoclonal antibody enable the monoclonal antibody to be widely applied to molecular experiments and clinical diagnosis.

The M1 protein of the influenza virus is the most abundant protein in the virus particles, and the M1 protein can maintain the integrity of the morphological structure of the influenza virus particles and plays an important role in replication and transcription of virus genomes and assembly and budding of viruses. Furthermore, the M1 protein is highly conserved between influenza virus subtypes. Therefore, the development of a broad-spectrum monoclonal antibody capable of specifically recognizing M1 protein of various subtypes of influenza viruses is beneficial to the diagnosis of influenza and the deep research of molecular mechanisms, and has important significance.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a broad-spectrum monoclonal antibody which has higher sensitivity and specificity and can specifically bind to the matrix protein M1 of a plurality of subtype influenza viruses and a hybridoma cell for producing the monoclonal antibody.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a monoclonal antibody against influenza virus matrix protein M1, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region, capable of specifically binding to influenza virus matrix protein M1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID nos. 1-3, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6.

Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 7;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 7;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.7 and has the same function; the homology is preferably at least 85%; more preferably at least 95%.

The amino acid sequence of the variable region of the light chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 8;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 8;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.8 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

The monoclonal antibody of the invention can specifically bind to a matrix protein M1 of a plurality of subtype influenza viruses, and the antigen recognition region of the monoclonal antibody is positioned in the 169-252 amino acid region of the M1 protein.

More preferably, the subtype of the monoclonal antibody is IgG1 with kappa chain light chain.

Specifically, the amino acid sequence of the heavy chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 9;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 9;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.9 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

The amino acid sequence of the light chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 10;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 10;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.10 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

In a second aspect, the present invention provides a hybridoma cell producing the monoclonal antibody to influenza virus matrix protein M1.

In a third aspect, the present invention provides a nucleic acid encoding the monoclonal antibody to influenza virus matrix protein M1.

Preferably, the nucleic acid sequence encoding the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.11 or is the complementary sequence thereof; the nucleic acid sequence of the light chain of the encoding monoclonal antibody is shown as SEQ ID NO.12 or is a complementary sequence thereof.

In a fourth aspect, the invention provides a labeling complex obtained by biochemical labeling of the monoclonal antibody of influenza virus matrix protein M1.

Preferably, the biochemical label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a radioactive label.

In a fifth aspect, the invention provides an application of the monoclonal antibody of the influenza virus matrix protein M1 or the hybridoma cell or the labeled complex of the monoclonal antibody of the influenza virus matrix protein M1 in preparation of a kit for detecting influenza virus or influenza virus matrix protein M1.

In a sixth aspect, the invention provides an application of the monoclonal antibody of the influenza virus matrix protein M1 or the labeled complex of the hybridoma cell or the monoclonal antibody of the influenza virus matrix protein M1 in preparing a kit for detecting influenza virus antibodies.

In a seventh aspect, the present invention provides a use of the monoclonal antibody against influenza virus matrix protein M1 or the hybridoma cell or the labeled complex against the monoclonal antibody against influenza virus matrix protein M1 for the preparation of a medicament for the prevention or treatment of influenza virus.

In an eighth aspect, the invention provides an application of the monoclonal antibody of the influenza virus matrix protein M1 or the hybridoma cell or the labeled complex of the monoclonal antibody of the influenza virus matrix protein M1 in quality control of influenza virus vaccines.

In a ninth aspect, the present invention provides a medicament comprising the monoclonal antibody to influenza virus matrix protein M1.

The medicine can also comprise other active ingredients or auxiliary materials allowed in the pharmaceutical field besides the monoclonal antibody of the influenza virus matrix protein M1.

In a tenth aspect, the present invention provides an influenza virus detection kit comprising the monoclonal antibody to the influenza matrix protein M1 or a labeled complex of the monoclonal antibody to the influenza matrix protein M1.

The detection kit for the influenza virus can also comprise other reagents or materials required for detection, including but not limited to buffer solution, secondary antibody and the like, besides the labeled complex of the monoclonal antibody of the influenza virus matrix protein M1 or the monoclonal antibody of the influenza virus matrix protein M1.

The invention has the beneficial effects that: the influenza virus base provided by the inventionThe monoclonal antibody of the cytoplasmic protein M1 can specifically recognize and combine with M1 proteins of various subtypes of influenza viruses, has broad spectrum among influenza virus subtypes, has high specificity among the influenza viruses and other viruses, and has high sensitivity, so the monoclonal antibody can be well applied to an immunoblotting experiment (Western Blot, the maximum dilution multiple can reach 8 multiplied by 10⁴Multiple), indirect immunofluorescence experiment (IFA, maximum dilution factor can reach 5X 10⁵Multiple) and immunoprecipitation (IP, the minimum usage amount is only 1 mu g), and the like, and has higher application value; meanwhile, the method lays a technical foundation for the deep research of the pathogenic mechanism of the influenza virus.

Drawings

FIG. 1 shows the result of the specificity analysis of the monoclonal antibody 2C5 for Western blot detection in example 2 of the present invention, wherein the ratio of the sequences in lane 1: a sample of cell lysate not infected with virus; lanes 2-5: cell lysate samples infected with H9N2, H7N9, H5N1, H1N1 subtype influenza virus were in order.

FIG. 2 shows the result of the sensitivity analysis of the monoclonal antibody 2C5 for Western blot detection in example 2 of the present invention, wherein lane 1 is the lysate of infected virus cells and lane 2 is the lysate of uninfected virus cells.

FIG. 3 shows the result of analysis of the specificity of monoclonal antibody 2C5 for IFA detection in example 3 of the present invention, wherein A, B, C, D represents the detection results of infection with influenza viruses of H9N2, H7N9, H5N1 and H1N1 subtypes, monoclonal antibody 2C5 as a primary antibody, and E represents a negative control well of uninfected virus.

FIG. 4 is a graph showing the results of analysis of the sensitivity of the monoclonal antibody 2C5 for IFA detection in example 3 of the present invention, in which A, B, C, D, E, F indicates the dilution levels of the monoclonal antibody 2C5 in the order of 1: 1X 10³、1:5×10³、1:1×10⁴、1:5×10⁴、1:1×10⁵、1:5×10⁵The detection result of (1).

FIG. 5 shows the analysis results of the specificity of monoclonal antibody 2C5 for IP assay detection in example 4 of the present invention; wherein, H1, H5, H7 and H9 respectively represent M1 protein enriched from different virus infected samples by the monoclonal antibody 2C5, Input is a cell lysis sample infected with virus, M1 is influenza virus M1 protein, and Actin is internal reference protein Actin.

FIG. 6 is the result of analysis of the sensitivity of monoclonal antibody 2C5 for IP experimental detection in example 4 of the present invention; wherein, IP is M1 protein enriched from different virus infected samples by monoclonal antibody 2C5, Input is cell lysis sample infected with virus, M1 is M1 protein of influenza virus, and Actin is reference protein Actin.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.