阅读说明：本技术 一种h1n1流感病毒抗体及其在h1n1病毒超微量检测方面的应用 (H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro-detection ) 是由 董金华 董航 单喜军 李海梅 陈丽梅 于 2019-11-14 设计创作，主要内容包括：本发明公开了一种H1N1流感病毒抗体及其在H1N1病毒超微量检测方面的应用,该H1N1流感病毒抗体包括重链可变区、第一重链超可变区、第二重链超可变区、第三重链超可变区、轻链可变区、第一轻链超可变区、第二轻链超可变区和第三轻链超可变区；本发明的H1N1流感病毒抗体能够与H1N1流感病毒特异性结合,用于H1N1流感的超微量检测,实现H1N1流感的尽早预防和治疗,具有非常重要的经济和社会意义。(The invention discloses an H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro detection, wherein the H1N1 influenza virus antibody comprises a heavy chain variable region, a first heavy chain hypervariable region, a second heavy chain hypervariable region, a third heavy chain hypervariable region, a light chain variable region, a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region; the H1N1 influenza virus antibody can be specifically combined with H1N1 influenza virus, is used for ultramicro detection of H1N1 influenza, realizes early prevention and treatment of H1N1 influenza, and has very important economic and social meanings.)

1. An influenza H1N1 virus antibody characterized by: comprises a heavy chain variable region, a first heavy chain hypervariable region, a second heavy chain hypervariable region, a third heavy chain hypervariable region, a light chain variable region, a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region;

wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2;

the first heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 3;

the second heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 4;

the third heavy chain hypervariable region has the amino acid sequence shown in SEQ ID NO. 5;

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 7;

the first light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 8;

the second light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 9;

the third light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 10.

2. A DNA fragment encoding the H1N1 influenza virus antibody of claim 1, wherein: the heavy chain variable region has a gene sequence shown in SEQ ID NO.1, and the light chain variable region has a gene sequence shown in SEQ ID NO. 6.

3. An expression vector, characterized in that: the expression vector comprising at least one copy of the DNA segment of claim 2.

4. A host cell, characterized in that: the host cell comprising the expression vector of claim 3.

5. The use of the H1N1 influenza virus antibody of claim 1, wherein: the application in the ultramicro detection of the H1N1 virus.

Technical Field

The invention relates to the technical field of influenza virus antibodies in biotechnology, in particular to an H1N1 influenza virus antibody and application thereof in ultra-micro detection of H1N1 viruses.

Background

Influenza virus (inflenzavirus) is an infectious disease pathogen that causes influenza and highly pathogenic influenza viruses can cause death of humans and animals. The virus belongs to the family Orthomyxoviridae (Orthomyxoviridae), the genus influenza. Influenza viruses are classified into A, B, C types according to nucleoprotein and matrix protein antigenicity. The surfaces of virus particles are provided with two important proteins of Hemagglutinin (HA) and Neuraminidase (NA), influenza viruses can be divided into a plurality of subtypes according to the difference of the two proteins, wherein the type A protein is taken as an example, currently found HA HAs 18 subtypes (H1-H18), and NA HAs 11 subtypes (NA 1-11). The surface protein of the influenza A virus is easy to have antigenic mutation and difficult to control, and a plurality of large influenza viruses are caused by the influenza A virus in history, and the large influenza viruses cause about 30 to 5 million deaths of people, thereby causing great threat to the life safety and the economic property of people.

The current methods for treating H1N1 influenza mainly comprise drug therapy and antibody therapy, wherein the drug therapy mainly adopts two types of ion channel inhibitors and neuraminidase inhibitors. Ion channel inhibitors including amantadine and rimantadine and derivatives thereof, which produce gastrointestinal and central nervous system side effects over prolonged periods of time and result in the production of resistant strains with undiminished virulence and transmission performance; the neuraminidase inhibitor mainly comprises zanamivir and oseltamivir, the medicine has good treatment effect on early-stage patients with H1N1 influenza, but the treated patients are easy to generate drug resistance to the neuraminidase inhibitor. In contrast, antibody therapy of H1N1 influenza has great advantages, because the active antibody can block the combination of virus and target cells, induce immune cells to kill virus-infected cells, and thus, the aim of rapidly treating H1N1 influenza is achieved.

Therefore, the development of antibodies against the H1N1 influenza virus is urgent to solve the problem that people can detect the H1N1 virus in an ultramicro manner and realize early prevention and treatment of the H1N1 viral influenza.

Disclosure of Invention

In order to solve the problems, the invention provides an H1N1 influenza virus antibody and application thereof in ultra-micro detection of H1N1 virus.

In order to achieve the purpose, the invention is realized by the following technical scheme:

an influenza H1N1 virus antibody comprising a unique and novel sequence of a heavy chain variable region, a first heavy chain hypervariable region, a second heavy chain hypervariable region, a third heavy chain hypervariable region, a light chain variable region, a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region;

wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2;

the first heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 3;

the second heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 4;

the third heavy chain hypervariable region has the amino acid sequence shown in SEQ ID NO. 5;

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 7;

the first light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 8;

the second light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 9;

the third light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 10.

The invention also includes a DNA fragment encoding the H1N1 influenza virus antibody, wherein the heavy chain variable region has a gene sequence shown in SEQ ID NO.1, and the light chain variable region has a gene sequence shown in SEQ ID NO. 6.

The invention also includes an expression vector comprising at least one copy of a DNA segment as described above.

The invention also includes a host cell comprising an expression vector as described above.

The invention also comprises the application of the H1N1 influenza virus antibody in the ultra-micro detection of the H1N1 virus.

Compared with the prior art, the invention has the following advantages:

the H1N1 influenza virus antibody can be specifically combined with H1N1 influenza virus, is used for ultramicro detection of H1N1 influenza virus, provides a new method for early prevention and treatment of H1N1 influenza, and has very important economic and social meanings.

Drawings

FIG. 1 is a diagram showing the comparison of the content of H1N1 influenza virus antibody in serum before and after the immunization of mice;

FIG. 2 is an agarose electrophoresis of PCR products of the amplified mouse antibody variable region gene;

FIG. 3 is a graph showing the binding performance of the antibodies in the antibody library with H1N1 virus and streptavidin;

FIG. 4 is a diagram showing the results of screening for monoclonal antibodies against H1N1 virus;

FIG. 5 is a diagram showing the result of polyacrylamide gel electrophoresis performed on the purified antibody fragment;

FIG. 6 is a schematic diagram showing the results of testing the binding properties of the H1N1 virus-resistant monoclonal antibody F1 against H1N 1;

FIG. 7 is a schematic diagram of the principle of detecting H1N1 influenza virus based on enzyme-linked immunosorbent assay of F1 antibody;

FIG. 8 is a standard curve for detecting H1N1 influenza virus in an enzyme-linked immunosorbent assay based on F1 antibody;

reference numerals

M：DNA marker；V_L: an antibody light chain variable region gene; v_H: antibody heavy chain variable region gene.

Detailed Description

The invention aims to provide an H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro detection, which are realized by the following technical scheme:

an influenza H1N1 antibody comprising a heavy chain variable region, a first heavy chain hypervariable region, a second heavy chain hypervariable region, a third heavy chain hypervariable region, a light chain variable region, a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region;

wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2;

the first heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 3;

the second heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 4;

the third heavy chain hypervariable region has the amino acid sequence shown in SEQ ID NO. 5;

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 7;

the first light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 8;

the second light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 9;

the third light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 10.

The invention also comprises a DNA segment for coding the H1N1 influenza virus antibody, wherein the heavy chain variable region has a gene sequence shown in SEQ ID NO.1, and the light chain variable region has a gene sequence shown in SEQ ID NO. 6.

The invention also includes an expression vector comprising at least one copy of a DNA segment as described above.

The invention also includes a host cell comprising the above-described expression vector.

The invention also comprises the application of the H1N1 influenza virus antibody in the ultra-micro detection of the H1N1 virus.

The invention is further described with reference to specific examples.