阅读说明：本技术 一种鸡源化抗禽流感神经氨酸酶单克隆抗体及其制备方法和应用 (Humanized avian influenza resistant neuraminidase monoclonal antibody and preparation method and application thereof ) 是由 秦爱建 王飞 王娅娟 林志娴 万志敏 钱琨 邵红霞 叶建强 于 2019-10-17 设计创作，主要内容包括：本发明属于生物医学技术领域,具体涉及一种鸡源化抗禽流感神经氨酸酶单克隆抗体及其制备方法和应用。通过PCR方法小鼠特异性的Fab与鸡Fc片段拼接并克隆入哺乳动物表达载体中,获得表达重组重链的质粒。两质粒共转染哺乳动物细胞,即可获得分泌表达的鸡源化抗禽流感神经氨酸酶单克隆抗体。本发明可广泛用于抗病毒、细菌单克隆抗体的鸡源化表达,获得的鸡源化抗禽流感神经氨酸酶单克隆抗体既能够保持Fab识别、中和抗原的能力,又具备在体内呈递抗原的潜力。(The invention belongs to the technical field of biomedicine, and particularly relates to a humanized avian influenza resistant neuraminidase monoclonal antibody and a preparation method and application thereof. The specific Fab of the mouse and the Fc fragment of the chicken are spliced and cloned into a mammal expression vector by a PCR method to obtain a plasmid for expressing the recombinant heavy chain. The two plasmids co-transfect mammalian cells to obtain the secreted and expressed chicken-derived anti-avian influenza neuraminidase monoclonal antibody. The invention can be widely used for the chicken source expression of the anti-virus and bacterial monoclonal antibody, and the obtained chicken source anti-avian influenza neuraminidase monoclonal antibody can not only keep the capability of Fab in recognizing and neutralizing antigen, but also has the potential of presenting antigen in vivo.)

1. The humanized avian influenza neuraminidase monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2 in the sequence table.

2. A preparation method of a humanized avian influenza resistant neuraminidase monoclonal antibody is characterized by comprising the following steps:

extracting and amplifying a full-length gene from a signal peptide start codon to a stop codon of a light chain of the monoclonal antibody from a hybridoma cell secreting the mouse monoclonal antibody, and cloning the full-length gene into a mammal expression vector to obtain a plasmid for expressing the light chain;

extracting and amplifying Fd fragment sequences from a signal peptide start codon to the end of a CH1 region of a heavy chain of the monoclonal antibody from a hybridoma cell secreting a mouse monoclonal antibody, extracting and amplifying Fc fragment gene sequences from a CH 2N-terminal start amino acid codon to a stop codon of the chicken IgY antibody from the spleen of an immunized chicken, splicing the Fd fragment and the Fc fragment and cloning the spliced Fd fragment and the Fc fragment into a mammalian expression vector to obtain a plasmid for expressing a recombinant heavy chain;

the two plasmids were co-transfected into serum-free mammalian cells and the supernatant was collected.

3. The method of claim 2, wherein the mammalian expression vector comprises pCDNA3, pCDNA3.1, pCMV, pCAGGS, and pIRES.

4. The method for preparing the humanized anti-avian influenza neuraminidase monoclonal antibody according to claim 2, wherein the mammalian cells comprise 293T cells, COS-1 cells, COS-7 cells, sp2/0 cells or CHO cells.

5. The method for preparing a humanized anti-avian influenza neuraminidase monoclonal antibody according to claim 2, wherein the nucleotide sequence of the Fc fragment of the chicken IgY antibody from the start amino acid codon to the stop codon of the N-terminal of CH2 is shown as SEQ ID No. 3.

6. A nucleotide molecule encoding the antibody of claim 1.

7. An expression vector comprising the nucleotide molecule of claim 6.

8. A host cell comprising the nucleotide molecule of claim 6 or the expression vector of claim 7.

9. A pharmaceutical composition comprising the monoclonal antibody of claim 1.

10. The use of the humanized anti-avian influenza neuraminidase monoclonal antibody of claim 1 for identifying antigens and/or neutralizing viruses in vitro.

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a humanized avian influenza resistant neuraminidase monoclonal antibody and a preparation method thereof, which can be widely used for the humanized expression of antiviral and bacterial monoclonal antibodies.

Background

Monoclonal antibody technology was invented in 1975 by the England scientists Milstein and Kohler. The sensitized B cells with the capacity of secreting specific antibodies and myeloma cells with the capacity of unlimited propagation are fused into B cell hybridomas. The obtained hybridoma secretes an antibody against a specific epitope, i.e., a monoclonal antibody. The monoclonal antibody obtained by the method has the characteristics of high purity, good specificity and high yield, and can be applied to research and development of clinical diagnostic reagents and therapeutic drugs.

Because most of the antibodies prepared at present are of murine origin, repeated clinical use can cause patients to generate anti-murine antibodies, thereby weakening or losing the curative effect of the antibodies and causing hypersensitivity. Therefore, in the early 80 s, people began to use genetic engineering to prepare antibodies to reduce the immunogenicity and functions of murine antibodies. At present, some of the sequences of murine antibodies are replaced by partial amino acid sequences of human antibodies, and the modified antibodies are used to prepare genetically engineered antibodies, called third-generation antibodies. However, few reports have been made on the utilization of genetic engineering techniques to transform murine monoclonal antibodies into chicken.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a humanized avian influenza resistant neuraminidase monoclonal antibody and a preparation method thereof.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a humanized anti-avian influenza neuraminidase monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2 in the sequence table.

A method for preparing a humanized avian influenza resistant neuraminidase monoclonal antibody comprises the following steps:

extracting and amplifying a full-length gene from a signal peptide start codon to a stop codon of a light chain of the monoclonal antibody from a hybridoma cell secreting the mouse monoclonal antibody, and cloning the full-length gene into a mammal expression vector to obtain a plasmid for expressing the light chain;

extracting and amplifying Fd fragment sequences from a signal peptide start codon to the end of a CH1 region of a heavy chain of the monoclonal antibody from a hybridoma cell secreting a mouse monoclonal antibody, extracting and amplifying Fc fragment gene sequences from a CH 2N-terminal start amino acid codon to a stop codon of the chicken IgY antibody from the spleen of an immunized chicken, splicing the Fd fragment and the Fc fragment and cloning the spliced Fd fragment and the Fc fragment into a mammalian expression vector to obtain a plasmid for expressing a recombinant heavy chain;

the two plasmids were co-transfected into serum-free mammalian cells and the supernatant was collected.

Further, the mammalian expression vector comprises pCDNA3, pCDNA3.1, pCMV, pCAGGS and pIRES.

Further, the mammalian cells include 293T cells, COS-1 cells, COS-7 cells, sp2/0 cells, or CHO cells.

Furthermore, the nucleotide sequence of the Fc fragment of the chicken IgY antibody from the N-terminal start amino acid codon to the stop codon of CH2 is shown as SEQ ID NO. 3.

The invention also provides nucleotide molecules encoding the antibodies.

The invention also provides an expression vector which contains the nucleotide molecule for coding the monoclonal antibody.

The invention also provides a host cell containing the expression vector.

The invention also provides a pharmaceutical composition, which comprises the monoclonal antibody and a pharmaceutically acceptable carrier.

The invention also provides a kit comprising the monoclonal antibody.

The invention also provides application of the humanized anti-avian influenza neuraminidase monoclonal antibody in-vitro antigen recognition and/or virus neutralization.

Has the advantages that:

fc fragment receptors of the avian IgY antibodies are widely distributed on the surfaces of antigen presenting cells such as DC cells, B cells, macrophages and the like, and assist the IgY in presenting antigens in humoral immunity. In addition, the Fc fragment receptor of the avian IgY antibody may also mediate maternal antibodies into the chick embryo. Therefore, the invention uses the Fc fragment of the chicken IgY antibody to replace the Fc fragment of the murine IgG antibody, constructs the humanized anti-avian influenza neuraminidase antibody and provides the humanized antibody which can be applied to passive immunization of chicken. Meanwhile, the method can be used for developing similar antibody of chicken source anti-other viruses such as chicken Newcastle disease virus, infectious bronchitis virus, chicken infectious bursal disease virus and the like, and is used for preventing corresponding epidemic diseases and the like.

Drawings

FIG. 1 is an electrophoresis diagram of Sac I and Nhe I double digestion products of a positive clone of an expression antibody light chain plasmid;

FIG. 2 is an electrophoresis diagram of Sac I and Nhe I double digestion products of a recombinant heavy chain plasmid positive clone expressing a humanized anti-avian influenza neuraminidase antibody;

FIG. 3 shows the results of IFA with the co-transfected supernatant as primary antibody;

FIG. 4 is the result of in vitro neutralization of H9N2 avian influenza virus by a humanized anti-avian influenza neuraminidase antibody, FabchFc.

Detailed Description

The embodiments of the present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

All the following examples use the Fab gene of the anti-H9N 2 avian influenza virus neuraminidase monoclonal antibody 1G8 as the object of chimeric antibody modification.