Anti-canine parvovirus genetic engineering antibody and application thereof
阅读说明：本技术 一种抗犬细小病毒的基因工程抗体及其应用 (Anti-canine parvovirus genetic engineering antibody and application thereof ) 是由 殷玉和 吴丛梅 李希辰 赫玉芳 李雪 石晶 雷欢 赵林烨 于 2019-12-23 设计创作，主要内容包括：本发明提供了一种抗犬细小病毒的基因工程抗体及其应用,涉及抗体工程技术领域,将已获得的犬细小病毒单克隆抗体的可变区序列与犬源抗体恒定区进行组装,得到抗犬细小病毒的基因工程嵌合抗体,经过试验验证,所述基因工程抗体的细胞上清液的血凝抑制效价为1：2<Sup>4</Sup>～2<Sup>5</Sup>；中和效价为1:203。可见所述基因工程抗体表现出良好的中和犬细小病毒的活性,且能抑制犬细小病毒对红细胞的凝集作用,可应用于单克隆抗体犬源化研究和犬细小病毒的防治等领域,对推动犬源化单克隆抗体药物的发展具有重要意义。(The invention provides a gene engineering antibody for resisting canine parvovirus and application thereof, relating to the technical field of antibody engineering, wherein a variable region sequence of an obtained canine parvovirus monoclonal antibody is assembled with a canine antibody constant region to obtain a gene engineering chimeric antibody for resisting the canine parvovirus, and tests prove that the hemagglutination inhibition titer of cell supernatant of the gene engineering antibody is 1:2 4 ～2 5 (ii) a The neutralization titer was 1: 203. Therefore, the genetic engineering antibody shows good activity of neutralizing canine parvovirus, can inhibit the agglutination of the canine parvovirus to erythrocytes, can be applied to the fields of monoclonal antibody caninization research, canine parvovirus prevention and treatment and the like, and promotes the canine parvovirusThe development of monoclonal antibody medicines has important significance.)
1. A genetic engineering antibody for resisting canine parvovirus is characterized in that the genetic engineering antibody comprises a constant region of a canine antibody and a variable region of a murine antibody;
the constant region comprises a heavy chain constant region and a light chain constant region, and the nucleotide sequence of the heavy chain constant region is shown as SEQ ID No. 5; the nucleotide sequence of the light chain constant region is shown as SEQ ID NO. 6;
the variable region comprises a heavy chain variable region and a light chain variable region, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No. 1; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 2.
2. The engineered antibody of claim 1, wherein the amino acid sequence encoding the heavy chain variable region is as shown in SEQ ID No. 3; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4.
3. The genetically engineered antibody of claim 1 or 2, wherein the species of the genetically engineered antibody comprises: single chain antibodies, diabodies or chimeric antibodies.
4. The engineered antibody of claim 3, wherein the species of the engineered antibody further comprises: derivatives, functional equivalents or homologues of said single-chain, diabodies or chimeric antibodies, as well as antibody fragments or any polypeptides comprising an antigen-binding domain.
5. Use of the genetically engineered antibody of any one of claims 1 to 4 in the preparation of a medicament for the prevention and treatment of canine parvovirus infection.
Technical Field
The invention belongs to the technical field of antibody engineering, and particularly relates to a canine parvovirus-resistant genetic engineering antibody and application thereof.
Background
Canine Parvovirus (CPV) belongs to the genus parvovirus of the family parvoviridae and is an important pathogen in domestic dogs and several wild carnivores, the primary natural host being dogs. The canine parvovirus disease is caused after the canine is infected, the canine parvovirus disease is divided into enteritis type and myocarditis type, has the characteristics of high morbidity, strong infectivity, high mortality and the like, can cause serious economic loss, and seriously harms the development of the canine industry in China.
Vaccines for preventing canine parvovirus diseases mainly include: inactivated vaccine, attenuated vaccine and multiple vaccine, but the investigation and research on the immune effect of the vaccine on the market find that only 20% of the vaccine can generate 100% protective antibody. For canine parvovirus infection, no specific medicine exists at present, and symptomatic treatment, supportive therapy and specific therapy are combined to be applied clinically. Among them, specific therapies generally employ canine parvovirus monoclonal antibodies and canine parvovirus antiserums in early stages. The commercially available antiserum comprises xenogeneic animal immune serum and canine serum, wherein the xenogeneic animal serum can protect young animals and adult animals from infection but cannot be continuously injected, otherwise severe anaphylactic reaction is caused, and animal death can be caused in severe cases; the dog-derived serum has the characteristics of dog source shortage and high preparation cost. The clinically adopted monoclonal antibody for resisting canine parvovirus is a murine monoclonal antibody, and the clinical application is influenced because the therapeutic effect is reduced due to the fact that the murine monoclonal antibody is easy to generate an anti-murine antibody when the murine monoclonal antibody is injected into other provenance animals. With the increase of the number of pet dogs, the research on the diagnosis and the prevention of canine parvovirus diseases has important significance.
At present, the canine parvovirus monoclonal antibody and antiserum on the market have stronger immunogenicity, and have side effects in the treatment process to reduce the curative effect, thereby limiting the clinical application of the canine parvovirus monoclonal antibody and antiserum. Therefore, the preparation of the monoclonal antibody with low immunogenicity, high affinity and strong specificity is more beneficial to promoting the large-scale clinical application of antibody drugs.
Disclosure of Invention
In view of the above, the present invention aims to provide a genetically engineered antibody against canine parvovirus and applications thereof, which can exhibit a good activity of neutralizing canine parvovirus, inhibit canine parvovirus from aggregating well on erythrocytes, and reduce immunogenicity and allergic reactions when used for treating canine parvovirus infection.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a genetic engineering antibody for resisting canine parvovirus, which comprises a constant region of a canine antibody and a variable region of a murine antibody;
the constant region comprises a heavy chain constant region and a light chain constant region, and the nucleotide sequence of the heavy chain constant region is shown as SEQ ID NO. 5; the nucleotide sequence of the light chain constant region is shown as SEQ ID NO. 6;
the variable region comprises a heavy chain variable region and a light chain variable region, and the nucleotide sequence of the heavy chain variable region is shown as SEQID NO. 1; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 2.
Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 3; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4.
Preferably, the species of the genetically engineered antibody include: single chain antibodies, diabodies or chimeric antibodies.
Preferably, the species of the genetically engineered antibody further comprises: derivatives, functional equivalents or homologues of said single-chain, diabodies or chimeric antibodies, as well as antibody fragments or any polypeptides comprising an antigen-binding domain.
The invention also provides application of the genetic engineering antibody in preparing a medicament for preventing and treating canine parvovirus infection.
Compared with the prior art, the invention has the following beneficial effects: the variable region sequence of the obtained canine parvovirus monoclonal antibody is assembled with the canine derived antibody constant region to obtain the genetic engineering chimeric antibody for resisting the canine parvovirus, which shows good activity for neutralizing the canine parvovirus, can be applied to the fields of monoclonal antibody caninization research, canine parvovirus prevention and the like, and has important significance for promoting the development of caninized monoclonal antibody medicaments.
The genetic engineering antibody has good neutralizing activity on canine parvovirus, can inhibit the agglutination of the canine parvovirus on erythrocytes, can be applied to the fields of prevention and treatment research on canine parvovirus diseases and the like, and has important significance for promoting the development of caninized monoclonal antibody medicaments.
In the embodiment of the invention, experiments prove that the hemagglutination inhibition titer of the cell supernatant of the genetically engineered antibody is 1:24～25(ii) a The neutralizing titer is 1:203, so the antibody is a neutralizing antibody, can be effectively combined with CPV to block the infection of cells, has a certain treatment effect, can be added with a pharmaceutically acceptable carrier, and is used for preparing a medicament for treating canine parvovirus infection.
Drawings
FIG. 1 is a schematic diagram of the structure of the caninized CPV engineered antibody of example 1, which comprises two light chains and two heavy chains, wherein the light chain is composed of an N-terminal variable region VLAnd C-terminal constant region CLThe heavy chain consists of an N-terminal variable region VHAnd C-terminal constant region CH
(CH1-CH2-CH3) Composition of, wherein VLAnd VHVariable region, C, from murine CPV monoclonal antibodyLAnd CHAn antibody constant region from canine origin;
FIG. 2 is the restriction enzyme map of the eukaryotic expression vector of the light and heavy chains of the caninized CPV genetically engineered antibody in example 1;
FIG. 3 is a graph showing the hemagglutination inhibition results of the caninized CPV genetically engineered antibody provided in example 2;
FIG. 4 is a virus regression control in neutralization test of caninized CPV genetically engineered antibodies with CPV provided in example 2;
FIG. 5 is a graph showing cytopathic effect of neutralization test of CPV with caninized CPV genetically engineered antibody provided in example 2;
FIG. 6 is a graph showing the results of indirect immunofluorescence assay of the caninized CPV genetically engineered antibody provided in example 2.
Detailed Description
The invention provides a genetic engineering antibody for resisting canine parvovirus, which has a structural schematic diagram shown in figure 1, wherein the genetic engineering antibody comprises a constant region of a canine antibody and a variable region of a murine antibody;
the constant region comprises a heavy chain constant region and a light chain constant region, and the nucleotide sequence of the heavy chain constant region is shown as SEQ ID NO. 5; the nucleotide sequence of the light chain constant region is shown as SEQ ID NO. 6;
the variable region comprises a heavy chain variable region and a light chain variable region, and the nucleotide sequence of the heavy chain variable region is shown as SEQID NO. 1; the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 2.
The genetic engineering antibody is caninized and transformed of a mouse CPV monoclonal antibody, the preparation method of the mouse CPV monoclonal antibody is not particularly limited, and the mouse CPV monoclonal antibody is prepared according to the method disclosed by the Chinese patent CN109627331A in the embodiment of the invention.
When the genetic engineering antibody is assembled, the heavy chain variable region gene sequence of the murine antibody is preferably assembled with the canine antibody heavy chain constant region gene sequence, the light chain variable region gene sequence is assembled with the canine antibody light chain constant region gene sequence, after codon optimization, the nucleotide sequence of the heavy chain is preferably shown as SEQ ID No.7, and the nucleotide sequence of the light chain is preferably shown as SEQ ID No. 8. The method of assembling is not particularly limited in the present invention, and the conventional assembling method of the present invention may be used.
In the present invention, the amino acid sequence encoding the heavy chain variable region is preferably as shown in SEQ ID NO. 3; the amino acid sequence of the light chain variable region is preferably shown as SEQ ID NO. 4.
In the present invention, the kind of the genetically engineered antibody preferably includes: a single chain antibody, diabody or chimeric antibody, as well as derivatives, functional equivalents or homologues of said single chain antibody, diabody or chimeric antibody, and antibody fragments or any polypeptide comprising an antigen binding domain.
The invention also provides application of the genetic engineering antibody in preparing a medicament for preventing and treating canine parvovirus infection.
The medicine also comprises pharmaceutically acceptable auxiliary materials, and the specific types of the auxiliary materials are not particularly limited.
The following examples are provided to describe the engineered anti-canine parvovirus antibody and the use thereof in detail, but they should not be construed as limiting the scope of the present invention.