Monoclonal antibody for resisting filovirus GP protein and application thereof
阅读说明：本技术 一种抗丝状病毒gp蛋白的单克隆抗体及其应用 (Monoclonal antibody for resisting filovirus GP protein and application thereof ) 是由 陈凌 王龙雨 冯立强 冯玉鹏 于 2019-10-11 设计创作，主要内容包括：本发明提供了一种抗丝状病毒GP蛋白的单克隆抗体及其应用,所述单克隆抗体的抗原结合片段的重链可变区包括如SEQ ID NO:7所示的氨基酸序列；所述抗原结合片段的轻链可变区包括如SEQ ID NO:8所示的氨基酸序列。本发明的抗原结合片段从成年中国猕猴体内获得,经人源化后对丝状病毒科的扎依尔型GP蛋白、苏丹型GP蛋白和马尔堡病毒GP蛋白均具有结合活性,得到的单克隆抗体与人基因的同源性高,实现了抗体的人源化,稳定性好,对GP蛋白具有较强的亲和力,所述单克隆抗体可潜在地应用于丝状病毒包括埃博拉病毒及马尔堡病毒的抗原检测、丝状病毒感染临床样本的检测与鉴定以及丝状病毒抑制剂研发等。(The invention provides a monoclonal antibody of anti-filovirus GP protein and application thereof, wherein a heavy chain variable region of an antigen binding fragment of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 7; the variable region of the light chain of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO 8. The antigen binding fragment is obtained from an adult Chinese macaque body, has binding activity on a zaire type GP protein, a Sudan type GP protein and a Marburg virus GP protein of a filoviridae after humanization, has high homology with a human gene, realizes humanization of an antibody, has good stability and strong affinity for the GP protein, and can be potentially applied to antigen detection of filoviruses including Ebola virus and Marburg virus, detection and identification of filovirus infection clinical samples, development of filovirus inhibitors and the like.)
1. An antigen-binding fragment, wherein the heavy chain variable region of the antigen-binding fragment comprises the heavy chain CDR3 as set forth in SEQ ID No. 1;
the light chain variable region of the antigen-binding fragment includes the light chain CDR3 shown in SEQ ID NO. 4.
2. The antigen-binding fragment of claim 1, wherein the heavy chain variable region of the antigen-binding fragment further comprises heavy chain CDR2 shown in SEQ ID NO. 2, heavy chain CDR1 shown in SEQ ID NO. 3;
the light chain variable region of the antigen-binding fragment further includes light chain CDR2 shown in SEQ ID NO. 5 and light chain CDR1 shown in SEQ ID NO. 6.
3. The antigen-binding fragment of claim 1 or 2, wherein the heavy chain variable region of the antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID No. 7;
the variable region of the light chain of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO 8.
4. A monoclonal antibody directed against a GP protein of a filovirus, said monoclonal antibody comprising an antigen-binding fragment according to any one of claims 1 to 3;
preferably, the monoclonal antibody further comprises any one of or a combination of at least two of human IgG1, IgG2, IgG3, or IgG4 constant regions, preferably human IgG1 constant regions.
5. A nucleic acid molecule comprising a DNA fragment encoding the antigen-binding fragment of any one of claims 1 to 3 and/or the heavy chain variable region and/or the light chain variable region of the monoclonal antibody of claim 4;
preferably, the heavy chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 9;
preferably, the light chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 10.
6. An expression vector comprising the nucleic acid molecule of claim 5;
preferably, the expression vector further comprises a nucleic acid molecule encoding a constant region of human IgG 1;
preferably, the expression vector is pCMV.
7. A host cell transfected with the nucleic acid molecule of claim 5 and/or the expression vector of claim 6;
preferably, the host cell comprises a 293T cell or a CHO cell.
8. A pharmaceutical composition comprising any one of the antigen binding fragment of any one of claims 1 to 3, the monoclonal antibody of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7, or a combination of at least two thereof;
preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
9. A kit comprising any one of the antigen binding fragment of any one of claims 1 to 3, the monoclonal antibody of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7, or a combination of at least two thereof;
preferably, the kit further comprises a washing solution.
10. Use of an antigen-binding fragment according to any one of claims 1 to 3, a monoclonal antibody according to claim 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6 or a host cell according to claim 7 for the preparation of a filovirus therapeutic and/or detection reagent.
Technical Field
The invention belongs to the technical field of biology, and relates to a monoclonal antibody for resisting a filovirus GP protein and application thereof.
Background
Filoviruses are single-stranded negative-strand RNA viruses, which are mainly classified into Ebola, Marburg and Quiver genera. Ebola virus genus includes 5 species of zaire type (ZEBOV), sudan type (SUDV), leston type (RESTV), tay forest Type (TAFV) and bendibotrey type (BDBV), wherein the lethality of zaire type, sudan type and bendibrey type viruses is up to 90%. Marburg virus includes a species of Marburg virus (MARAV), and the lethality rate is as high as 83-90%. In 2014, zaire-type virus had a major outbreak in west africa, with a total of 11372 deaths by 2016. By 2019, 3 and 10 months, ebola hemorrhagic fever outbreaks in the 10 th cycle of a congo outbreak had caused 603 deaths. At present, no specific therapeutic drug is on the market internationally, so the research on the diagnosis and treatment of the filars is particularly important.
In recent years, intensive studies have been conducted on the pathogenic mechanism of a filamentous virus, and it has been found that GP protein is a unique protein on the surface of a viral envelope and plays an important role in viral invasion, cell membrane fusion and the like. Therefore, GP protein is mainly used as a main antigen target in vaccine design and drug development. Monoclonal or combinatorial antibodies to individual species of filovirus are currently validated in animal models or are in clinical stage. However, there is no report of monoclonal antibodies against multiple pathogenic strains of the filovirus family.
CN107922939A discloses a monoclonal antibody neutralizing the infectivity of all kinds of ebola viruses, which is able to recognize the internal membrane fusion loop of GP of ebola virus and has a biological activity neutralizing ebola virus, but which is not biologically active against marburg virus and kukukutavirus.
CN104168903A discloses methods and compositions for treating diseases caused by enveloped viruses selected from the group consisting of retroviruses, preferably HIV, and viruses of the filoviridae family, which are less effective in targeting filamentous viruses, by inhibiting the binding of sialyllactose molecules contained in the virus' enveloped gangliosides to the CD 169/sialoadhesin receptor, to prevent the virus from entering cells expressing the CD 169/sialoadhesin surface receptor.
Therefore, the monoclonal antibody against multiple pathogenic strains of the filovirus family is of great significance in the field of diagnosis and treatment of filovirus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a monoclonal antibody of anti-filovirus GP protein and application thereof, wherein the monoclonal antibody is prepared based on a macaque experiment technology platform, solves the problems of practical diagnosis and treatment of the filovirus monoclonal antibody, and provides a new scheme for establishing filovirus detection, diagnosis, prevention and treatment.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an antigen-binding fragment, the heavy chain variable region of which comprises the heavy chain CDR3 shown in SEQ ID No. 1;
the light chain variable region of the antigen-binding fragment comprises the light chain CDR3 shown in SEQ ID NO. 4;
the amino acid sequence shown in SEQ ID NO. 1 is: AMRYSNYRTWFNV, respectively;
the amino acid sequence shown in SEQ ID NO. 4 is: QLWDSSSDYPL are provided.
Preferably, the heavy chain variable region of the antigen-binding fragment further comprises heavy chain CDR2 shown in SEQ ID NO. 2, heavy chain CDR1 shown in SEQ ID NO. 3;
the light chain variable region of the antigen-binding fragment further comprises light chain CDR2 shown in SEQ ID NO. 5, light chain CDR1 shown in SEQ ID NO. 6;
the amino acid sequence shown in SEQ ID NO. 2 is: IDLSDSET;
the amino acid sequence shown in SEQ ID NO. 3 is: GYSFPTYW;
the amino acid sequence shown in SEQ ID NO. 5 is: YNR;
the amino acid sequence shown in SEQ ID NO. 6 is: NIGSEA.
Preferably, the heavy chain variable region of the antigen binding fragment comprises the amino acid sequence shown as SEQ ID NO. 7;
the variable region of the light chain of the antigen-binding fragment comprises an amino acid sequence shown as SEQ ID NO. 8;
the amino acid sequence shown in SEQ ID NO. 7 is:
EVQLVQSGAEVKRPGESLKISCKTSGYSFPTYWITWVRQMPGKGLEWMGTIDLSDSETKYSPSFDGQVTISADKSINTAYLQWSSLKASDSATYYCAMRYSNYRTWFNVWGPGVLVTVSS；
the amino acid sequence shown in SEQ ID NO. 8 is:
SYDLTQPPSVSVSPGQTARITCGGDNIGSEAVHWYQQQPPQAPVLVIYYNRERPSGIPERFSGSKSGNTATLTISGVEAGDEADYYCQLWDSSSDYPLFGGGTRLTVL.
in the invention, adult Chinese macaque is selected as an experimental animal, and the Zaier type GP protein, the Sudan type GP protein and the Marburg virus GP protein are used as antigens for immunization, so that the obtained monoclonal antibody has high homology with human genes.
In a second aspect, the present invention provides a monoclonal antibody against the GP protein of a filovirus, said monoclonal antibody comprising an antigen-binding fragment as described in the first aspect.
Preferably, the monoclonal antibody further comprises any one of or a combination of at least two of human IgG1, IgG2, IgG3, or IgG4 constant regions, preferably human IgG1 constant regions.
In the invention, the variable region gene and the human IgG1 constant region sequence are connected through homologous recombination to construct an antibody expression vector, thereby realizing the humanization of the antibody.
In a third aspect, the present invention provides a nucleic acid molecule comprising a DNA fragment encoding the antigen-binding fragment of the first aspect and/or the heavy chain variable region and/or the light chain variable region of the monoclonal antibody of the second aspect.
Preferably, the heavy chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 9;
the nucleic acid molecule shown in SEQ ID NO. 9 is:
GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAGGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGACTTCTGGATACAGCTTTCCCACCTACTGGATCACCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGAACAATTGATCTTAGTGATTCTGAAACCAAATACAGCCCGTCCTTCGATGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACTCCGCCACGTATTACTGTGCGATGAGGTACAGTAATTACCGGACCTGGTTCAATGTCTGGGGCCCGGGAGTCCTGGTCACCGTCTCCTCAG.
preferably, the light chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in
the nucleic acid molecule shown in SEQ ID NO. 10 is:
TCCTATGATCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGTGGGGGAGACAATATTGGAAGTGAAGCTGTACACTGGTACCAGCAGCAGCCACCGCAGGCCCCTGTGTTGGTCATCTATTATAATAGGGAACGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAAATCAGGGAACACCGCCACCCTGACCATCAGCGGGGTCGAGGCCGGGGATGAGGCTGACTATTACTGTCAGCTGTGGGATAGTAGTAGTGATTATCCGCTTTTCGGAGGAGGGACCCGGCTCACCGTCCTAG.
in a fourth aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described in the third aspect.
Preferably, the expression vector further comprises a nucleic acid molecule encoding a constant region of
Preferably, the expression vector is pCMV.
In a fifth aspect, the present invention provides a host cell transfected with a nucleic acid molecule according to the third aspect and/or an expression vector according to the fourth aspect.
Preferably, the host cell comprises a 293T cell or a CHO cell.
In a sixth aspect, the present invention provides a method of preparing an antigen-binding fragment according to the first aspect and/or a monoclonal antibody according to the second aspect, the method comprising the steps of:
(1) performing RT-PCR on the obtained B cells to obtain nucleic acid molecules of the heavy chain variable region and the light chain variable region of the antibody;
(2) respectively connecting the nucleic acid molecules of the heavy chain variable region and the light chain variable region in the step (1) into an expression vector, transferring the expression vector into competent cells, and selecting monoclonal cells for screening after culturing;
(3) and transferring the screened expression vector into a host cell, culturing, collecting supernatant, and separating and purifying to obtain the monoclonal antibody.
Preferably, said B cells of step (1) are derived from cynomolgus monkey.
In the invention, the macaque experiment technical platform is used for preparing the monoclonal antibody, the defect of low homology between a mouse and a human gene is overcome, and the problem of the filovirus monoclonal antibody in the aspects of actual diagnosis and treatment is solved through the humanization process of the antibody.
Preferably, the method for obtaining B cells in step (1) comprises the following steps:
(1') immunizing the macaque by using a mixture of the zaire type GP protein, the sudan type GP protein and the Marburg virus GP protein as antigens;
(2') obtaining peripheral blood mononuclear cells of the immunized macaque, carrying out antibody marking on the B cells, and sorting by using a flow cytometry sorting method to obtain the B cells.
In the invention, GP proteins of filovirus type zaire, Sudan type (GenBank: KT878488.1) and Marburg type virus (GenBank: JX458858.1) are used as antigens, and affinity mature antibody genes are separated from Chinese macaque through multiple immunizations by utilizing immunology and molecular biology techniques, so that a stable humanized anti-GP protein monoclonal antibody expression vector is constructed.
Preferably, the molar ratio of the zaire-type GP protein, sudan-type GP protein and marburg virus GP protein in step (1') is (0.5-1): 0.8-1, and may be, for example, 0.5:0.5:0.8, 0.5:0.5:1, 0.5:1:0.8, 0.5:1:1, 1:0.5:0.8, 1:0.5:1, 1:1:0.8 or 1:1:1, preferably 1:1: 1.
Preferably, the antibody labeling of step (2') is performed using a CD3 antibody, a CD20 antibody, a CD27 antibody, an IgG antibody, and an anti-histidine antibody.
In the present invention, RT-PCR was performed using a reverse transcription kit (cat: A5000/A5001) from Premega.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising any one of or a combination of at least two of the antigen-binding fragment of the first aspect, the monoclonal antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect.
Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
In an eighth aspect, the present invention provides a kit comprising any one or a combination of at least two of the antigen-binding fragment of the first aspect, the monoclonal antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect.
Preferably, the kit further comprises a washing solution.
In a ninth aspect, the present invention provides the use of an antigen binding fragment according to the first aspect, a monoclonal antibody according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect or a host cell according to the fifth aspect for the manufacture of a medicament for the treatment and/or detection of a filovirus.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, macaques are selected as experimental animals to prepare monoclonal antibodies, so that the defect of low homology between mice and human genes is overcome;
(2) the invention takes the Zaier type GP protein, the Sudan type GP protein and the Marburg virus GP protein as antigens for immunization, the obtained variable region gene is connected with the human IgG1 constant region sequence to construct an antibody expression vector, the obtained monoclonal antibody has high homology with the human gene, and the humanization of the antibody is realized;
(3) the monoclonal antibody prepared by the invention has good stability and stronger affinity to three GP proteins, namely Zaire type GP protein (Zaire GP), Sudan type GP protein (Sudan GP) and Marburg virus GP protein (Marburg GP);
(4) the invention prepares the single gramThe monoclonal antibody has good neutralizing effect on three pseudo viruses of Zaire type, Sudan type and Marburg type, and has maximum half Inhibition Concentration (IC) on the three pseudo viruses50) 25.276 μ g/mL, 3.115 μ g/mL, and 14.477 μ g/mL, respectively;
(5) the monoclonal antibody prepared by the invention can be potentially applied to antigen detection of filoviruses including Ebola virus and Marburg virus, detection and identification of clinical samples infected by filoviruses, research and development of filoviruses inhibitors and the like.
Drawings
FIG. 1 shows the binding of monoclonal antibody to GP protein in serum ELISA after 3 rd booster immunization;
FIG. 2(A) is physical data of all cells, for example, FIG. 2(B) shows a sorted lymphocyte population, FIG. 2(C) shows a sorted single cell population, FIG. 2(D) shows a sorted live cell population, FIG. 2(E) shows a sorted B cell population, for example, FIG. 2(F) shows a sorted memory B cell population capable of secreting IgG, and FIG. 2(G) shows a sorted memory B cell capable of specifically binding to an antigen;
FIG. 3 shows nested PCR amplification of heavy and light chain variable region genes of an antibody, wherein
FIG. 4(A) is a humanized plasmid map of heavy chain of monoclonal antibody, and FIG. 4(B) is a humanized plasmid map of Lambda chain of monoclonal antibody;
FIG. 5 shows the results of the detection of the binding activity of the monoclonal antibody 40C1L to 3 GP proteins;
FIG. 6 shows the results of the detection of the neutralizing activity of the monoclonal antibody 40C1L against 3 pseudoviruses.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.