CN108017715B

CN108017715B - Zaire type Ebola virus detection antibody and preparation method and application thereof

Info

Publication number: CN108017715B
Application number: CN201711497938.9A
Authority: CN
Inventors: 杨光; 高福; 黄超宾; 校海霞; 曲志虎; 路希山; 郭天玲; 庞雪飞
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS; ShanghaiTech University
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS; ShanghaiTech University
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2021-11-12
Anticipated expiration: 2037-12-29
Also published as: CN108017715A

Abstract

The invention provides a Zaire type Ebola virus detection antibody and a preparation method and application thereof. The Zaire type Ebola virus detection antibody is characterized in that the Zaire type Ebola virus detection antibody is a human monoclonal antibody; the amino acid sequences of the light chain hypervariable regions CDR1, CDR2 and CDR3 are respectively SEQ ID No.3, DDS and SEQ ID No.5, and the amino acid sequences of the heavy chain hypervariable regions CDR1, CDR2 and CDR3 are respectively SEQ ID No.6, SEQ ID No.7 and SEQ ID No. 8. The antibody provided by the invention can well detect Zaire type Ebola virus envelope protein GP, and has important economic and social significance.

Description

Zaire type Ebola virus detection antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and in particular relates to a Zaire type Ebola virus detection antibody, and a preparation method and application thereof.

Background

Ebola virus (Ebolavirus) was discovered in the democratic republic of sudan and congo, respectively, in 1976. Currently, 5 subtypes have been developed: zaire (Zaire), Sudan (Sudan), bundbury (bundbugyo), cotdetdewar (Cote d' Ivoire), and Reston (Reston), of which the first 4 subtypes are pathogenic to humans. The last outbreak of ebola virus infection started in 2 months 2014, the mortality rate can reach as high as 90%, and the causes of death are mainly stroke, myocardial infarction, hypovolemic shock or organ failure.

The origin and host of Ebola virus in nature are not determined, and evidence shows that the virus is of animal origin, and bat can be a potential natural host. Ebola virus has strong infectivity, blood, semen, sweat, saliva and secretion of infected patients have infectivity, and can infect closely contacted people through skin, conjunctiva or respiratory tract and other ways. The Ebola virus infected cells are mediated by a membrane protein GP, the GP is processed by furin or furin-like protease to form two subunits of GP1 and GP2, and GP1 and GP2 are connected by a pair of disulfide bonds to form a heterodimer to form a mature trimer form. The GP1 subunit is responsible for anchoring the virus to the target cell, and the GP2 subunit is responsible for fusion of the viral envelope with the cell membrane, thereby releasing the viral genome into the host cell, a process that requires the involvement of an endogenous cysteine protease.

GP plays an important role in the pathogenicity of ebola viruses, and can selectively reduce the expression of macromolecules on the cell surface related to cell adhesion and immune function, thereby causing the shedding and death of cells. In addition, sGP binds to neutrophils via CD16b, interacts with the host immune system, altering the physical and functional interaction between Fc γ RIIIB and CR3, thereby inhibiting early neutrophil clearance of the virus, thereby causing immune escape.

Currently, only two companies, namely Taibei Abnova and Beijing Yiwangzhou, produce and sell related mouse-derived and rabbit-derived single (multiple) antibiotics in the market. The commonly used antibody screening methods comprise phage display antibody library screening, single B cell sequencing technology, B cell immortalization, humanized mouse hybridoma cell screening and the like, and the Zaire type Ebola virus detection antibody is obtained by using the phage display antibody library screening method. The invention utilizes the phage display antibody library constructed by PBMC of normal people to obtain the gene sequence of the monoclonal antibody with affinity by adopting an affinity screening mode, simplifies the screening process of the antibody and provides a shortcut for the research and development of antibody medicaments.

Disclosure of Invention

Aiming at the shortage and actual demand of the existing market supply, the invention aims to provide a Zaire type Ebola virus detection antibody, a preparation method and application thereof. The Zaire type Ebola virus detection antibody provided by the invention provides a new choice for diagnosis of Ebola virus infection and detection of an Ebola virus antigen GP, and has important economic and social significance.

In order to achieve the above object, the present invention provides a Zaire type ebola virus detection antibody, which is characterized in that it is a human monoclonal antibody; the amino acid sequences of the light chain hypervariable regions CDR1, CDR2 and CDR3 are respectively the amino acid sequence with the same function formed by SEQ ID No.3 or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence, DDS (E-1C1CDR-L2, Asp Ser) or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence, and the amino acid sequence with the same function formed by SEQ ID No.5 or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence, the amino acid sequences of the heavy chain hypervariable regions CDR1, CDR2 and CDR3 are respectively the amino acid sequence with the same function formed by SEQ ID No.6 or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence, the amino acid sequence formed by SEQ ID No.7 or the amino acid sequence with one or more amino acids replaced, deleted or added in the sequence, and SEQ ID No.8 or an amino acid sequence with one or more amino acids replaced, deleted or added and with the same function.

Preferably, the amino acid sequence of the light chain variable region of the Zaire-type Ebola virus detection antibody is SEQ ID No.1 or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence, and the amino acid sequence of the heavy chain variable region is SEQ ID No.2 or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence.

Preferably, the amino acid sequences of the light chain framework regions FR1, FR2 and FR3 of the Zaire-type ebola virus detection antibody are respectively the amino acid sequence with the same function formed by SEQ ID No.9 or the sequence with one or more amino acids replaced, deleted or added, the amino acid sequence with the same function formed by SEQ ID No.10 or the sequence with one or more amino acids replaced, deleted or added, and the amino acid sequence with the same function formed by SEQ ID No.11 or the sequence with one or more amino acids replaced, deleted or added; the amino acid sequences of the heavy chain framework regions FR1, FR2 and FR3 are respectively an amino acid sequence with equivalent function formed by SEQ ID No.12 or the sequence with one or more amino acids replaced, deleted or added, an amino acid sequence with equivalent function formed by SEQ ID No.13 or the sequence with one or more amino acids replaced, deleted or added, and an amino acid sequence with equivalent function formed by SEQ ID No.14 or the sequence with one or more amino acids replaced, deleted or added.

Preferably, the nucleotide sequences of the light chain variable region and the heavy chain variable region of the Zaire-type Ebola virus detection antibody are respectively the nucleotide sequence with the same function formed by SEQ ID No.15 or the sequence with one or more nucleotides replaced, deleted or added, and the nucleotide sequence with the same function formed by SEQ ID No.16 or the sequence with one or more nucleotides replaced, deleted or added.

The invention also provides an expression vector, which is characterized by comprising at least one of a nucleotide sequence with equivalent function formed by replacing, deleting or adding one or more nucleotides in SEQ ID No.15 or the sequence and a nucleotide sequence with equivalent function formed by replacing, deleting or adding one or more nucleotides in SEQ ID No.16 or the sequence.

The invention also provides a host cell, which is characterized by comprising the expression vector.

The invention also provides a preparation method of the Zaire type Ebola virus detection antibody, which is characterized by comprising the following steps: connecting at least one of the nucleotide sequences with the same function formed by replacing, deleting or adding one or more nucleotides in SEQ ID No.15-SEQ ID No.20 and the sequences with an expression vector, amplifying, transfecting 293T cells, collecting the supernatant of the 293T cells, hanging a proA column, and eluting to obtain the Zaire type Ebola virus detection antibody.

Preferably, the expression vector is a mammalian expression vector.

More preferably, the mammalian expression vector is a pFase-Fc and pCAGGS mammalian expression vector.

The invention also provides application of the Zaire type Ebola virus detection antibody in preparation of a detection antibody which has affinity to the GP antigen of the Ebola virus and is used for detecting the Ebola virus.

In a first aspect of the invention, the invention provides a Zaire type ebola virus detection antibody, which is a human monoclonal antibody. According to the embodiment of the invention, the amino acid sequences of the hypervariable regions CDR1, CDR2 and CDR3 of the light chain of the antibody are shown in SEQ ID No.3, DDS (E-1C1CDR-L2, Asp Asp Asp Ser) and SEQ ID No.5 respectively; the amino acid sequences of the heavy chain hypervariable region CDR1, CDR2 and CDR3 are respectively shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No. 8.

The amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID No.1, or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence; the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID No.2, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence.

Sequences may contain certain biologically functionally equivalent amino acids or "conservative substitutions" and other sequences may contain functionally non-equivalent amino acids or "non-conservative substitutions" that are engineered to improve the properties of the CDRs or CDR-containing antibodies.

The amino acid sequences described above are as follows:

SEQ ID No.1(E-1C1 L)：

YVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVLG。

SEQ ID No.2(E-1C1 H)：

MAQVQLVQSGAEVKKPGASVKASCKASGYTFTSYYISWVRQAPGQGLEWMGIINPGDAGTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTGVYYCARGRSGWYGMDVWGQGTTVTVSS。

SEQ ID No.3(E-1C1 CDR-L1)：NIGSKS。

SEQ ID No.5(E-1C1 CDR-L3)：QVWDSSSDHWV。

SEQ ID No.6(E-1C1 CDR-H1)：GYTFTSYY。

SEQ ID No.7(E-1C1 CDR-H2)：INPGDAGT。

SEQ ID No.8(E-1C1 CDR-H3)：ARGRSGWYGMDV。

the amino acid sequences of the light chain framework regions FR1, FR2 and FR3 of the antibody are shown as SEQ ID No.9, SEQ ID No.10 and SEQ ID No. 11; the amino acid sequences of the heavy chain framework regions FR1, FR2 and FR3 are shown as SEQ ID No.12, SEQ ID No.13 and SEQ ID No. 14.

According to an embodiment of the invention, the amino acid sequence as described above is as follows:

SEQ ID No.9(E-1C1 FR-L1)：YVLTQPPSVSVAPGQTARITCGGN。

SEQ ID No.10(E-1C1 FR-L2)：VHWYQQKPGQAPVLVVY。

SEQ ID No.11(E-1C1FR-L3)：DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC。

SEQ ID No.12(E-1C1FR-H1)：MAQVQLVQSGAEVKKPGASVKASCKAS。

SEQ ID No.13(E-1C1 FR-H2)：ISWVRQAPGQGLEWMGI。

SEQ ID No.14(E-1C1FR-H3)：TYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTGVYYC。

the antibodies are human monoclonal antibodies.

The antibody is a single chain antibody, a double chain antibody, a chimeric antibody or a derivative thereof.

The antibody or the antigen binding fragment thereof can well bind the unique antigen-envelope protein GP on the surface of the Ebola virus.

In a second aspect of the invention, the invention provides two DNA fragments. According to an embodiment of the invention, these DNA fragments encode an antibody as described in the first aspect.

According to an embodiment of the present invention, the DNA fragment described above, comprises a heavy chain variable region encoding sequence and a light chain variable region encoding sequence; the nucleotide sequences of the light chain variable region and the heavy chain variable region are respectively shown as SEQ ID No.15 and SEQ ID No. 16.

According to an embodiment of the invention, the nucleotide sequence as described above is as follows:

SEQ ID No.15(E-1C1 L chain)：

TATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT。

SEQ ID No.16(E-1C1 H chain)：

ATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGCATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAGTGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA。

by adopting the polynucleotide of the invention, the Zaire Ebola virus detection antibody or antigen binding fragment can be effectively synthesized.

In a third aspect of the invention, the invention also provides two expression vectors comprising at least one copy of a DNA fragment according to the second aspect.

In a fourth aspect of the invention, the invention provides a host cell comprising an expression vector according to the third aspect.

By using the method according to the present invention, the aforementioned polynucleotides can be used to efficiently synthesize the Zaire-type ebola virus detection antibody or antigen binding fragment according to the embodiments of the present invention. The features and advantages described above with respect to the Zaire-type ebola virus detection antibody or antigen binding fragment apply equally to the polynucleotide and are not described in detail herein.

In a fifth aspect of the present invention, there is provided a method for producing an ebola virus detection antibody according to the first aspect, comprising the steps of:

(1) screening a monoclonal capable of specifically binding Zaire type Ebola virus envelope protein GP from a phage display antibody library by using an affinity screening method, and sequencing the monoclonal to obtain the nucleotide sequence information of the highly variable region sequences of the heavy chain and the light chain of the monoclonal;

(2) constructing an antibody expression vector by PCR amplification, enzyme digestion and connection methods.

According to the embodiments of the present invention, the preparation method of the antibody may be performed by using techniques well known to those skilled in the art, and is not particularly limited herein.

According to an embodiment of the present invention, the vector of step (2) is a mammalian expression vector, preferably a mammalian expression vector of pFase-Fc and pCAGGS.

In consideration of the degeneracy of codons, the gene sequence encoding the above antibody can be modified, for example, in the coding region thereof without changing the amino acid sequence, to obtain a gene encoding an antibody having the same function. One skilled in the art can artificially synthesize and modify genes according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

Further, the present invention recombines the light chain variable region and the heavy chain variable region of the aforementioned antibody to obtain a single chain antibody (ScFv) with a smaller molecular weight, which is also capable of recognizing Zaire type Ebola virus antigen GP. The single-chain antibody has strong penetrating power and is easy to enter local tissues to play a role. The antibody and the single-chain antibody can be obtained by cloning the gene encoding the antibody and the ScFv gene into an expression vector, and further transforming or transfecting host cells.

In addition, the light chain variable region encoding gene and the heavy chain variable region gene of the antibody can be cloned into a full-antibody expression vector and introduced into a host cell to obtain the full-antibody immunoglobulin for expressing and detecting the Ebola virus.

Compared with the prior art, the invention has the following beneficial effects:

the antibody is derived from a human body, can better reflect the interaction between the virus and the immune system of the human body, and has the affinity KD of 3.93nM between the antibody (ScFv-Fc form) and GP protein. The antibody provided by the invention can well detect Zaire type Ebola virus envelope protein GP, and has important economic and social significance.

Drawings

FIG. 1 shows the results of ELISA binding of both ScFv-Fc and IgG antibody formats of E-1C1 prepared according to the invention to Ebola GP proteins;

FIG. 2 shows the result of IFC binding of ScFv-Fc antibody of the antibody E-1C1 of the present invention to GP protein expressed on the surface of 293T cells.

FIG. 3 shows the results of detection of Ebola GP protein by the IgG antibody Western Blot method of the antibody E-1C1 of the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

The phage display antibody libraries used in the following examples were supplied by Richard Lerner, GAO's Lib, according to Zhang H, Wilson IA, Lerner RA: selection of antibodies which are present in a predetermined pattern from an intercellular synthetic antibody library, Proc Natl Acad Sci USA2012, 109 (39): 15728-15733. the construction method described in the above paragraph.

The Zaire type Ebola virus envelope protein GP (NCBI GenBank: AHX24658.2, constructed to be expressed on insect expression plasmids) used in the following examples was as described in Wang H, Shi Y, Song J, Qi J, Lu G, Yan J, Gao GF: ebola Viral Glycoprotein Bound to Its Endosomal ReceptorNiemann-Pick C1.cell 2016, 164 (1-2): 258 ℃ and 268.

sGP (NCBI GenBank: U23187.1, obtained by constructing to express insect expression plasmids) the expression vectors described in Mohan GS, Li W, Ye L, companies RW, Yang C: antibiotic Subversion: a Novel Mechanism of Host Immune evolution by Ebola virus. plos Pathogens 2012, 8 (12): e 1003065.

ssGP (NCBI GenBank: NP-066248, constructed to express insect expression plasmids) was prepared as described in Radoshitzky SR, Warfield KL, Chi X, Dong L, Kota K, Bradfute SB, Gearhart JD, Rettterer C, Kranzusch PJ, Misasi JN et al: journal of virology 2011, 85 (17): 8502-8513.

EXAMPLE 1 preparation of antibodies

The preparation method of the Zaire type Ebola virus GP protein detection antibody comprises the following steps:

1.1 screening of a monoclonal antibody capable of specifically binding to Zaire type Ebola virus envelope protein GP from a phage display antibody library using an affinity screening method:

1.1.1 biotinylation of amino acids containing primary amino groups in antigen (GP) molecules with NHS-PEG4-Biotin (Thermo Fisher, 21955) to give biotinylated antigen Bio-GP:

1.1.1.1 the molar quantity of NHS-PEG4-Biotin required for the calculation of the molar quantity of the antigen GP, so that the molar quantity of NHS-PEG4-Biotin is 50 times that of GP, the calculation method is as follows:

mL protein*(mg protein/mL protein)*(mmol protein/mg protein)*(50mmol Biotin/mmol protein)＝mmol Biotin；

50-recommended molar ratio of NHS-PEG4-Biotin to antigenic molecule;

1.1.1.2. the volume of 20mM HS-PEG4-Biotin at the desired concentration was calculated from 1.1.1.1 as follows:

mmol Biotin*(589mg/mmol Biotin)*170μL/2.0mg)＝μL Biotin Solution

589. NHS-PEG4-Biotin molecular weight;

170.0 mg of NHS-PEG4-Biotin dissolved in a molar concentration of 20 mM;

1.1.1.3 dissolve 200 μ g GP into 700 μ L PBS;

1.1.1.4 NHS-PEG4-Biotin was dissolved to 20mM using 170. mu.L of solvent in the kit;

1.1.1.5 adding 4 μ L NHS-PEG4-Biotin to GP, mixing well, incubating for 2h on ice;

1.1.1.6 protein samples incubated were added to the center of desalting column (Thermo Fisher, 89882) media to allow protein to infiltrate into the desalting column;

1.1.1.71000 g, centrifuging for 2min to obtain a river fluid which is biotinylated Bio-GP;

1.1.2 screening using the magnetic bead method:

1.1.2.1 day one:

the 1.5ml centrifuge tubes were blocked with 5% BSA (AMRESCO, 0332) -PBST and incubated for 1h at room temperature;

the magnetic beads (200. mu.L each, Thermo Fisher, 11206D) were washed 4 times with PBS;

5% BSA-PBST blocking library (200. mu.L), incubated for 30min at room temperature;

the library was added to PBS washed magnetic beads (1/4 volume beads) and incubated at room temperature for 30 min;

adding 5 mu g of biotinylated antigen Bio-GP to the supernatant of the previous step, and incubating for 2h at room temperature;

slightly centrifuging, adding the obtained product in the previous step into 3/4-volume magnetic beads remained in the previous step, and incubating for 20min at room temperature;

recovering the magnetic beads, washing with PBST for 4 times, and washing with PBS for 2 times;

recovering the magnetic beads, adding 300 mu L of Glycine-HCl with pH of 2.2, and reacting for 10min at room temperature;

adding the supernatant to a new centrifuge tube, and immediately adding 115 mu L of Tris with the pH of 8.0 for neutralization;

adding the obtained supernatant into 9mL XL1-Blue (Agilent, 200228) with OD600 of 0.5-0.7, mixing, and keeping at 37 deg.C for 30 min;

titration:

OutPut OutPut: taking the LB solution from 1 mu L to 200 mu L, then using the LB solution to carry out 100-time concentration gradient dilution, and coating an ampicillin plate;

input: taking 1 mu L to 1mL LB of the library, using LB to carry out 1000-fold gradient dilution, adding the diluted solution into 200 mu L XL1-Blue, and coating an ampicillin plate;

removing supernatant from the above step at 3000RPM at 4 deg.C for 15min, adding 200 μ L LB, mixing, and coating Amp large plate;

the plate was placed at 37 ℃ overnight and the large plate at 30 ℃ overnight;

1.1.2.2 the next day

Scraping a large plate by using a coating rod, scraping by using 25mL of LB, and washing by using 5mL of LB;

measuring an OD value;

diluting appropriate volume of scraper solution with LB (LB containing 10% Glucose, 100. mu.g/mL Amp, 5. mu.g/mL Tet) to 100mL, OD600 of about 0.1, 37 ℃ for 2h to OD 6000.5;

adding 1.5mL of Helper phase (Thermo Fisher, 18311019), shaking at 37 deg.C for 0.5h every 10 min;

measuring OD600 at 37 deg.C and 200RPM for 1.5h, and diluting to OD 6000.8;

resuspending 50mL of LB at 4 ℃ and 3000RPM for 15min, and centrifuging again;

50mLLB, taking 40mL into a new bottle (LB containing 100. mu.g/mL Amp, 5. mu.g/mL Tet, 35. mu.g/mL Kan);

standing overnight at 30 ℃;

1.1.2.3 day three

Taking the bacteria shaken every yesterday, at 4 ℃, 5000RPM, for 15 min;

adding the supernatant into a new centrifugal barrel, adding PEG-NaCl (1: 4), and freezing for 1 h;

at 4 ℃, 14000g for 30min, and discarding the supernatant;

14000g at 4 ℃ for 5min, and discarding the supernatant;

2mL of 1% BSA-PBS for resuspension, 14000g, 5min, 2 times, and taking 200 mu L for the next round of screening, which is one round of complete screening;

repeating the previous screening step;

1.1.2.4 day four

Repeating a new round of screening;

1.1.2.5 fifth day

Repeating a new round of screening;

1.1.2.6 day six

And repeating a new round of screening, and completing four rounds of screening in six days.

The yield and input rate were calculated and the results are shown in the following table:

TABLE 1 output to input ratio

Sequencing the obtained clone phagemid to obtain the nucleotide sequence information of the variable region sequences of the heavy chain and the light chain, wherein the nucleotide sequences of the variable region of the light chain and the variable region of the heavy chain are respectively SEQ ID No.15 and SEQ ID No.16, and the sequencing results of E-1C1 are shown as follows:

B09_S148806_S625854a_EGP-1-1-C1_PGMF，(SEQ ID NO：18)：

TGCATTAGGAGGATTTAAATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGCATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAGTGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGCGGCGGCGGCTCTGGCGGAGGTGGCAGCGGCGGTGGCGGATCCTCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTGGCCTCGGGGGCCTGGTCGACTACAAAGATGACGATGACAAATAGACTAGTGGCCAGGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAGATGGCAACGCTAATAAGGGGCTATGACCGAA

1.2a the cloned phagemid (VH-VL, SEQ. ID No.17) and the target vector plasmid pFase-Fc (InvivoGen, pFuse-hg1Fc2) were digested with the endonuclease SfiI (Thermo Fisher, FD1824, 1. mu.L/reaction) for 30min at 37 ℃;

1.2b using the screened phagemid (SEQ. ID No.18) as template, and performing gene synthesis (GENEray) on the expression sequence according to the conventional method (EcoRI and XhoI restriction sites are introduced on both sides) to obtain a heavy chain variable region-heavy chain constant region (VH-CH, SEQ. ID No.19), a light chain variable region-light chain constant region (VL-CL, SEQ. ID No.20), using the endonuclease EcoRI (Thermo Fisher, FD0274, 1 uL/reaction), XhoI (Thermo Fisher, FD0694, 1 uL/reaction) to cleave VH-CH, VL-CL and the target plasmid pCAGGS (Addge, 12445), 37 ℃, 30 min;

1.3 performing DNA gel electrophoresis, cutting the target antibody fragment (ScFv form VH-VL, SEQ. ID No.17 and IgG form heavy chain full length VH-CH, SEQ. ID No.19, light chain full length VL-CL, SEQ. ID No.20) and target vector plasmids (pFase-Fc and pCAGGS, wherein pFase-Fc is used for connecting with VH-VL, and pCAGGS is used for connecting with VH-CH and VL-CL) and recovering the target antibody fragment and the vector fragment by using a gel recovery kit (CWBIO, 2302 CW);

SEQ ID No.17(E-1C1 ScFv，VH-VL)：

ATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGCATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAGTGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGCGGCGGCGGCTCTGGCGGAGGTGGCAGCGGCGGTGGCGGATCCTCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT。

SEQ.ID No.19(E-1C1VH-CH)：

GAATTCGCCACCatggagttcggcctgagctgggtgttcctggtggccatcatcaagggcgtgcaatgccagATGGCACAGGTCCAGCTTGTGCAGTCTGGGGCTGAAGTCAAGAAGCCTGGGGCCTCAGTGAAGGCTTCCTGCAAGGCATCTGGATATACCTTCACCAGCTACTATATCTCCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAATCCTGGTGATGCTGGCACAACCTACGCACAGAAGTTTCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGGCGTGTATTACTGTGCGCGGGGTAGGAGTGGCTGGTACGGTATGGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAgcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaacgcgtgGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGACTCGAG。

SEQ.ID No.20(E-1C1 VL-CL)：

GAATTCGCCACCatggcotgggctctgctattcctcaccctcctcactcagggcacagggtcctgggccTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCAcaccaccaceaccaccacTAACTCGAG。

1.4 use the ligation kit (Thermo Fisher, 15224041) to ligate the antibody fragment of interest to the vector fragment at 22 ℃ for 60 min;

1.5 the ligation product obtained was transformed into DH 5. alpha. competent (CWBIO, CW0808, 100. mu.L) and placed on ice for 30 min;

heat shock at 1.642 deg.C for 1 min;

1.7 standing on ice for 1 min;

1.8 adding LB culture medium to 1mL, shaking bacteria at 37 deg.C and 200RPM for 60 min;

1.9 taking 200. mu.L of the suspension and coating on an LB plate (ampicillin resistance) at 37 ℃ overnight;

1.10 selecting a yesterday plate monoclonal, adding glycerol to perform monoclonal bacteria preservation (the final concentration of the glycerol is 10%) and sequencing, and comparing the monoclonal sequences to obtain a monoclonal (SEQ.ID NO.17, SEQ.ID NO.19 and SEQ.ID NO.20) with a correct sequence;

1.11 inoculating the correctly cloned glycerol bacteria into LB culture medium according to the volume ratio of 1: 1000, shaking the bacteria, and standing overnight at 37 ℃;

1.12 extraction of plasmids using the Large upgrade plasmid kit (TIANGEN, DP 117);

1.13 transfection of 293T cells (purchased from PEI-Aldrich, P3143, Mass: PEI: plasmid, 3: 1) with PEI

CRL-3216)；

After 1.14 days, the supernatant of 293T cells was collected, filtered through a 0.22 μm filter, and suspended on a proA column (GE Healthcare, 17-0403-01) at 2 mL/min;

1.15 elution of antibody with AKTA pure (GE Healthcare, 17-0403-01) followed by neutralization with 1M Tris (pH8.0);

1.16 concentrating the obtained protein, and changing the solution into a PBS solvent;

1.17SDS-PAGE electrophoresis to determine whether the obtained protein (IgG form as well as ScFv-Fc form) is an antibody protein.

The monoclonal antibody of the strain is named as E-1C1(SEQ.ID NO.17, SEQ.ID NO.19 and SEQ.ID NO.20) and is a human monoclonal antibody; the amino acid sequences of the light chain hypervariable regions CDR1, CDR2 and CDR3 are respectively SEQ ID No.3, DDS (E-1C1CDR-L2, AspPasp Ser) and SEQ ID No.5, and the amino acid sequences of the heavy chain hypervariable regions CDR1, CDR2 and CDR3 are respectively SEQ ID No.6, SEQ ID No.7 and SEQ ID No. 8; the amino acid sequence of the light chain variable region is SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is SEQ ID No. 2; the amino acid sequences of the light chain framework regions FR1, FR2 and FR3 are SEQ D No.9, SEQ ID No.10 and SEQ ID No.11, respectively; the amino acid sequences of the heavy chain framework regions FR1, FR2 and FR3 are SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14, respectively.

1.18ELISA

Coating 200 ng/well of antigen protein in 96-well ELISA plate, 100. mu.L/well, 4 ℃ overnight; washing with 200 μ L/well PBS 3 times; sealing with 200 μ L/hole of 5% skimmed milk powder-PBST at room temperature for 1 h; E-1C1(ScFv-Fc format and IgG format) diluted in a 10-fold concentration gradient, 100. mu.L/well (starting concentration 100. mu.g/ml) was added and incubated at room temperature for 1 h; washing with 200 μ L/hole PBST for 3 times; adding 100 mu L/well of HRP-goat anti-human secondary antibody (ZSBB-BIO, ZB-2304), and incubating at room temperature for 1 h; washing with 200 μ L/hole PBST for 3 times; adding TMB color development liquid (Beyotime, P0209) at 50 μ L/hole for 2 min; 2M H was added₂Stopping the reaction by SO 450 mu L/hole; reading an OD450 value; data analysis was performed using Graphpad Prism 5 and EC50 was calculated.

As a result, as shown in FIG. 1, both forms of the antibody were able to bind GP proteins well, and the IgG form of the antibody was able to bind secreted GP proteins (sGP, NCBI GenBank: U23187.1, and ssGP, NCBI GenBank: NP-066248, constructed by expression on insect expression plasmids), and Table 2 shows ScFv-Fc and half effect concentration EC50 of IgG form and Ebola virus GP of E-1C 1.

TABLE 2

Example 2 antigen antibody affinity assay

The ScFv-Fc form of E-1C1 (4.125. mu.g/mL) obtained in example 1 was immobilized by affinity to a Protein A probe (PALL forteBio, 18-5010) for 500s using OCTET technique to reach a response value of 1-2nM and the liquid phase antigen Protein Ebola virus GP was diluted 2-fold (concentration range 3.125nM-200 nM). Kinetic parameter measurements were performed with an association time of 500s and an dissociation time of 500 s. Then utilize

Affinity was calculated by Software, and Table 3 shows the results of affinity determination of ScFv-Fc form of E-1C1 with Ebola virus GP, each at 3.93 nM.

TABLE 3

Example 3 IFC assay of antibodies with cell surface GP proteins

In 293T cells (

CRL-3216) was transiently transfected with GP protein expression vector pEGFP-N1-GP (pEGFP-N1, BD Biosciences, #6085-1) (GP sequence synthesized by GENEray, NCBI GenBank: AHX24658.2 was cut with XhoI and BamHI sites at both ends, inserted into pEGFP-N1, SEQ. ID No.4), and 24 hours later, ScFv-Fc form of E-1C1 obtained in example 1 was used as a primary antibody (10. mu.g/mL, 200. mu.L), incubated with 293T cells transfected to express GP protein, and then Goat anti Human-PE fluorescent antibody (eBioscience) was used^TM12-4998-82, 1: 200, 200. mu.L), and observing and shooting by using a fluorescence microscope, and determining the in situ binding activity of the screened antibody and GP, the result is shown in figure 1 that E-1C1 has the binding activity with GP protein expressed on the surface of the cell.

SEQ.ID NO.4(EGFP-N1-GP)：

ctcgagaccaccATGGGCGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGATGTCGACAAACTAGTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTGAATCTCGAAGGGAATGGAGTGGCAACTGACGTGCCATCTGCAACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGTGAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTCCATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAGGCTACCGGTTTTGGAACCAATGAGACAGAGTACTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAACTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCCTTCTGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTGTCTTTCACAGTTGTATCAAACGGAGCCAAAAACATCAGTGGTCAGAGTCCGGCGCGAACTTCTTCCGACCCAGGGACCAACACAACAACTGAAGACCACAAAATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGCACAGTCAAGGAAGGGAAGCTGCAGTGTCGCATCTAACAACCCTTGCCACAATCTCCACGAGTCCCCAATCCCTCACAACCAAACCAGGTCCGGACAACAGCACCCATAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAGTTGAACAACATCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCTCTGCCACGACCGCAGCCGGACCCCCAAAAGCAGAGAACACCAACACGAGCAAGAGCACTGACTTCCTGGACCCCGCCACCACAACAAGTCCCCAAAACCACAGCGAGACCGCTGGCAACAACAACACTCATCACCAAGATACCGGAGAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAAIACTATTGCTGGAGTCGCAGGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAAGCAATTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGACTGGCCTGGATACCATATTTCGGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGTTTAATCTGTGGGTTGAGACAGCTGGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACTGAGCTACGCACCTTTTCAATCCTCAACCGTAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGGCACATGCCACATTCTGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATTGCAGTTATCGCTTTATTCTGTATATGCAAATTTGTCTTTggggatccACCGGTCGCCACCATGGTG。

EXAMPLE 4 Western Blot assay for the detection of GP proteins with antibodies

GP protein samples (4: 1 volume ratio mixed with 5 Loading buffer (Beyotime, P0015), boiling water bath 10min), SDS-PAGE gel (5. mu.g/mL, 10. mu.L), membrane transfer (nitrocellulose membrane, Thermo Fisher, IB301001), incubation at room temperature for 1h using the IgG form of E-1C1 (10. mu.g/mL, 1mL) obtained in example 1 as primary antibody, followed by incubation at room temperature for 1h using Goat anti Human-HRP (ZSBB-BIO, ZDR-5301, 1: 2000, 1mL) as secondary antibody, incubation at room temperature for 1h, and finally visualization using CN/DAB Substrate Kit (Thermo Fisher, 34000, 2mL) resulted in the detection of GP protein at Western Blot level using E-1C1 as shown in FIG. 3.

The embodiments show that the antibody is derived from normal human PBMC, and can well bind to Ebola virus envelope protein GP in ELISA, OCTET, IFC and Western Blot tests, so that the antibody has certain economic and social significance.

The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Sequence listing

<110> Shanghai science and technology university

<120> Zaire type Ebola virus detection antibody, preparation method and application thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 108

<212> PRT

<213> human (Homo sapiens)

<400> 1

Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr

1 5 10 15

Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr Asp

35 40 45

Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn

50 55 60

Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His Trp

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105

<210> 2

<211> 121

<212> PRT

<213> human (Homo sapiens)

<400> 2

Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

1 5 10 15

Gly Ala Ser Val Lys Ala Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

20 25 30

Ser Tyr Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

35 40 45

Trp Met Gly Ile Ile Asn Pro Gly Asp Ala Gly Thr Thr Tyr Ala Gln

50 55 60

Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr

65 70 75 80

Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Gly Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Arg Ser Gly Trp Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 3

<211> 6

<212> PRT

<213> human (Homo sapiens)

<400> 3

Asn Ile Gly Ser Lys Ser

1 5

<210> 4

<211> 2067

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctcgagacca ccatgggcgt tacaggaata ttgcagttac ctcgtgatcg attcaagagg 60

acatcattct ttctttgggt aattatcctt ttccaaagaa cattttccat cccacttgga 120

gtcatccaca atagcacatt acaggttagt gatgtcgaca aactagtttg tcgtgacaaa 180

ctgtcatcca caaatcaatt gagatcagtt ggactgaatc tcgaagggaa tggagtggca 240

actgacgtgc catctgcaac taaaagatgg ggcttcaggt ccggtgtccc accaaaggtg 300

gtcaattatg aagctggtga atgggctgaa aactgctaca atcttgaaat caaaaaacct 360

gacgggagtg agtgtctacc agcagcgcca gacgggattc ggggcttccc ccggtgccgg 420

tatgtgcaca aagtatcagg aacgggaccg tgtgccggag actttgcctt ccataaagag 480

ggtgctttct tcctgtatga tcgacttgct tccacagtta tctaccgagg aacgactttc 540

gctgaaggtg tcgttgcatt tctgatactg ccccaagcta agaaggactt cttcagctca 600

caccccttga gagagccggt caatgcaacg gaggacccgt ctagtggcta ctattctacc 660

acaattagat atcaggctac cggttttgga accaatgaga cagagtactt gttcgaggtt 720

gacaatttga cctacgtcca acttgaatca agattcacac cacagtttct gctccagctg 780

aatgagacaa tatatacaag tgggaaaagg agcaatacca cgggaaaact aatttggaag 840

gtcaaccccg aaattgatac aacaatcggg gagtgggcct tctgggaaac taaaaaaaac 900

ctcactagaa aaattcgcag tgaagagttg tctttcacag ttgtatcaaa cggagccaaa 960

aacatcagtg gtcagagtcc ggcgcgaact tcttccgacc cagggaccaa cacaacaact 1020

gaagaccaca aaatcatggc ttcagaaaat tcctctgcaa tggttcaagt gcacagtcaa 1080

ggaagggaag ctgcagtgtc gcatctaaca acccttgcca caatctccac gagtccccaa 1140

tccctcacaa ccaaaccagg tccggacaac agcacccata atacacccgt gtataaactt 1200

gacatctctg aggcaactca agttgaacaa catcaccgca gaacagacaa cgacagcaca 1260

gcctccgaca ctccctctgc cacgaccgca gccggacccc caaaagcaga gaacaccaac 1320

acgagcaaga gcactgactt cctggacccc gccaccacaa caagtcccca aaaccacagc 1380

gagaccgctg gcaacaacaa cactcatcac caagataccg gagaagagag tgccagcagc 1440

gggaagctag gcttaattac caatactatt gctggagtcg caggactgat cacaggcggg 1500

agaagaactc gaagagaagc aattgtcaat gctcaaccca aatgcaaccc taatttacat 1560

tactggacta ctcaggatga aggtgctgca atcggactgg cctggatacc atatttcggg 1620

ccagcagccg agggaattta catagagggg ctaatgcaca atcaagatgg tttaatctgt 1680

gggttgagac agctggccaa cgagacgact caagctcttc aactgttcct gagagccaca 1740

actgagctac gcaccttttc aatcctcaac cgtaaggcaa ttgatttctt gctgcagcga 1800

tggggcggca catgccacat tctgggaccg gactgctgta tcgaaccaca tgattggacc 1860

aagaacataa cagacaaaat tgatcagatt attcatgatt ttgttgataa aacccttccg 1920

gaccaggggg acaatgacaa ttggtggaca ggatggagac aatggatacc ggcaggtatt 1980

ggagttacag gcgttataat tgcagttatc gctttattct gtatatgcaa atttgtcttt 2040

ggggatccac cggtcgccac catggtg 2067

<210> 5

<211> 11

<212> PRT

<213> human (Homo sapiens)

<400> 5

Gln Val Trp Asp Ser Ser Ser Asp His Trp Val

1 5 10

<210> 6

<211> 8

<212> PRT

<213> human (Homo sapiens)

<400> 6

Gly Tyr Thr Phe Thr Ser Tyr Tyr

1 5

<210> 7

<211> 8

<212> PRT

<213> human (Homo sapiens)

<400> 7

Ile Asn Pro Gly Asp Ala Gly Thr

1 5

<210> 8

<211> 12

<212> PRT

<213> person (ARGRSGWYGMDV)

<400> 8

Ala Arg Gly Arg Ser Gly Trp Tyr Gly Met Asp Val

1 5 10

<210> 9

<211> 24

<212> PRT

<213> human (Homo sapiens)

<400> 9

Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr

1 5 10 15

Ala Arg Ile Thr Cys Gly Gly Asn

20

<210> 10

<211> 17

<212> PRT

<213> human (Homo sapiens)

<400> 10

Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val

1 5 10 15

Tyr

<210> 11

<211> 36

<212> PRT

<213> human (Homo sapiens)

<400> 11

Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly

1 5 10 15

Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala

20 25 30

Asp Tyr Tyr Cys

35

<210> 12

<211> 27

<212> PRT

<213> human (Homo sapiens)

<400> 12

Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

1 5 10 15

Gly Ala Ser Val Lys Ala Ser Cys Lys Ala Ser

20 25

<210> 13

<211> 17

<212> PRT

<213> human (Homo sapiens)

<400> 13

Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10 15

Ile

<210> 14

<211> 38

<212> PRT

<213> human (Homo sapiens)

<400> 14

Thr Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr

1 5 10 15

Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Gly Val Tyr Tyr Cys

35

<210> 15

<211> 324

<212> DNA

<213> human (Homo sapiens)

<400> 15

tatgtgctga ctcagccacc ctcggtgtca gtggccccag gacagacggc caggattacc 60

tgtgggggaa acaacattgg aagtaaaagt gtgcactggt accagcagaa gccaggccag 120

gcccctgtgc tggtcgtcta tgatgatagc gaccggccct cagggatccc tgagcgattc 180

tctggctcca actctgggaa cacggccacc ctgaccatca gcagggtcga agccggggat 240

gaggccgact attactgtca ggtgtgggat agtagtagtg atcattgggt gttcggcgga 300

gggaccaagc tgaccgtcct aggt 324

<210> 16

<211> 363

<212> DNA

<213> human (Homo sapiens)

<400> 16

atggcacagg tccagcttgt gcagtctggg gctgaagtca agaagcctgg ggcctcagtg 60

aaggcttcct gcaaggcatc tggatatacc ttcaccagct actatatctc ctgggtgcga 120

caggcccctg gacaagggct tgagtggatg ggaataatca atcctggtga tgctggcaca 180

acctacgcac agaagtttca gggcagagtc accatgacca gggacacgtc cacgagcaca 240

gtctacatgg agctgagcag cctgagatct gaagacacgg gcgtgtatta ctgtgcgcgg 300

ggtaggagtg gctggtacgg tatggatgtc tggggccaag ggaccacggt caccgtctcc 360

tca 363

<210> 17

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atggcacagg tccagcttgt gcagtctggg gctgaagtca agaagcctgg ggcctcagtg 60

aaggcttcct gcaaggcatc tggatatacc ttcaccagct actatatctc ctgggtgcga 120

caggcccctg gacaagggct tgagtggatg ggaataatca atcctggtga tgctggcaca 180

acctacgcac agaagtttca gggcagagtc accatgacca gggacacgtc cacgagcaca 240

gtctacatgg agctgagcag cctgagatct gaagacacgg gcgtgtatta ctgtgcgcgg 300

ggtaggagtg gctggtacgg tatggatgtc tggggccaag ggaccacggt caccgtctcc 360

tcaggcggcg gcggctctgg cggaggtggc agcggcggtg gcggatcctc ctatgtgctg 420

actcagccac cctcggtgtc agtggcccca ggacagacgg ccaggattac ctgtggggga 480

aacaacattg gaagtaaaag tgtgcactgg taccagcaga agccaggcca ggcccctgtg 540

ctggtcgtct atgatgatag cgaccggccc tcagggatcc ctgagcgatt ctctggctcc 600

aactctggga acacggccac cctgaccatc agcagggtcg aagccgggga tgaggccgac 660

tattactgtc aggtgtggga tagtagtagt gatcattggg tgttcggcgg agggaccaag 720

ctgaccgtcc taggt 735

<210> 18

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgcattagga ggatttaaat gaaataccta ttgcctacgg cagccgctgg attgttatta 60

ctcgcggccc agccggccat ggcacaggtc cagcttgtgc agtctggggc tgaagtcaag 120

aagcctgggg cctcagtgaa ggcttcctgc aaggcatctg gatatacctt caccagctac 180

tatatctcct gggtgcgaca ggcccctgga caagggcttg agtggatggg aataatcaat 240

cctggtgatg ctggcacaac ctacgcacag aagtttcagg gcagagtcac catgaccagg 300

gacacgtcca cgagcacagt ctacatggag ctgagcagcc tgagatctga agacacgggc 360

gtgtattact gtgcgcgggg taggagtggc tggtacggta tggatgtctg gggccaaggg 420

accacggtca ccgtctcctc aggcggcggc ggctctggcg gaggtggcag cggcggtggc 480

ggatcctcct atgtgctgac tcagccaccc tcggtgtcag tggccccagg acagacggcc 540

aggattacct gtgggggaaa caacattgga agtaaaagtg tgcactggta ccagcagaag 600

ccaggccagg cccctgtgct ggtcgtctat gatgatagcg accggccctc agggatccct 660

gagcgattct ctggctccaa ctctgggaac acggccaccc tgaccatcag cagggtcgaa 720

gccggggatg aggccgacta ttactgtcag gtgtgggata gtagtagtga tcattgggtg 780

ttcggcggag ggaccaagct gaccgtccta ggtggcctcg ggggcctggt cgactacaaa 840

gatgacgatg acaaatagac tagtggccag gagggtggtg gctctgaggg tggcggttct 900

gagggtggcg gctctgaggg aggcggttcc cggtggtggc tctggttccg gtgattttga 960

ttatgaaaga tggcaacgct aataaggggc tatgaccgaa 1000

<210> 19

<211> 1434

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gaattcgcca ccatggagtt cggcctgagc tgggtgttcc tggtggccat catcaagggc 60

gtgcaatgcc agatggcaca ggtccagctt gtgcagtctg gggctgaagt caagaagcct 120

ggggcctcag tgaaggcttc ctgcaaggca tctggatata ccttcaccag ctactatatc 180

tcctgggtgc gacaggcccc tggacaaggg cttgagtgga tgggaataat caatcctggt 240

gatgctggca caacctacgc acagaagttt cagggcagag tcaccatgac cagggacacg 300

tccacgagca cagtctacat ggagctgagc agcctgagat ctgaagacac gggcgtgtat 360

tactgtgcgc ggggtaggag tggctggtac ggtatggatg tctggggcca agggaccacg 420

gtcaccgtct cctcagcgag caccaaaggc ccgagcgtgt ttccgctggc gccgagcagc 480

aaaagcacca gcggcggcac cgcggcgctg ggctgcctgg tgaaagatta ttttccggaa 540

ccggtgaccg tgagctggaa cagcggcgcg ctgaccagcg gcgtgcatac ctttccggcg 600

gtgctgcaga gcagcggcct gtatagcctg agcagcgtgg tgaccgtgcc gagcagcagc 660

ctgggcaccc agacctatat ttgcaacgtg aaccataaac cgagcaacac caaagtggat 720

aaacgcgtgg agcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 780

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 840

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 900

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 960

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1020

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1080

gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1140

ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1200

tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1260

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1320

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1380

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatgact cgag 1434

<210> 20

<211> 738

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gaattcgcca ccatggcctg ggctctgcta ttcctcaccc tcctcactca gggcacaggg 60

tcctgggcct atgtgctgac tcagccaccc tcggtgtcag tggccccagg acagacggcc 120

aggattacct gtgggggaaa caacattgga agtaaaagtg tgcactggta ccagcagaag 180

ccaggccagg cccctgtgct ggtcgtctat gatgatagcg accggccctc agggatccct 240

gagcgattct ctggctccaa ctctgggaac acggccaccc tgaccatcag cagggtcgaa 300

gccggggatg aggccgacta ttactgtcag gtgtgggata gtagtagtga tcattgggtg 360

ttcggcggag ggaccaagct gaccgtccta ggtggtcagc ccaaggctgc cccctcggtc 420

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 480

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 540

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 600

agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 660

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc acaccaccac 720

caccaccact aactcgag 738

Claims

1. A Zaire type Ebola virus detection antibody, which is characterized in that the Zaire type Ebola virus detection antibody is a human monoclonal antibody; the amino acid sequences of the light chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ ID No.3, DDS and SEQ ID No.5, and the amino acid sequences of the heavy chain hypervariable region CDR1, CDR2 and CDR3 are respectively SEQ ID No.6, SEQ ID No.7 and SEQ ID No. 8.

2. The Zaire-type ebola virus detection antibody of claim 1, wherein the amino acid sequence of the light chain variable region of said Zaire-type ebola virus detection antibody is SEQ ID No.1 and the amino acid sequence of the heavy chain variable region is SEQ ID No. 2.

3. The Zaire-type ebola virus detection antibody of claim 1, wherein the amino acid sequences of light chain framework regions FR1, FR2 and FR3 of said Zaire-type ebola virus detection antibody are SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, respectively; the amino acid sequences of the heavy chain framework regions FR1, FR2 and FR3 are SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14, respectively.

4. The Zaire-type ebola virus detection antibody of claim 1, wherein the nucleotide sequences encoding the light chain variable region and the heavy chain variable region of said Zaire-type ebola virus detection antibody are SEQ ID No.15 and SEQ ID No.16, respectively.

5. An expression vector, characterized in that the expression vector comprises the nucleotide sequence of SEQ ID No.15 and the nucleotide sequence of SEQ ID No. 16.

6. A host cell comprising the expression vector of claim 5.

7. The method for producing the Zaire-type ebola virus detection antibody of claims 1-4, comprising the steps of: connecting the nucleotide sequences containing SEQ ID No.15 and SEQ ID No.16 with an expression vector, amplifying, transfecting 293T cells, collecting the supernatant of the 293T cells, hanging a proA column, and eluting to obtain the Zaire type Ebola virus detection antibody.

8. The method of claim 7, wherein the expression vector is a mammalian expression vector.

9. The method of claim 8, wherein the mammalian expression vector is a pFuse-Fc or pCAGGS mammalian expression vector.

10. Use of the Zaire-type ebola virus detection antibody of claims 1-4 in the preparation of single chain antibodies with affinity for the GP antigen of ebola virus and in the preparation of detection reagents for detecting ebola virus.