CN110894236B

CN110894236B - Anti-aspergillus galactomannan monoclonal antibody and application thereof

Info

Publication number: CN110894236B
Application number: CN201911305161.0A
Authority: CN
Inventors: 刘春龙; 翟栓柱; 付成华; 张舟; 盛长忠; 周泽奇; 粟艳
Original assignee: Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2021-01-01
Anticipated expiration: 2039-12-17
Also published as: CN110894236A

Abstract

The invention provides a monoclonal antibody of anti-aspergillus galactomannan and application thereof, wherein a heavy chain variable region of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 7; the variable region of the light chain of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 8. The monoclonal antibody is obtained from New Zealand big ear rabbits, has strong binding activity and neutralizing activity on galactomannan of aspergillus, has good stability, and can be potentially applied to detection and identification of clinical samples infected by aspergillus, development of aspergillus inhibitors and the like.

Description

Anti-aspergillus galactomannan monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a monoclonal antibody of aspergillus-resistant galactomannan and application thereof.

Background

In recent years, the incidence and mortality of deep fungal infections have increased year by year due to the large number of uses of broad-spectrum antibacterial drugs, and the proliferation of patients with tumors and organ transplants. Aspergillus fumigatus (Aspergillus fumigatus) is a opportunistic pathogen that often infects immunocompromised or immunocompromised patients, and is becoming an important pathogenic fungus. The detection gold standard of invasive aspergillus infection is tissue biopsy and sterile body fluid culture, however, after the infection of aspergillus, the disease condition is developed quickly, the traditional culture method has a long period and low positive detection rate, the phenomena of missed diagnosis and misdiagnosis are often caused, and the disease condition is easily delayed. Therefore, there is a need to establish a new diagnostic method for fungal infection.

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone and directed only to a specific epitope, and are generally prepared by using hybridoma cells, and after sensitized B cells having the ability to secrete specific antibodies and myeloma cells having an unlimited reproductive ability are fused into B cell hybridomas based on a cell fusion technique, and cultured into a cell population, specific antibodies directed to one epitope, i.e., monoclonal antibodies, can be prepared.

The antibody detection method is becoming a new diagnosis method for fungal infection due to its high detection speed and high accuracy. CN109609466A discloses a mouse aspergillus-resistant polysaccharide hybridoma cell strain, a monoclonal antibody and application thereof; CN109628410A discloses a mouse anti-aspergillus polysaccharide hybridoma cell strain, monoclonal antibodies and application, wherein the obtained mouse-derived aspergillus polysaccharide monoclonal antibodies and aspergillus polysaccharide have high titer and strong binding specificity and can be used for detecting aspergillus polysaccharide, but the two monoclonal antibodies are mouse-derived monoclonal antibodies, and although the mouse-derived monoclonal antibodies belong to the most widely used antibodies, the problem of weak affinity still exists.

Therefore, the construction of a new monoclonal antibody against aspergillus has wide application prospect in the field of diagnosis and treatment of fungal infection.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a monoclonal antibody of aspergillus-resisting galactomannan and a preparation method and application thereof, wherein the monoclonal antibody is a rabbit-derived monoclonal antibody, has the characteristics of multiple antigen recognition sites, good specificity and high affinity, solves the problem of the practical application of the mouse-derived monoclonal antibody, and provides a new scheme for establishing aspergillus 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. 3;

the light chain variable region of the antigen-binding fragment comprises the light chain CDR3 shown in SEQ ID NO. 6;

SEQ ID NO:3：SKTSTTVDLKM；

SEQ ID NO:6：YSNNRLANCQ。

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. 1;

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. 4;

SEQ ID NO:2：TGYANWTSPTTED；

SEQ ID NO:1：YITNYYYVRQ；

SEQ ID NO:5：DAATYYTPSSTIDSQKP；

SEQ ID NO:4：QSEACAGYKYTGTWY。

in the invention, a new zealand big ear rabbit is selected as an experimental animal, and the aspergillus galactomannan is used as an antigen for immunization, so that the obtained monoclonal antibody has good specificity, strong affinity and high homology with human.

In a second aspect, the present invention provides a monoclonal antibody against a aspergillus galactomannan, the monoclonal antibody comprising an antigen binding fragment as described in the first aspect.

Preferably, the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 7;

the variable region of the light chain of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 8;

SEQ ID NO:7：

METGLRWLLLVAVLKGVQCQSVEEVSGFSLSSYITNYYYVRQAPGKSGGRLTGLEYIGMISGANTGYANWTSPTTEDTANGRFDMNWVTYVTPGTPLTLTCFCARTISKTSTTVDLKMYGMDLWGPGTLVTVSS；

SEQ ID NO:8：

MDTRAPTQLLGLLLLWLPGATFAIVMTQSEACAGYKYTGTWYQFTLTISDGSVPVCDQDAATYYTPSSTIDSQKPGSTLASGVPSRFKGSGSGTQVGDTVPPKLLIYQSVYSNNRLANCQAVIAFGGGTEVVVK。

preferably, the monoclonal antibody further comprises any one of or a combination of at least two of rabbit IgG1, IgG2, IgG3, or IgG4 constant regions, preferably rabbit IgG1 constant regions.

In a third aspect, the present invention provides a hybridoma cell which produces a monoclonal antibody as described in the second aspect.

Preferably, the hybridoma cell is named as DNK-GM2, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC No.18891 and the preservation date of 2019, 11 months and 25 days.

In a fourth 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;

SEQ ID NO:9：

ATGGAGACCGGCCTGAGGTGGCTGCTGCTGGTGGCCGTGCTGAAGGGCGTGCAGTGCCAGAGCGTGGAGGAGGTGAGCGGCTTCAGCCTGAGCAGCTACATCACCAACTACTACTACGTGAGGCAGGCCCCCGGCAAGAGCGGCGGCAGGCTGACCGGCCTGGAGTACATCGGCATGATCAGCGGCGCCAACACCGGCTACGCCAACTGGACCAGCCCCACCACCGAGGACACCGCCAACGGCAGGTTCGACATGAACTGGGTGACCTACGTGACCCCCGGCACCCCCCTGACCCTGACCTGCTTCTGCGCCAGGACCATCAGCAAGACCAGCACCACCGTGGACCTGAAGATGTACGGCATGGACCTGTGGGGCCCCGGCACCCTGGTGACCGTGAGCAGC。

preferably, the light chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 10;

SEQ ID NO:10：

ATGGACACCAGGGCCCCCACCCAGCTGCTGGGCCTGCTGCTGCTGTGGCTGCCCGGCGCCACCTTCGCCATCGTGATGACCCAGAGCGAGGCCTGCGCCGGCTACAAGTACACCGGCACCTGGTACCAGTTCACCCTGACCATCAGCGACGGCAGCGTGCCCGTGTGCGACCAGGACGCCGCCACCTACTACACCCCCAGCAGCACCATCGACAGCCAGAAGCCCGGCAGCACCCTGGCCAGCGGCGTGCCCAGCAGGTTCAAGGGCAGCGGCAGCGGCACCCAGGTGGGCGACACCGTGCCCCCCAAGCTGCTGATCTACCAGAGCGTGTACAGCAACAACAGGCTGGCCAACTGCCAGGCCGTGATCGCCTTCGGCGGCGGCACCGAGGTGGTGGTGAAG。

in a fifth aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the fourth aspect.

Preferably, the expression vector further comprises a nucleic acid molecule encoding the constant region of rabbit IgG 1.

In a sixth aspect, the present invention provides a host cell transfected with a nucleic acid molecule according to the fourth aspect and/or an expression vector according to the fifth aspect.

Preferably, the host cell comprises a 293T cell or a CHO cell.

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 hybridoma cell of the third aspect, the nucleic acid molecule of the fourth aspect, the expression vector of the fifth aspect or the host cell of the sixth 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 of, or a combination of at least two of, an antigen-binding fragment according to the first aspect, a monoclonal antibody according to the second aspect, a hybridoma cell according to the third aspect, a nucleic acid molecule according to the fourth aspect, an expression vector according to the fifth aspect, or a host cell according to the sixth aspect.

Preferably, the kit further comprises any one or combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a developing solution, a stop solution, a blocking solution or a washing solution.

In a ninth aspect, the present invention provides a use of the antigen binding fragment according to the first aspect, the monoclonal antibody according to the second aspect, the hybridoma cell according to the third aspect, the nucleic acid molecule according to the fourth aspect, the expression vector according to the fifth aspect, the host cell according to the sixth aspect, the pharmaceutical composition according to the seventh aspect, or the kit according to the eighth aspect, in the preparation of a drug for treating and/or detecting an aspergillus infection disease.

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

(1) the invention selects New Zealand big ear rabbit as experimental animal, prepares monoclonal antibody, overcomes the weak affinity defect of mouse source antibody;

(2) the rabbit-derived monoclonal antibody is obtained by taking aspergillus galactomannan as an antigen for immunization and performing cell fusion on the obtained spleen cells and myeloma cells, has good stability and strong affinity to the aspergillus galactomannan;

(3) the monoclonal antibody prepared by the invention has good specificity and strong affinity with galactomannan, but does not generate cross reaction with mannan, capsular polysaccharide, lipopolysaccharide and other saccharides, and can be potentially applied to antigen detection of aspergillus, detection and identification of clinical samples infected by aspergillus, and the like.

Drawings

FIG. 1 is an electrophoretogram of monoclonal antibody, in which lane M₁Lane 1 shows the results of monoclonal antibody electrophoresis;

FIG. 2 shows the potency assay of monoclonal antibodies against GM;

FIG. 3 is a cross-reaction diagram.

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.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 animal immunization

(1) Preparation of Aspergillus Galactomannan (GM)

Inoculating aspergillus fumigatus to a Sabouraud's liquid culture medium (containing 1% of peptone and 4% of D-glucose per liter of culture medium), culturing at 30 ℃ for about 46 hours, wherein the rotation speed of a shaking table is 200rpm/min, and stopping culturing when the pH value of a culture solution is reduced to 4.2-4.5; sterilizing at 121 deg.C for 40min to kill thallus and possible spore, filtering the obtained bacterial liquid with qualitative filter paper at room temperature, and filtering with 0.22 μm filter membrane to remove thallus;

transferring the filtrate to a new centrifuge tube, adding 4 times of volume of absolute ethyl alcohol, and standing overnight at 4 ℃; centrifuging at 4 deg.C for 15min at 16,000g, dissolving the precipitate in deionized water, adding 4 times volume of anhydrous ethanol, and standing for 1 hr;

centrifuging, washing the precipitate with anhydrous ethanol for three times, centrifuging at 4 deg.C for 15min at 16,000g, and removing the supernatant; dissolving the precipitate in deionized water, adding activated carbon powder, and stirring at room temperature for 2 h; filtering the filtrate with 0.22 μm filter membrane, transferring to 10KD centrifugal ultrafiltration tube, and centrifuging for 10min at 5000g to obtain galactomannan pure product.

(2) Animal immunization

Selecting a new zealand big ear white rabbit with the age being suitable and the weight being about 1.5 kg, feeding the new zealand big ear white rabbit in a standard animal house for 3 days, and starting to immunize if no abnormal condition exists:

adding 30 mu g of GM antigen into 0.5mL of autoclaved physiological saline, fully and uniformly mixing by using a micro vortex oscillator, adding 0.5mL of Freund's complete adjuvant, fully mixing and emulsifying by mutually pushing and pulling an injector, and carrying out back subcutaneous multi-point injection immunization on the New Zealand big ear rabbits;

boosting is carried out after two weeks, then boosting is carried out once every other week for six times, and 200-500 mu L of auricular vein blood of the white rabbits is taken after one week from the third immunization, and the titer and the affinity are measured;

after the last immunization, spleen was taken for cell fusion for preparation of hybridoma cells.

Example 2 preparation and screening of hybridoma cells

Performing titer detection on the prepared rabbit antiserum, and performing cell fusion on rabbit spleen under the qualified condition to prepare a monoclonal hybridoma cell strain by the following method:

(1) preparation of

Killing immunized New Zealand big ear rabbit, taking out spleen under aseptic condition, washing with cell culture solution for 1 time, grinding, sieving with stainless steel sieve, centrifuging the obtained cell, and washing with cell culture solution for 2 times;

mixing SP2/0 myeloma cells and spleen cells in logarithmic growth phase, washing with cell culture solution without fetal calf serum, centrifuging, removing supernatant, adding polyethylene glycol solution, and treating at 37 deg.C for about 90 s;

terminating the reaction with a cell culture medium containing no fetal calf serum, centrifuging, resuspending the cells in HAT selection medium containing 20% fetal calf serum, adding the cells to a 96-well plate, incubating at 37 deg.C and 5.0% CO₂Medium culture;

diluting cells with good growth state in 96-well plate with cell culture solution to 1-3 cells/mL, adding into 96-well plate, placing into cell culture box at 37 deg.C and 5.0% CO₂Culturing under the condition, numbering each cell strain, selecting the cell strain with positive culture solution supernatant, and performing expanded culture to obtain a hybridoma cell strain;

(2) screening

Screening the obtained hybridoma cells by adopting an ELISA method, observing the growth condition of the cells on the 5 th day after fusion, detecting the titer of cell culture supernatant by adopting an indirect ELISA method on the 10 th to 14 th days, carrying out expanded culture on the positive hybridoma cells with the strongest titer until the cell positive rate reaches 100%, carrying out strain determination to obtain a hybridoma cell strain DNK-GM2, and freezing and storing in liquid nitrogen for later use.

The hybridoma cell strain DNK-GM2 is preserved in China general microbiological culture Collection center (CGMCC) on 25.11.2019, with the address of No. 3 of Xilu No.1 of Beijing, Chaoyang, and the preservation number of 100101 of CGMCC No. 18891.

Example 3 isolation of antibody variable region genes from hybridoma cells Using RT-PCR

Homogenizing hybridoma cells, adding cell lysate to perform RNA extraction, precipitating RNA from an aqueous phase layer by using isopropanol, washing the precipitated RNA after centrifugation, removing impurities, and performing reverse transcription after heavy suspension to obtain cDNA;

PCR was carried out using specific primers from New Zealand big ear rabbits, heavy and light chain variable region genes of the antibody were amplified using hybridoma cDNA as a template, and 50. mu.L of the system contained 5. mu.L of cDNA, HotStarTaq Plus enzyme, dNTPs and 0.5. mu.M specific primers, and PCR amplification was carried out under the following conditions: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 55 ℃, 50s at 72 ℃ and 35 cycles; 7min at 72 ℃; the obtained PCR product is identified by 1% agarose gel electrophoresis, the target fragment is recovered, the sample is sent for sequencing, and the sequencing result is compared with an IMGT database (http:// www.imgt.org/IMGT _ vquest/vquest) to obtain the antibody variable region gene fragment shown as SEQ ID NO. 9-10.

EXAMPLE 4 construction of expression vectors for monoclonal antibodies

Respectively adding homologous recombination arms at two ends of the heavy chain variable region gene and two ends of the light chain variable region gene of the antibody by using homologous recombination primers, and linearizing expression plasmids containing rabbit antibody heavy and light chain IgG1 constant regions by using double enzymes to generate homologous recombination arms; the variable region gene segment added with the homologous recombination arm and the linearized plasmid are connected by a homologous recombination mode to form a complete expression vector, the recombinant product is transformed into TOP10 escherichia coli competence, and the plasmid is amplified.

EXAMPLE 5 expression and purification of monoclonal antibodies

Adding the heavy-chain expression plasmid and the light-chain expression plasmid of the monoclonal antibody obtained in the example 4 into an Opti-Mem transfection medium according to the proportion of 1:1, fully mixing, adding a transfection reagent PEI with the mass 4 times that of DNA, mixing, placing for 30min at room temperature in a dark place, and then adding into 293T cells;

after 6h incubation the transfection system was removed and FreeStyle was added^TM293 expression culture medium, using AKTA Protein purification system, adopting affinity purification (Protein A) method to purify the expressed antibody supernatant, obtaining the monoclonal antibody of the anti-aspergillus galactomannan, the concrete steps are:

(1) centrifuging the expressed antibody supernatant at 2500 Xg for 10min at room temperature, and removing the precipitate;

(2) the affinity purification column containing Protein A was extensively washed with 10 volumes of Binding Buffer;

(3) passing the expression supernatant through a purification column at a flow rate of 5 mL/min;

(4) washing the purification column thoroughly with 20 times the volume of the purification column in Binding Buffer;

(5) eluting the column with 0.1M citric acid buffer solution at pH 3.0-3.5 until the peak is at equilibrium, and adjusting pH to 7.0 with 1M Tris-HCl buffer solution at pH 9.0;

(6) and (3) concentrating the purified monoclonal antibody by using a concentration centrifugal column, using PBS as a buffer for storing the antibody, and finally determining the concentration of the concentrated antibody by using a BSA protein concentration detection method.

Example 6 molecular weight determination

The molecular weight of the monoclonal antibody is identified by SDS-PAGE electrophoresis, the sample adding amount of each electrophoresis channel is 5 mug, meanwhile, a standard series of known molecular weight is used as a reference, electrophoresis is carried out for 20min under the voltage of 90V firstly, electrophoresis is carried out under the voltage of 140V until the indicator is completely run out, the gel is taken down, Coomassie brilliant blue is used for staining, and the gel analyzes the molecular weight of the biological raw material after staining. As can be seen from FIG. 1, the heavy and light chains of the monoclonal antibody proteins are mainly distributed around 55kD and 25 kD.

Example 7 potency assay

The ELISA method is adopted to detect the affinity activity (titer) of the monoclonal antibody to aspergillus Galactomannan (GM), and the main steps are as follows:

(1) diluting GM antigen to 1 ng/mu L with PBS, adding 100 mu L of GM antigen into a 96-well enzyme label plate per well, and coating for 2h at 37 ℃;

(2) discarding the supernatant, washing the plate 3 times with 0.01M PBST, preparing a blocking solution containing 3% BSA with PBST, adding 100 μ L of the blocking solution into each well, and blocking for 2h at 37 ℃;

(3) discarding supernatant, washing with PBST for 5 times, performing gradient dilution on the purified and concentrated antibody to the concentration of 1:2560000 from 1:1000 times, adding 100 μ L of the antibody into each well, and incubating at 37 deg.C for 1 h;

(4) discarding the antibody diluent, washing with PBST for 6 times, diluting goat anti-rabbit IgG-HRP with 1:5000 blocking solution, adding 100 μ L per well, and incubating at 37 deg.C for 1 h;

(5) discarding the secondary antibody diluent, washing with PBST for 6 times, adding TMB (tetramethylbenzidine) at 100 μ L/hole, and standing at 37 ℃ in a dark place for 15 min;

(6) the reaction was stopped by adding 50. mu.L of 1M dilute sulfuric acid to each well, and the absorbance was measured at 450 nm.

As shown in FIG. 2 and Table 1, the monoclonal antibody has a strong binding ability to GM and a potency of 1:1280000(OD > 0.5) against GM antigen.

TABLE 1

Example 8 Cross-reaction

Coating the enzyme label plate with galactomannan, mannan, capsular polysaccharide, lipopolysaccharide, peptidoglycan, 1, 3-beta-D-glucan and BSA respectively, wherein the coating amount of each hole is 50 ng; diluting the monoclonal antibody to 10ng/mL, adding the diluted monoclonal antibody into each enzyme label plate, adding 100 mu L of monoclonal antibody into each hole, and incubating for 1h at 37 ℃; adding HRP (horse radish peroxidase) -labeled goat anti-rabbit secondary antibody after washing, adding 100 mu L of HRP-labeled goat anti-rabbit secondary antibody into each hole, and incubating for 0.5h at 37 ℃; after washing, TMB was added and incubation was carried out at 37 ℃ for 15min, and the reading was terminated. The results are shown in FIG. 3, which shows that the monoclonal antibody does not cross-react with other saccharides and has strong specificity.

In conclusion, the monoclonal antibody is prepared by selecting a New Zealand big ear rabbit as an experimental animal and taking the aspergillus galactomannan as an antigen for immunization, so that the defect of weak affinity of a mouse source antibody is overcome, and the obtained rabbit source monoclonal antibody has good stability and stronger affinity to the aspergillus galactomannan, and can be potentially applied to antigen detection of aspergillus, detection and identification of clinical samples infected by aspergillus, and the like.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Dana (Tianjin) Biotechnology Ltd

<120> monoclonal antibody of anti-aspergillus galactomannan and application thereof

<130> 20191217

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 10

<212> PRT

<213> Rabbit Source

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Tyr Ile Thr Asn Tyr Tyr Tyr Val Arg Gln

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<210> 2

<211> 13

<212> PRT

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<400> 2

Thr Gly Tyr Ala Asn Trp Thr Ser Pro Thr Thr Glu Asp

1 5 10

<210> 3

<211> 11

<212> PRT

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<400> 3

Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met

1 5 10

<210> 4

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Gln Ser Glu Ala Cys Ala Gly Tyr Lys Tyr Thr Gly Thr Trp Tyr

1 5 10 15

<210> 5

<211> 17

<212> PRT

<213> Rabbit Source

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Asp Ala Ala Thr Tyr Tyr Thr Pro Ser Ser Thr Ile Asp Ser Gln Lys

1 5 10 15

Pro

<210> 6

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<212> PRT

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Tyr Ser Asn Asn Arg Leu Ala Asn Cys Gln

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<210> 7

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Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly

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Tyr Ile Thr Asn Tyr Tyr Tyr Val Arg Gln Ala Pro Gly Lys Ser Gly

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Gly Arg Leu Thr Gly Leu Glu Tyr Ile Gly Met Ile Ser Gly Ala Asn

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Thr Gly Tyr Ala Asn Trp Thr Ser Pro Thr Thr Glu Asp Thr Ala Asn

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Gly Arg Phe Asp Met Asn Trp Val Thr Tyr Val Thr Pro Gly Thr Pro

85 90 95

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Ser Asp Gly Ser Val Pro Val Cys Asp Gln Asp Ala Ala Thr Tyr Tyr

50 55 60

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atggagaccg gcctgaggtg gctgctgctg gtggccgtgc tgaagggcgt gcagtgccag 60

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aggcaggccc ccggcaagag cggcggcagg ctgaccggcc tggagtacat cggcatgatc 180

agcggcgcca acaccggcta cgccaactgg accagcccca ccaccgagga caccgccaac 240

ggcaggttcg acatgaactg ggtgacctac gtgacccccg gcacccccct gaccctgacc 300

tgcttctgcg ccaggaccat cagcaagacc agcaccaccg tggacctgaa gatgtacggc 360

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tggtaccagt tcaccctgac catcagcgac ggcagcgtgc ccgtgtgcga ccaggacgcc 180

gccacctact acacccccag cagcaccatc gacagccaga agcccggcag caccctggcc 240

agcggcgtgc ccagcaggtt caagggcagc ggcagcggca cccaggtggg cgacaccgtg 300

ccccccaagc tgctgatcta ccagagcgtg tacagcaaca acaggctggc caactgccag 360

gccgtgatcg ccttcggcgg cggcaccgag gtggtggtga ag 402

Claims

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. 3;

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. 1;

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. 4.

2. A monoclonal antibody against aspergillus galactomannan comprising the antigen binding fragment of claim 1.

3. The monoclonal antibody of claim 2, wherein the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO. 7;

the variable region of the light chain of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 8.

4. The monoclonal antibody of claim 3, further comprising any one of or a combination of at least two of rabbit IgG1, IgG2, IgG3, or IgG4 constant regions.

5. The monoclonal antibody of claim 4, further comprising a rabbit IgG1 constant region.

6. A hybridoma cell that produces the monoclonal antibody of claim 3;

the hybridoma cell is named as DNK-GM2 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18891 and the preservation date of 2019, 11 months and 25 days.

7. A nucleic acid molecule comprising a DNA fragment encoding the antigen-binding fragment of claim 1 and/or the heavy chain variable region and the light chain variable region of the monoclonal antibody of any one of claims 2-5;

8. the nucleic acid molecule of claim 7, wherein the heavy chain variable region of said monoclonal antibody is encoded by the nucleic acid molecule set forth in SEQ ID NO 9;

the variable region of the light chain of the monoclonal antibody is encoded by a nucleic acid molecule shown as SEQ ID NO. 10.

9. An expression vector comprising the nucleic acid molecule of claim 7 or 8.

10. The expression vector of claim 9, further comprising a nucleic acid molecule encoding a rabbit IgG1 constant region.

11. A host cell transfected with a nucleic acid molecule according to claim 7 or 8 and/or an expression vector according to claim 9 or 10.

12. The host cell of claim 11, wherein the host cell comprises a 293T cell or a CHO cell.

13. A composition comprising any one of the antigen-binding fragment of claim 1, the monoclonal antibody of any one of claims 2-5, the hybridoma cell of claim 6, the nucleic acid molecule of claim 7 or 8, the expression vector of claim 9 or 10, or the host cell of claim 11 or 12, or a combination of at least two thereof.

14. The composition of claim 13, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

15. A kit comprising any one of the antigen binding fragment of claim 1, the monoclonal antibody of any one of claims 2-5, the hybridoma cell of claim 6, the nucleic acid molecule of claim 7 or 8, the expression vector of claim 9 or 10, or the host cell of claim 11 or 12, or a combination of at least two thereof.

16. The kit according to claim 15, wherein the kit further comprises any one or a combination of at least two of a positive control, a negative control, an antibody diluent, a developing solution, a stop solution, a blocking solution, or a washing solution.

17. Use of the antigen-binding fragment of claim 1, the monoclonal antibody of any one of claims 2-5, the hybridoma cell of claim 6, the nucleic acid molecule of claim 7 or 8, the expression vector of claim 9 or 10, the host cell of claim 11 or 12, the composition of claim 13 or 14, or the kit of claim 15 or 16 for the preparation of a reagent for the detection of an aspergillus infection.