阅读说明：本技术 一种抗曲霉菌半乳甘露聚糖的单克隆抗体及其应用 (Anti-aspergillus galactomannan monoclonal antibody and application thereof ) 是由 刘春龙 翟栓柱 付成华 张舟 盛长忠 周泽奇 粟艳 于 2019-12-17 设计创作，主要内容包括：本发明提供了一种抗曲霉菌半乳甘露聚糖的单克隆抗体及其应用,所述单克隆抗体的重链可变区包括如SEQ ID NO:7所示的氨基酸序列；所述单克隆抗体的轻链可变区包括如SEQ ID NO:8所示的氨基酸序列。本发明的单克隆抗体从新西兰大耳兔内获得,对曲霉菌的半乳甘露聚糖具有较强的结合活性和中和活性,稳定性好,可潜在地应用于曲霉菌感染的临床样本的检测与鉴定以及曲霉菌抑制剂研发等。(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.)

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

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. 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.

3. A monoclonal antibody against aspergillus galactomannan, wherein the monoclonal antibody comprises the antigen binding fragment of claim 1 or 2;

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;

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.

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

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.

5. A nucleic acid molecule comprising a DNA fragment encoding the antigen-binding fragment of claim 1 or 2 and/or the heavy chain variable region and/or the light chain variable region of the monoclonal antibody of claim 3;

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 the constant region of rabbit IgG 1.

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 claim 1 or 2, the monoclonal antibody of claim 3, the hybridoma cell 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, or a combination of at least two of, the antigen-binding fragment of claim 1 or 2, the monoclonal antibody of claim 3, the hybridoma cell of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7;

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.

10. Use of the antigen-binding fragment of claim 1 or 2, the monoclonal antibody of claim 3, the hybridoma cell of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, the pharmaceutical composition of claim 8, or the kit of claim 9 for the preparation of a medicament and/or a detection reagent for the treatment of an aspergillus infection disease.

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 as set forth in SEQ ID NO. 5, light chain CDR1 as set forth 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.