阅读说明：本技术 一种抗隐球菌夹膜多糖的兔源单克隆抗体及其应用 (Rabbit-derived monoclonal antibody for resisting cryptococcus neoformans tunica polysaccharide and application thereof ) 是由 刘春龙 付成华 付彦凯 翟栓柱 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隐球菌荚膜多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,对隐球菌荚膜多糖具有较强的结合活性和中和活性,稳定性好,可潜在地应用于隐球菌感染的临床样本的检测与鉴定以及隐球菌抑制剂研发等。(The invention provides a monoclonal antibody against cryptococcus capsular polysaccharide 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 stronger binding activity and neutralizing activity on cryptococcus capsular polysaccharide, has good stability, and can be potentially applied to detection and identification of clinical samples of cryptococcus infection, research and development of cryptococcus 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 cryptococcus capsular polysaccharide comprising 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.

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 for the treatment and/or detection of cryptococcus infection.

Technical Field

The invention belongs to the technical field of biology, and relates to a rabbit-derived monoclonal antibody for resisting cryptococcus neoformans tunica polysaccharide and application thereof.

Background

Cryptococcus neoformans is an important opportunistic pathogenic fungus that often invades the meninges, lungs and skin, causing fungal infections. Cryptococcus Neoformans Meningitis (CNM), abbreviated as cryptobrain, is the most common fungal infection type of the central nervous system, and the incidence and mortality of CNM have a trend of obvious increase in recent years. Statistically, approximately 5% to 10% of AIDS patients in the United states have cryptoencephalic involvement, with higher rates of cryptoencephalic involvement in AIDS patients in some developing countries. Meanwhile, the number of cryptococcosis in lung caused by cryptococcus neoformans infecting respiratory system is increasing year by year.

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. CN109879961A discloses an anti-cryptococcus capsular polysaccharide monoclonal antibody and a hybridoma cell strain preparation and application thereof, wherein the antibody can be specifically combined with cryptococcus capsular polysaccharide, can be used for in vitro detection of cryptococcus infection, has antibody titer as high as more than one million, has excellent affinity, and has good specific binding capacity; the detection limit of the colloidal gold labeled immunodiagnostic reagent developed by taking the antibody as a raw material is 0.5ng/ml, and the detection reagent has better performances in the aspects of sensitivity, specificity, stability and antibody, but the monoclonal antibody is a mouse-derived monoclonal antibody, and although the mouse-derived monoclonal antibody belongs to the most widely used antibodies, the problems of weak affinity and poor specificity still exist.

Therefore, the construction of a novel monoclonal antibody against cryptococcus 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 rabbit-derived monoclonal antibody for resisting cryptococcus neoformans tunica polysaccharide and application thereof, wherein the monoclonal antibody is the 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 candida 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：KTSTSTYITNM；

SEQ ID NO:6：SSQKPATANIDDPQ。

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：PTEGYANWDTTTS；

SEQ ID NO:1：VDYYYLKVRQ；

SEQ ID NO:5：KYTGTWYCAYNNRL；

SEQ ID NO:4：AYTQSSEATSCGYY。

in the invention, a new zealand big ear rabbit is selected as an experimental animal, cryptococcus neoformans tunica polysaccharide is used as an antigen for immunization, and 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 cryptococcus neoformans capsular polysaccharide, said 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：

MEVSGFSLSSYITAPGKSGRWLVDLKMGRLTGLETGLYGMDLDTANGWGPGTLVTVSSLLVAVLKGVQCQSVEEYIGMISGANTGYANWTSPTTERFDMNWVTYVTPGTPLTLNYYYVRQTCFCARTISKTSTT；

SEQ ID NO:8：

MLLPSEACAGDQDAATYYGATLIVMGTQLWFSNNVPLTLLPKLLIYARLANCQAVIAFGGGTEVVVKTWYQFTLTISDGSVPVCYKYTGVYDSQKPSGSGTQVGSSTGSTLASGVDTQSDTRAPTPPSRFKGIQ。

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.

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：

ATGGAAGTTTCTGGTTTTTCCTTATCATCGTATATTACTGCTCCTGGCAAAAGTGGACGTTGGTTGGTCGATCTTAAGATGGGGCGCCTCACCGGTCTAGAGACAGGCCTGTACGGAATGGACTTAGATACGGCCAATGGGTGGGGTCCCGGCACTTTGGTAACCGTGAGCTCTCTTCTCGTTGCAGTCCTAAAAGGAGTACAATGTCAGTCCGTGGAAGAGTATATCGGGATGATATCAGGTGCGAACACAGGCTACGCTAATTGGACGTCGCCAACTACCGAACGATTCGACATGAACTGGGTTACATATGTCACGCCGGGAACTCCTCTGACCTTAAATTACTATTACGTACGGCAAACATGCTTTTGTGCCAGAACGATTAGTAAGACTAGCACCACA。

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：

ATGTTATTGCCTTCTGAAGCTTGTGCCGGTGATCAAGACGCAGCGACTTATTACGGCGCTACCCTTATTGTTATGGGAACACAGCTCTGGTTTTCCAATAACGTCCCCCTAACGCTGTTACCAAAATTGCTTATCTATGCCCGTCTCGCAAATTGCCAAGCGGTAATAGCTTTCGGGGGTGGCACTGAGGTGGTTGTCAAGACCTGGTACCAGTTTACACTAACGATTTCAGATGGATCGGTACCGGTGTGTTATAAATACACTGGGGTTTATGACAGTCAAAAGCCTAGCGGTTCTGGCACCCAGGTCGGATCCTCAACAGGGTCGACGCTGGCCAGTGGTGTAGATACTCAAAGCGAC361ACCCGCGCACCCACACCACCGTCTCGATTCAAAGGCATCCAG。

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 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, the host cell of the sixth aspect, the pharmaceutical composition of the seventh aspect, or the kit of the eighth aspect, in the preparation of a medicament and/or a detection reagent for treating cryptococcus 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 cryptococcus envelope polysaccharide is used as an antigen for immunization, and the obtained spleen cells are subjected to cell fusion with myeloma cells to obtain the rabbit-derived monoclonal antibody, so that the stability is good, and the cryptococcus envelope polysaccharide has strong affinity;

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

Drawings

FIG. 1 is an electrophoresis diagram of a monoclonal antibody, wherein lane M shows the molecular weight of the protein, and lane 1 shows the result of the electrophoresis of the monoclonal antibody;

FIG. 2 shows the potency detection of GXM by monoclonal antibody;

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.