阅读说明：本技术 人源降钙素原的单克隆抗体其制备方法和用途 (Monoclonal antibody of human procalcitonin and preparation method and application thereof ) 是由 叶森 唐勇 何仕钊 汪金凤 于 2015-07-16 设计创作，主要内容包括：本发明涉及与人源降钙素原蛋白特异性结合的单克隆抗体。具体地,该单克隆抗体可以特异地、高效地识别人源降钙素原的蛋白。本发明提供了分泌该单克隆抗体的保藏号为CGMCC No.10417的杂交瘤细胞系,其能够稳定分泌高效价的抗降钙素原的单克隆抗体。此外,本发明还涉及一种以降钙素原突变抗原为免疫原制备抗降钙素原抗体的方法。另外,本发明还涉及上述单克隆抗体在制备检测或者定量降钙素原或其片段的免疫检测试剂的用途,该单克隆抗体可以应用于疾病的诊断或者相关领域。(The present invention relates to monoclonal antibodies that specifically bind to human procalcitonin protein. Specifically, the monoclonal antibody can specifically and efficiently recognize the protein of the human procalcitonin. The invention provides a hybridoma cell line which secretes the monoclonal antibody and has the preservation number of CGMCC No.10417, and the hybridoma cell line can stably secrete the high-titer anti-procalcitonin monoclonal antibody. In addition, the invention also relates to a method for preparing anti-procalcitonin antibody by using the procalcitonin mutant antigen as immunogen. In addition, the invention also relates to the application of the monoclonal antibody in preparing an immunoassay reagent for detecting or quantifying procalcitonin or fragments thereof, and the monoclonal antibody can be applied to the diagnosis of diseases or related fields.)

1. A method of making an anti-procalcitonin antibody comprising the steps of:

(1) mutating N-terminal amino acid residues of procalcitonin to obtain a nucleotide sequence corresponding to the mutated amino acid residue sequence;

(2) inserting the nucleotide sequence into a prokaryotic expression vector for induced expression and protein purification;

(3) immunizing animals by taking the purified protein as an antigen, fusing spleen cells of the animals with mouse myeloma cells to prepare hybridoma cells, screening to obtain the hybridoma cells capable of stably secreting anti-procalcitonin antibodies, and obtaining corresponding monoclonal antibodies;

wherein the mutated procalcitonin amino acid sequence is SEQ ID NOs:2-4, and the anti-procalcitonin antibody is obtained from a hybridoma cell line with the preservation number of CGMCC No. 10417.

2. The method of claim 1, wherein said anti-procalcitonin antibody is a monoclonal antibody.

3. The method of claim 1, wherein the anti-procalcitonin antibody specifically binds to an epitope having amino acid sequence RLLLAALVQDYV (SEQ ID NO: 1).

Technical Field

The present invention relates to the field of medical diagnostics. Specifically, the invention relates to a hybridoma cell line capable of generating a monoclonal antibody specific to human procalcitonin and a preparation method thereof. The monoclonal antibody prepared by the hybridoma can be applied to diagnosis of diseases or related fields.

Background

Procalcitonin (PCT) is a specific marker of bacterial and fungal infections discovered only in the nineties of the twentieth century, and is a calcitonin propeptide without hormonal activity, consisting of 116 amino acids and having a molecular weight of about 13 KD. It can be cleaved stepwise by the action of an enzyme into amino procalcitonin (N-PCT) of 57 amino acids, Calcitonin (CT) of 32 amino acids and calcitonin protein of 21 amino acids. Procalcitonin is the expression product of the calcitonin I gene (CALC-I) on chromosome 11. In the absence of infection, extra-thyroid CALC-I expression is suppressed, while neuroendocrine cells, primarily confined to the thyroid and lungs, express to some extent; bacterial infection induces the expression of CALC-I and the continuous release of procalcitonin from various cell types in various tissues throughout the body. Recent studies have shown that procalcitonin is a new indicator with high sensitivity and specificity in the diagnosis of severe systemic bacterial, fungal, parasitic, acute malaria infection, Systemic Inflammatory Response Syndrome (SIRS), multiple organ failure syndrome (MODS).

Procalcitonin has a total of 116 amino acids, which can be divided into three segments according to domain and function. Bits 1-57 are its N segments. In vivo, the N-terminus will be cleaved starting at position 57 to yield amino acids 60-116 containing calcitonin. At the same time, it is also cleaved by proteases at position 91 to form calcitonin and procalcitonin (amino acids katacalcin at positions 96-116). Thus, there is a whole body of procalcitonin and its various cleaved fragments in human serum, which fragments exert physiological functions in humans different from procalcitonin.

In order to specifically and accurately diagnose and identify complete procalcitonin fragments, there are three current schemes for determining procalcitonin content. First, a polyclonal antibody against fragments 1-57 was selected for procalcitonin determination. Second, monoclonal antibodies against fragments 1-57 and monoclonal antibodies against fragments 60-116 were selected for sandwich assays. Third, monoclonal antibodies against the N-segment and calcitonin-binding region and monoclonal antibodies against the 60-116 segment were selected for sandwich assays.

Because the N-terminal of procalcitonin is unstable and easy to degrade, when the pure human procalcitonin antigen is used for immunization, the unstable antigen can generate weaker immune response, and the preparation condition of the high-efficiency antibody cannot be achieved. Therefore, there is a need in the art to solve the problem of weak immune response due to unstable antigens when immunising with pure human procalcitonin antigens.

Disclosure of Invention

In order to solve the problem that unstable antigens can generate weaker immune response when pure human procalcitonin antigen is immunized, the invention aims to provide a monoclonal antibody specifically combined with human procalcitonin protein and a preparation method thereof. The inventors have expressed this mutated protein recombinantly in vitro by mutating individual amino acids from 1 to 57 amino acids, primarily to individual amino acids from positions 19 to 40. The inventors have found that the above treatment improves the stability of the procalcitonin protein, making it less susceptible to degradation when injected into animals after co-emulsification with an adjuvant, thereby producing more potent antibodies. The inventors found that the final epitope analysis was determined to be amino acids 20-35.

The invention provides an anti-procalcitonin antibody specifically bound with human procalcitonin, wherein the anti-procalcitonin antibody specifically binds to the same procalcitonin epitope with a procalcitonin monoclonal antibody produced by a hybridoma cell line with the preservation number of CGMCC No. 10417.

In one aspect of the invention, the anti-procalcitonin antibody comprises a humanized sequence of a procalcitonin monoclonal antibody produced by a hybridoma cell line with a collection number of CGMCC No. 10417.

In another aspect of the invention, the anti-procalcitonin antibody is a monoclonal antibody.

In yet another aspect of the invention, the anti-procalcitonin antibody specifically binds to an epitope having amino acid sequence RLLLAALVQDYV (SEQ ID NO: 1).

In one embodiment of the invention, the anti-procalcitonin antibody is obtained from a hybridoma cell line having accession number cgmccno. 10417.

The invention also provides an immunoassay reagent containing the anti-procalcitonin antibody, which is used for detecting or quantifying procalcitonin or fragments thereof.

The invention also provides the application of the anti-procalcitonin antibody in preparing an immunoassay reagent for detecting or quantifying procalcitonin or fragments thereof.

The invention also provides a kit containing the anti-procalcitonin antibody, which is used for detecting or quantifying procalcitonin or fragments thereof. In an embodiment of the invention, the kit according to the invention comprises at least the above-mentioned anti-procalcitonin antibody, and an antibody directed against an epitope in the amino acid sequence from position 50 to 116 of procalcitonin; preferably, the antibody directed against an epitope in the amino acid sequence from position 50 to 116 of procalcitonin may be an antibody directed against an epitope in the amino acid sequence from position 50 to 116 of procalcitonin as disclosed in the prior art. More preferably, the method for detecting or quantifying procalcitonin or the fragments thereof according to the present invention employs an immunoassay, and further, the immunoassay comprises methods conventional in the art, such as chemical methods, fluorescence immunoassay, enzyme-linked immunoassay, immunochromatography, etc., and the specific determination steps of the above detection methods can be obtained by the prior art.

The invention also provides the application of the anti-procalcitonin antibody in preparing a kit for detecting or quantifying procalcitonin or fragments thereof.

The invention provides a hybridoma cell line for producing procalcitonin monoclonal antibody, which has the preservation number of CGMCCNo.10417.

In addition, the present invention provides a method for preparing an anti-procalcitonin antibody, comprising the steps of:

(1) mutating N-terminal amino acid residues of procalcitonin to obtain a nucleotide sequence corresponding to the mutated amino acid residue sequence;

(2) inserting the nucleotide sequence into a prokaryotic expression vector for induced expression and protein purification;

(3) taking the purified protein as an antigen to immunize animals, taking spleen cells of the animals to fuse with mouse myeloma cells to prepare hybridoma cells, obtaining the hybridoma cells capable of stably secreting anti-procalcitonin antibodies through screening, and obtaining corresponding monoclonal antibodies.

In one aspect of the invention, the mutation is made to amino acid residues 1 to 57 of procalcitonin.

In one embodiment of the invention, the mutated procalcitonin amino acid sequence is SEQ ID NOs: 2-4.

In the present invention, there is also provided an antibody produced by the above method.

In one embodiment of the invention, the antibody is a monoclonal antibody.

The invention has the advantages that after the human-derived procalcitonin is subjected to mutation modification, the stability is higher than that of a common antigen, and the human-derived procalcitonin can be used as a calibrator in a quantitative detection kit for the human procalcitonin and is not easy to degrade; meanwhile, the recombinant protein is high-purity recombinant protein and can be used as immunogen and screening antigen for antibody preparation. In addition, the antibody generated by the human procalcitonin modified by the mutation can be specifically bound to the human procalcitonin, and can obtain better titer and higher specific binding compared with the existing human procalcitonin antibody.

The invention is explained in more detail below with reference to the drawings. The above aspects of the invention and other aspects of the invention will be apparent from the detailed description below.

Drawings

FIG. 1 shows the results of agarose gel electrophoresis identification of three mutants. TB 1: mutant 1, M: DL2000 DNAMarker, TB 2: mutant 2, TB 3: mutant 3, the arrow points to the positions of 500bp and 300bp Marker respectively.

FIG. 2 shows the result of double-restriction agarose gel electrophoresis of pET 34.

FIG. 3 shows the result of agarose gel electrophoresis identification of PCR of bacterial liquid. TB 1: mutant 1, TB 2: mutant 2, TB 3: mutant 3, the arrow points to the positions of 500bp and 300bp Marker respectively.

FIG. 4 shows the results of agarose gel electrophoresis identification of plasmids pET34-PCT-TB1, pET34-PCT-TB2, and pET34-PCT-TB 3.

FIG. 5 shows the results of SDS-PAGE electrophoretic identification of protein purification of pET34-PCT-TB1, pET34-PCT-TB2, and pET34-PCT-TB 3. The arrow indicates a 15kd protein band. L: sample loading, M: protein Marker, P: leak, 80: 80mM imidazole, 250: 250mM imidazole.

Deposit description

The Balb/c mouse hybridoma obtained by the invention is preserved in the China general microbiological culture Collection center in the preservation center of the culture Collection of microorganisms with the preservation number of CGMCC No.10417 in 1/4 of 2015, and the preservation center address is as follows: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101.

DESCRIPTION OF THE SEQUENCES

The sequences referred to in the present invention, including nucleotide and amino acid sequences, have been organized into an order list, which is appended to the specification, and the inventors have also filed a computer-readable form of the sequence listing.

Detailed Description

The invention is not limited to the particular methodology, protocols, antibodies or cell lines described herein, as these may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless defined differently, all technical and scientific terms and any abbreviations herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the illustrative methods, devices, and materials are described herein.

The terms of the present invention have meanings commonly used in the art unless otherwise specified.

The term "antibody", as used herein, refers to any immunoglobulin or intact molecule that binds a particular epitope, as well as fragments thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or portions of intact antibodies, provided that such fragments or portions retain the antigen binding ability of the parent antibody. For example, in the present invention, "anti-procalcitonin antibody" refers to monoclonal antibodies, polyclonal antibodies, single chain antibodies and immunologically active fragments or portions thereof that specifically bind to procalcitonin protein, or a functional variant or functional fragment thereof. In the present invention, terms such as "procalcitonin antibody", "anti-procalcitonin antibody" and "antibody against procalcitonin" are used interchangeably.

The term "binding" or "specific binding" as used herein refers to the binding of an antibody to an epitope of an antigen in an in vitro assay with a purified wild-type antigen, preferably in a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden). Binding affinity is defined by the terms ka (the association rate constant of an antibody from an antibody/antigen complex), kD (dissociation constant) and kD (kD/ka). Binding or specificityBonded means 10^-8mol/l or less, preferably 10^-9To 10^-13Binding affinity (KD) in mol/l. Thus, the procalcitonin antibodies according to the invention are represented by 10^-8mol/l or less, preferably 10^-9To 10^-13The binding affinity (Kd) of mol/l binds specifically to the antigen.

The term "human monoclonal antibody" as used herein refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a transgenic non-human animal, e.g., a B cell obtained from a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell.