CN116693677B - Antibodies against Chlamydia trachomatis, reagents and kits for detecting Chlamydia trachomatis - Google Patents

Antibodies against Chlamydia trachomatis, reagents and kits for detecting Chlamydia trachomatis Download PDF

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CN116693677B
CN116693677B CN202210192835.6A CN202210192835A CN116693677B CN 116693677 B CN116693677 B CN 116693677B CN 202210192835 A CN202210192835 A CN 202210192835A CN 116693677 B CN116693677 B CN 116693677B
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antibody
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antigen
thr
binding fragment
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CN116693677A (en
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孟媛
钟冬梅
唐丽娜
曹慧方
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Dongguan Pengzhi Biotechnology Co Ltd
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract

The invention discloses an anti-chlamydia trachomatis antibody, a reagent and a kit for detecting chlamydia trachomatis, and relates to the technical field of antibodies. The antibodies against Chlamydia trachomatis disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for the detection of chlamydia trachomatis.

Description

Antibodies against Chlamydia trachomatis, reagents and kits for detecting Chlamydia trachomatis
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-chlamydia trachomatis antibody, a reagent for detecting chlamydia trachomatis and a kit.
Background
Chlamydia trachomatis (CHLAMYDIA TRACHOMATIS, CT) is a pathogen parasitic in obligate cells, can cause transmission of trachoma and venereal diseases, has increasingly significant influence in Sexually Transmitted Diseases (STDs) exceeding gonorrhea, becomes STDs with highest global morbidity, and accounts for about 40-60% of non-gonococcal urethritis (NUG), can cause male urethritis, prostatitis, epididymitis, female cervicitis endometritis, salpingitis, pelvic inflammatory disease, infertility, ectopic pregnancy, abortion and the like. Most of the cases in clinical practice develop with concealment, it is reported that 50% of men and 70% of women are infected asymptomatic, and asymptomatic infection can last from 1 year to several years. Chlamydia trachomatis can establish chronic infections in the body for months or years, which can damage the female reproductive organs, standard antimicrobial therapy does not completely destroy such pathogens, and Chlamydia trachomatis tends to develop a pattern of resistance, thereby causing longer chronic infections. Chlamydia trachomatis can also transmit neonatal conjunctivitis and pneumonia through a mother and infant, and can cause premature rupture of fetal membranes, premature birth, death of neonates, and the like. Therefore, routine testing of Chlamydia trachomatis is of great clinical significance.
Currently, the main detection methods of chlamydia trachomatis include direct smear microscopy, separation culture, serological test, PCR test, rapid detection, etc. in microbiology. The direct smear microscopic examination, separation culture and the like based on microbiology are mainly tissue cell culture methods, are more traditional diagnosis methods, but the method has large workload, requires special facilities and experienced technicians, is time-consuming and expensive, and cannot meet clinical requirements. The nucleic acid detection based on the PCR test is mainly used for PCR amplification detection of templates obtained by washing, centrifuging and other treatment of samples such as throat swabs, sputum and alveolar lavage liquid of patients, has the advantages of short time, high sensitivity and specificity and the like, but has high requirements on operators, the samples are easy to pollute, and the instruments and equipment are expensive, so that the development of the nucleic acid detection in primary hospitals is limited.
Quick detection of Chlamydia trachomatis is characterized by qualitative and quantitative quick detection. Gold calibration rapid detection (colloidal gold method) is popular. The colloidal gold immunochromatography is an immunological detection method based on the specific reaction of antibodies and antigens, and simultaneously utilizes colloidal gold to amplify and display detected signals, and the gold-labeled method for rapidly detecting the chlamydia trachomatis has the advantages of rapidness, convenience and high accuracy. Saving much time for the auxiliary diagnosis of the clinician and the like. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunoassay, chemiluminescent method, etc., which all require antibodies against Chlamydia trachomatis. There is therefore a strong need in the art for antibodies that are effective and bind to and detect chlamydia trachomatis.
In view of this, the present invention has been made.
Disclosure of Invention
Aiming at the fact that the sources of anti-chlamydia trachomatis antibodies in the market are few, the application provides the anti-chlamydia trachomatis antibody with improved activity, and provides an important raw material source for accurately detecting the chlamydia trachomatis.
In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising HCDR1, HCDR2, HCDR3 having amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 and LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6.
In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody against Chlamydia trachomatis or a functional fragment thereof, which comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody against chlamydia trachomatis comprising a heavy chain comprising the heavy chain variable region described above and/or a light chain comprising the light chain variable region described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit for detecting chlamydia trachomatis, comprising the above antibody or a functional fragment thereof or the above antibody conjugate.
In order to achieve the above object, according to a sixth aspect of the present invention, there is provided the use of the above antibody or a functional fragment thereof, the above antibody conjugate or the above reagent or kit for detection of chlamydia trachomatis.
In order to achieve the above object, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of reducing SDS-PAGE of Anti-CT-3C10mut1 and Anti-CT-3C10mut 2.
Detailed Description
The invention provides an antibody or a functional fragment thereof, which comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6. The antibodies have improved affinity and activity.
In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.
In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.
In other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In an alternative embodiment, HFR3 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 21.
In another aspect, embodiments of the present invention provide an anti-chlamydia trachomatis antibody or a functional fragment thereof, comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, HFR2, LFR3, r4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR4, or the amino acid sequence of HCDR1, HCDR 3.
In an alternative embodiment, the heavy chain variable region amino acid sequence is set forth in SEQ ID NO. 15 or 22;
In an alternative embodiment, the light chain variable region amino acid sequence is set forth in SEQ ID NO. 16.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of species origin of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 17 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 18.
In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the above-described constant region (SEQ ID NO:17 or 18).
In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In another aspect, the invention provides an antibody against Chlamydia trachomatis comprising a heavy chain comprising the heavy chain variable region described above and the heavy chain constant region described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO. 19 or 23.
In an alternative embodiment, the amino acid sequence of the light chain is shown in SEQ ID NO. 20.
In another aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).
In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.
In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18 F.
In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid phase is a nitrocellulose membrane.
In another aspect, the invention provides a reagent or kit for detecting chlamydia trachomatis comprising an antibody or functional fragment thereof or an antibody conjugate as described above. The reagent or the kit containing the antibody or the functional fragment thereof or the antibody conjugate has improved detection sensitivity, specificity and accuracy.
In another aspect, the invention provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate, or a reagent or kit as described above in the detection of chlamydia trachomatis.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the invention provides a cell comprising the nucleic acid or vector described above.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.
On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 preparation of Anti-CT-3C10 monoclonal antibody
Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.
1 Construction of recombinant plasmid
(1) Antibody Gene production
MRNA is extracted from hybridoma cell strain secreting monoclonal antibody against Chlamydia trachomatis, DNA product is obtained through RT-PCR process, the product is inserted into pMD-18T vector after adding A reaction with rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are taken after colony growth, and 4 clones are sent to gene sequencing company for sequencing.
(2) Sequence analysis of Anti-CT-3C10 antibody variable region Gene
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI 11.5.5 software is utilized for analysis to determine that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct, wherein in the LIGHT CHAIN amplified gene fragment, the VL gene sequence is 324bp, and the front side of the VL gene sequence is 57bp leader peptide sequence; in the gene fragment amplified by HEAVY CHAIN primer pair, the VH gene sequence is 360bp, belonging to VH1 gene family, and the front of the gene fragment has 57bp leader peptide sequence.
(3) Construction of recombinant antibody expression plasmids
pcDNAT3.4Vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, and is hereinafter abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the VL and VH gene specific primers are respectively provided with HindIII, ecoRI digestion sites and protective bases, and a LIGHT CHAIN gene fragment of 0.72kb and a HEAVY CHAIN gene fragment of 1.43kb are amplified by a PCR amplification method.
The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.
2 Stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to 40ug/100ul with ultrapure water, CHO cells were adjusted to 1.43X10 7 cells/ml in centrifuge tubes, 100. Mu.L of plasmid was mixed with 700. Mu.L of cells, transferred to an electrotransfer cup, electrotransferred, sampled and counted on days 3,5, 7, and collected on day 7.
Coating (main ingredient NaHCO 3) diluted 100-fold of chlamydia trachomatis culture (purchased from the organism fepeng) 100 μl per well overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50 mu L/hole, EDTA.2Na+ concentrated H 2SO4); OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that the antibodies produced after transient transformation of the plasmid were active against Chlamydia trachomatis.
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puvI enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
Diluting the plasmid to 40ug/100ul with ultrapure water, regulating CHO cells to 1.43X10 7 cells/ml in a centrifuge tube, mixing 100 mu L of plasmid with 700 mu L of cells, transferring into an electrorotating cup, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation batch culture is carried out after 3 days, cell density is adjusted to be 0.5X10 6 cells/ml, batch culture is carried out by 2.2ml, and seed preservation is carried out by 2ml, wherein the cell density is 0.3X10 6 cells/ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 Recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding is started every day when the culture is carried out in a shake flask for 72 hours, hyCloneTM Cell BoostTM Feed a is fed with 3% of the initial culture volume every day, and the feeding amount of Feed 7b is one thousandth of the initial culture volume every day until the 12 th day (feeding on 12 th day). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 3 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.
Example 2 optimization of antibody Activity
The Anti-CT-3C10 monoclonal antibody obtained in example 1 was not satisfactory in antibody activity although it had the ability to bind Chlamydia trachomatis, and thus, the applicant had performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. The monoclonal antibody with remarkably improved antibody activity is obtained through screening and is named as: anti-CT-3C10mut1 and Anti-CT-3C10mut2.
The heavy chain and light chain amino acid sequences of the Anti-DNP-6G1mut1 monoclonal antibody are shown in SEQ ID NO. 19 and 20 respectively;
the heavy chain and light chain amino acid sequences of the Anti-DNP-6G1mut2 monoclonal antibody are shown in SEQ ID NO. 23 and 20 respectively.
EXAMPLE 3 Activity and Performance identification
1 Activity assay
Coating (main ingredient NaHCO 3) diluted 100-fold of chlamydia trachomatis culture (purchased from the organism fepeng) 100 μl per well overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu l/hole; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L concentrated H 2SO4) was added; OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in Table 1 below.
TABLE 1 Activity data
Sample concentration (ng/ml) 40 20 10 5 2.5 0
Control 1.834 1.201 0.725 0.501 0.292 0.015
Anti-CT-3C10mut1 2.164 1.704 1.198 0.712 0.499 0.031
Anti-CT-3C10mut2 2.031 1.661 1.193 0.784 0.543 0.028
2 Evaluation of Performance
The antibodies are respectively used as coating antibodies, the other anti-CT monoclonal antibody is used as a labeling antibody to be coupled with colloidal gold, the performance difference of the group of coating antibodies and a control antibody is compared on a colloidal gold platform, the specific performance is shown in the following table, and the group of antibodies has better performance from the color development result:
table 2 performance evaluation data
3 Stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 3 below shows the results of the detection of OD after 21 days of antibody examination.
Table 3 stability data
Sample concentration (ng/ml) 20 2.5 0
4 ℃,21 Days sample 1.653 0.386 0.025
Sample at-80℃for 21 days 1.605 0.377 0.031
37 ℃ And 21 days of sample 1.638 0.367 0.022
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The partial amino acid sequence related to the application is as follows:
Numbering device Sequence fragments
SEQ ID NO:1 DYYMS
SEQ ID NO:2 FIRDKPNGYTTEYNASVKG
SEQ ID NO:3 DRRAYAM
SEQ ID NO:4 KASQDVSTTVA
SEQ ID NO:5 SASYRYT
SEQ ID NO:6 QQHYSTPP
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> Antibody against Chlamydia trachomatis, reagent and kit for detecting Chlamydia trachomatis
<130> P2022025CN01
<160> 23
<170> PatentIn version 3.3
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Asp Tyr Tyr Met Ser
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Phe Ile Arg Asp Lys Pro Asn Gly Tyr Thr Thr Glu Tyr Asn Ala Ser
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Val Lys Gly
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Asp Arg Arg Ala Tyr Ala Met
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Lys Ala Ser Gln Asp Val Ser Thr Thr Val Ala
1 5 10
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Ser Ala Ser Tyr Arg Tyr Thr
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Gln Gln His Tyr Ser Thr Pro Pro
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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr
20 25 30
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Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly
1 5 10
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Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile Leu Tyr Leu Gln
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Met Asn Thr Leu Arg Val Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Arg
20 25 30
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Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
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Asp Arg Val Ser Ile Thr Cys
20
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Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
1 5 10 15
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Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
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Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys
20 25 30
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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
1 5 10
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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asp Lys Pro Asn Gly Tyr Thr Thr Glu Tyr Asn Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Val Glu Asp Ser Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Arg Arg Ala Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Thr
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
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Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
100 105 110
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
130 135 140
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
180 185 190
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
210 215 220
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
225 230 235 240
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
245 250 255
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
260 265 270
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
275 280 285
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
305 310 315 320
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
325 330
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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
1 5 10 15
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
20 25 30
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
35 40 45
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
65 70 75 80
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
85 90 95
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asp Lys Pro Asn Gly Tyr Thr Thr Glu Tyr Asn Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Val Glu Asp Ser Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Arg Arg Ala Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser
180 185 190
Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile
210 215 220
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile
245 250 255
Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp
260 265 270
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
275 280 285
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg
290 295 300
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
305 310 315 320
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
325 330 335
Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
340 345 350
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
355 360 365
Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp
370 375 380
Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu
405 410 415
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His
420 425 430
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro
435 440 445
Gly Lys
450
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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Thr
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
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Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Leu Leu Tyr Leu Gln
1 5 10 15
Met Asn Thr Leu Arg Val Glu Asp Ser Ala Thr Tyr Tyr Cys Ala Arg
20 25 30
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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asp Lys Pro Asn Gly Tyr Thr Thr Glu Tyr Asn Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Leu
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Val Glu Asp Ser Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Arg Arg Ala Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Phe Ile Arg Asp Lys Pro Asn Gly Tyr Thr Thr Glu Tyr Asn Ala
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Leu
65 70 75 80
Leu Tyr Leu Gln Met Asn Thr Leu Arg Val Glu Asp Ser Ala Thr Tyr
85 90 95
Tyr Cys Ala Arg Asp Arg Arg Ala Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser
180 185 190
Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile
210 215 220
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile
245 250 255
Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp
260 265 270
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
275 280 285
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg
290 295 300
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
305 310 315 320
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
325 330 335
Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
340 345 350
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
355 360 365
Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp
370 375 380
Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu
405 410 415
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His
420 425 430
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro
435 440 445
Gly Lys
450

Claims (34)

1. An antibody against chlamydia trachomatis or an antigen-binding fragment thereof, characterized in that the antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 having the amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 and LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID No. 7 to SEQ ID No. 10 and LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID No. 11 to SEQ ID No. 14, or an amino acid sequence having at least 90% homology with each of said sequences.
3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR3 is replaced with the sequence shown in SEQ ID No. 21.
4. An antibody against chlamydia trachomatis or an antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, LFR1, HFR2, LFR3, LFR4 of the amino acid sequence of HFR1, HCDR2, LCDR3, LFR4 of the amino acid sequence of HFR2 or HFR3 of claim 1, HFR2, HFR3, LFR 4.
5. An antibody or antigen-binding fragment thereof against chlamydia trachomatis, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID No. 15 or 22; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.
7. The antibody or antigen-binding fragment thereof of claim 6, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.
8. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
9. The antibody or antigen-binding fragment thereof of claim 7, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
10. The antibody or antigen-binding fragment thereof of claim 9, wherein the constant region is of murine species origin.
11. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 17 or has at least 80% homology thereto and the light chain constant region sequence is as shown in SEQ ID No. 18 or has at least 80% homology thereto.
12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antigen-binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
13. An antibody against chlamydia trachomatis comprising a heavy chain and a light chain, wherein said heavy chain comprises a heavy chain variable region as claimed in claim 4 or 5 and a heavy chain constant region as claimed in any one of claims 7 to 11; the light chain comprises the light chain variable region of claim 4 or 5 and the light chain constant region of any one of claims 7 to 11.
14. An antibody against chlamydia trachomatis comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID No. 19 or 23; the amino acid sequence of the light chain is shown as SEQ ID NO. 20.
15. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the antibody of claim 13 or 14.
16. The antibody conjugate of claim 15, wherein the antibody or antigen binding fragment thereof is labeled with a label.
17. The antibody conjugate of claim 16, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
18. The antibody conjugate of claim 17, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof, rhodamine-based dyes and derivatives thereof, cy-based dyes and derivatives thereof, alexa-based dyes and derivatives thereof, and protein-based dyes and derivatives thereof.
19. The antibody conjugate of claim 17, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
20. The antibody conjugate of claim 17, wherein the radioisotope is selected from the group consisting of 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18 F.
21. The antibody conjugate of claim 17, wherein the chemiluminescent reagent is selected from the group consisting of luminol and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, ruthenium bipyridine and derivatives thereof, acridinium esters and derivatives thereof, dioxane and derivatives thereof, lotensine and derivatives thereof, and peroxyoxalate and derivatives thereof.
22. The antibody conjugate of claim 17, wherein the nanoparticle-based label is selected from the group consisting of a nanoparticle, a colloid.
23. The antibody conjugate of claim 22, wherein the nanoparticle is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
24. The antibody conjugate of claim 22, wherein the colloid is selected from the group consisting of colloidal selenium, latex, colloidal metal, disperse dye, and dye-labeled microsphere.
25. The antibody conjugate of claim 24, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
26. The antibody conjugate of claim 15, wherein the antibody or antigen binding fragment thereof is coated onto a solid phase.
27. The antibody conjugate of claim 26, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
28. The antibody conjugate of claim 27, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
29. A reagent or kit for the detection of chlamydia trachomatis, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or an antibody according to claim 13 or 14 or an antibody conjugate according to any one of claims 15 to 28.
30. Use of the antibody or antigen binding fragment thereof of any one of claims 1 to 12 or the antibody of claim 13 or 14 or the antibody conjugate of any one of claims 15 to 28 in the preparation of a reagent or kit for detecting chlamydia trachomatis.
31. A nucleic acid encoding the antibody or antigen binding fragment thereof of any one of claims 1 to 12 or the antibody of claim 13 or 14.
32. A vector comprising the nucleic acid of claim 31.
33. A cell comprising the nucleic acid of claim 31 or the vector of claim 32.
34. A method of preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or an antibody according to claim 13 or 14, comprising: culturing the cell of claim 33.
CN202210192835.6A 2022-02-28 2022-02-28 Antibodies against Chlamydia trachomatis, reagents and kits for detecting Chlamydia trachomatis Active CN116693677B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0348725A2 (en) * 1988-06-29 1990-01-03 Miles Inc. Species-specific epitode of chlamydia trachomatis and antibodies that recognize the same
JP2001286295A (en) * 2000-01-31 2001-10-16 Asahi Kasei Corp Antibody for detecting chlamydia trachomatis
CN1372142A (en) * 2002-04-02 2002-10-02 孙克江 Method for testing chlamydia trachomatis by using monclonal antibody of chlamydia trachomatis
CN112920270A (en) * 2021-02-09 2021-06-08 杭州隆基生物技术有限公司 Anti-chlamydia trachomatis recombinant antibody and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0348725A2 (en) * 1988-06-29 1990-01-03 Miles Inc. Species-specific epitode of chlamydia trachomatis and antibodies that recognize the same
JP2001286295A (en) * 2000-01-31 2001-10-16 Asahi Kasei Corp Antibody for detecting chlamydia trachomatis
CN1372142A (en) * 2002-04-02 2002-10-02 孙克江 Method for testing chlamydia trachomatis by using monclonal antibody of chlamydia trachomatis
CN112920270A (en) * 2021-02-09 2021-06-08 杭州隆基生物技术有限公司 Anti-chlamydia trachomatis recombinant antibody and application thereof

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