Recombinant humanized anti-Cpn IgM monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical fields of gene engineering, antibody engineering and immunodiagnosis. The invention provides a recombinant humanized anti-Cpn IgM monoclonal antibody. The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises three complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the three complementary determining regions are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; the heavy chain variable region comprises three complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the three complementary determining regions are shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively. The invention also provides a method for preparing the monoclonal antibody. The recombinant humanized anti-Cpn IgM monoclonal antibody provided by the invention is expressed in mammalian cells by utilizing a gene recombination technology, has the characteristics of high purity, small batch difference and the like, and can be applied to a Cpn IgM antibody detection kit as a quality control product; as a quality control product, the recombinant humanized anti-Cpn IgM monoclonal antibody has the advantages of high sensitivity, high safety, inter-batch stability and the like.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, antibody engineering, and immunodiagnostic technology, and particularly to a recombinant humanized anti-Cpn IgM monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Chlamydia pneumoniae ( C. pneumoniae, Cpn Chlamydia pneumoniae (Chlamydia pneumoniae) is an obligate intracellular parasite and a major pathogen causing community-acquired pneumonia, bronchitis, pharyngitis, and other respiratory infections. Its infection is widespread globally, with a particularly high incidence in children, adolescents, and the elderly. Timely and accurate diagnosis of acute Chlamydia pneumoniae infection is crucial for guiding clinical medication (such as using macrolides or tetracyclines instead of β-lactams), preventing prolonged illness, and preventing complications. In serological diagnosis, the detection of specific immunoglobulin M (IgM) antibodies is a key indicator for determining whether an infection is acute or primary. IgM antibodies are the first antibodies produced by the body in response to initial infection, typically reaching their peak 2-3 weeks after infection. Therefore, detecting Chlamydia pneumoniae IgM antibodies in serum has become an important clinical tool for the auxiliary diagnosis of acute Chlamydia pneumoniae infection.

[0003] Currently, most mainstream Chlamydia pneumoniae IgM detection kits on the market employ technologies such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), or immunochromatography. The common principle of these methods is to use Chlamydia pneumoniae antigen coated on a solid phase to capture specific IgM antibodies in the serum of the test subject, and then detect and amplify the signal using anti-human IgM (μ-chain specific) secondary antibodies labeled with enzymes or luminescent substances. In these detection systems, quality control products are the core element ensuring the validity and reliability of each test result. An ideal IgM detection quality control product should be able to react stably and specifically with the key reagents in the detection system (especially the anti-human IgM secondary antibody) to verify whether the entire detection process (from sample addition and incubation to signal reading) is functioning correctly. Currently, the commonly used IgM detection quality control products in the industry mainly exist in the following two forms, and both have obvious disadvantages: (1) Human positive serum: It is the most traditional source of quality control products; however, its source is scarce and unstable: It depends on the voluntary donation of recovered patients infected with Chlamydia pneumoniae, and the source is very limited, making it difficult to meet the needs of large-scale industrial production. Human positive serum has complex components and large batch-to-batch differences: Positive serum not only contains the target IgM antibody, but also contains a variety of heterogeneous antibodies such as IgG and IgA. Moreover, the antibody titers and compositions of different donors and different collection times are different, resulting in significant batch-to-batch differences in quality control products, which seriously affects the stability and standardization of reagent kit quality control. In addition, it has potential biosafety risks: Human blood products may be contaminated with unknown pathogens (such as hepatitis virus, HIV, etc.), posing safety hazards to producers and users. Human serum is subject to ethical and regulatory restrictions. With increasingly strict regulations, the acquisition and use of human materials face more and more ethical and compliance challenges. (2) Animal-derived (e.g., mouse-derived) monoclonal antibodies: To address the issue of human serum, mouse-derived anti-Chlamydia pneumoniae monoclonal antibodies have been attempted as alternatives. However, they have a significant drawback: incompatibility with detection systems. The constant region (Fc region) of mouse-derived monoclonal antibodies is mouse-derived, while the secondary antibody used in the detection kit is anti-human IgM (μ chain). Anti-human IgM secondary antibodies have high specificity for the Fc region of human IgM, but extremely low affinity or no binding to the Fc region of mouse IgM. This results in mouse-derived monoclonal antibodies being unable to be effectively recognized by the detection system, producing false-negative quality control results, thus incorrectly determining the entire batch of tests as failed, defeating the purpose of quality control. Therefore, while human-derived positive serum is compatible, its stability and safety are poor; mouse-derived monoclonal antibodies have good stability but are incompatible with detection systems.

[0004] It is crucial to develop a Cpn IgM monoclonal antibody that simultaneously meets the requirements of high affinity, perfect compatibility with anti-human IgM detection systems, high sensitivity, no biosafety risks, and the ability to achieve standardized, large-scale, and stable production.

[0005] Therefore, existing technologies urgently need improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a recombinant humanized anti-Cpn IgM monoclonal antibody, its preparation method, and its applications. The recombinant humanized anti-Cpn IgM monoclonal antibody provided by this invention is expressed in mammalian cells using gene recombination technology, exhibiting characteristics such as high purity and small batch-to-batch variation. It can be used as a quality control material in Cpn IgM antibody detection kits. This recombinant monoclonal antibody, as a quality control material, offers advantages such as high sensitivity, high safety, and batch-to-batch stability.

[0007] Specifically, the technical solution provided in this application is as follows: In a first aspect, the present invention provides a recombinant humanized anti-Cpn IgM monoclonal antibody, the monoclonal antibody comprising a light chain variable region and a heavy chain variable region; The light chain variable region includes three complementarity-determining regions CDR1, CDR2 and CDR3, whose amino acid sequences are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; The heavy chain variable region includes three complementarity-determining regions CDR1, CDR2 and CDR3, whose amino acid sequences are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0008] The monoclonal antibody described above has the amino acid sequence of the light chain variable region as shown in SEQ ID NO.1; and the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO.2.

[0009] The monoclonal antibody as described above further includes a J chain; the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.13; the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.14; and the amino acid sequence of the J chain of the monoclonal antibody is shown in SEQ ID NO.12.

[0010] Optionally, a human light chain constant region (hIgκC) sequence is added to the C-terminus of the light chain variable region: the sequence reference is Genbank: P01834.2, as shown in SEQ ID NO.10; a human heavy chain constant region (hIgM-CH) is added to the C-terminus of the heavy chain variable region: the sequence reference is Genbank: AIC63051.1, as shown in SEQ ID NO.11, and the C-terminus is connected to 6*H via linker: GGGGSGGGG.

[0011] Optionally, the same signal peptide sequence may be added to the light chain variable region, the heavy chain variable region, and the N-terminus of the human J chain, as shown in SEQ ID NO.9.

[0012] In this invention, the variable region sequence of the recombinant monoclonal antibody was obtained by immunizing mice with Cpn natural antigen, screening and preparing hybridoma cells, and sequencing them using hybridoma gene sequencing technology. The recombinant monoclonal antibody was humanized by modifying its light and heavy chain constant regions, and finally constructed into the cell expression vector pCDNA3.4 after codon optimization. It was then transfected into HEK293 suspension cells of mammals for recombinant expression. The expression product was purified by NI column to finally obtain the recombinant humanized anti-Cpn IgM monoclonal antibody, which was named R-Anti-Cpn-IgM.

[0013] Specifically, the amino acid sequence of the light chain of the recombinant humanized anti-Cpn IgM monoclonal antibody is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain from the N-terminus to the C-terminus is Signal-VL(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)-CL(hIgκC); the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO. 1.

[0014] The light chain variable region also includes four FR light chain variable region backbone regions, wherein the amino acid sequences of the light chain variable region backbone regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

[0015] The light chain variable region (VL) includes one signal peptide sequence, three light chain complementarity-determining regions (CDR1, CDR2, CDR3), and four backbone regions (FR1-FR4).

[0016] In this context, Signal is the variable region of the light chain, the variable region of the heavy chain, and the signal peptide sequence added to the N-terminus of the J chain. CDR1, CDR2, and CDR3 are the complementarity-determining regions of the light chain, FR1-FR4 are the four backbone regions of the light chain, and CL (hIgκC) is the constant region of the human light chain.

[0017] The three complementarity-determining regions (CDR1, CDR2, and CDR3) of the light chain have amino acid sequences as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The signal peptide sequence of the light chain is shown in SEQ ID NO.9. The human light chain constant region sequence is shown in SEQ ID NO.10. The amino acid sequence of the light chain is shown in SEQ ID NO.13.

[0018] The amino acid sequence of the heavy chain of the recombinant humanized anti-Cpn IgM monoclonal antibody is shown in SEQ ID NO. 14. The amino acid sequence of the heavy chain, from the N-terminus to the C-terminus, is Signal-VH(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)-CH-Linker-6H (HIgM). The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO. 2. Analysis of the light chain complementarity-determining region (CDR1, CDR2, CDR3) sequence fragments is shown in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively; the heavy chain complementarity-determining region (CDR1, CDR2, CDR3) sequence fragments are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively; the light chain, heavy chain, and J chain signal peptide sequences are all shown in SEQ ID NO. 9; the humanized constant region sequence of the heavy chain is shown in SEQ ID NO. 11; and the amino acid sequence of the J chain of the polypeptide chain is shown in SEQ ID NO. 12. The heavy chain variable region also includes four FR heavy chain variable region backbone regions, wherein the amino acid sequences of the heavy chain variable region backbone regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25.

[0019] Secondly, the present invention also provides a nucleic acid molecule encoding a recombinant humanized anti-Cpn IgM monoclonal antibody as described above.

[0020] Optionally, the nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.15. The nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO.16. The nucleotide sequence encoding the J chain of the polypeptide is shown in SEQ ID NO.17. The above sequences are preferred nucleotide sequences after codon optimization for expression in mammalian cells.

[0021] Thirdly, the present invention also provides a recombinant expression vector comprising the nucleic acid molecules described above.

[0022] Optionally, the expression vector is: inserting the nucleotide sequences optimized by the codons shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17 into the pCDNA3.4 vector to successfully obtain light chain recombinant plasmid, heavy chain recombinant plasmid, and J chain recombinant plasmid.

[0023] Fourthly, the present invention also provides a host cell containing the nucleic acid molecules described above or transformed with the recombinant expression vector described above.

[0024] Optionally, the host cell is HEK293 suspension cells.

[0025] Fifthly, the present invention provides a method for preparing the monoclonal antibody as described above, comprising the following steps: S1. Immunize mice with Cpn natural antigen and screen to obtain hybridoma cells; specifically, BALB / C mice were immunized with Cpn-Ag (N0210) from Qingdao Shuojing Biotechnology Co., Ltd. S2. Extract mRNA from hybridoma cells, reverse transcribe it into cDNA, amplify the variable regions of the light and heavy chains, and sequence it. Specifically, culture the prepared hybridoma cells, extract their mRNA, prepare cDNA through reverse transcription, use the prepared cDNA as a template, perform PCR amplification on the variable regions of the light and heavy chains, construct the amplification products into a vector, and send the constructed recombinant vector for sequencing. S3. Ligate the light and heavy chain variable region sequences obtained from sequencing with the human constant region sequences to construct recombinant expression vectors; specifically, analyze the sequenced light and heavy chain variable regions, ligate the successfully sequenced light and heavy chain variable regions with the human light and heavy chain constant regions, perform codon optimization synthesis, and construct them into the pCDNA3.4 vector; at the same time, codon optimization synthesis of the human J chain is constructed into the pCDNA3.4 vector; prepare light chain, heavy chain, and J chain recombinant vectors; S4. Transfect HEK293 suspension cells with light chain, heavy chain and J chain recombinant plasmids in a certain proportion and culture them for secretion expression. S5. Purify to obtain the monoclonal antibody; specifically, collect the supernatant of HEK293 suspension cell culture, purify using an NI column, and finally obtain the monoclonal antibody.

[0026] Sixthly, the present invention also provides an anti-Cpn IgM quality control sample, which comprises the monoclonal antibody as described above. The quality control sample provided by the present invention can accurately assess the sensitivity of Cpn IgM antibody detection reagents, solving the problems of difficulty in obtaining, instability, and low sensitivity of anti-Cpn IgM positive serum quality control samples on the market.

[0027] In a seventh aspect, the present invention provides the application of the monoclonal antibody as described above or the anti-Cpn IgM quality control material as described above in a Cpn IgM antibody detection kit.

[0028] As described above, the detection kit is an indirect gold chromatography, ELISA, or chemiluminescence detection kit that relies on an anti-human IgM (μ chain) detection system.

[0029] The present invention has the following beneficial effects: 1. The present invention provides a recombinant humanized anti-Cpn IgM monoclonal antibody, which utilizes the natural Cpn antigen for immune screening. This natural Cpn antigen is extracted and purified from the actual cultured pathogen. It contains all naturally occurring proteins, carbohydrates, lipids and other molecules on the surface of the pathogen, and maintains their original three-dimensional spatial conformation and post-translational modifications. It can induce B cell immune responses against a variety of different antigens and epitopes, thereby making it easier to screen out monoclonal antibodies with high affinity that can be humanized.

[0030] 2. This invention provides a recombinant humanized anti-Cpn IgM monoclonal antibody. This antibody retains the murine variable region to ensure high affinity and specificity for Chlamydia pneumoniae antigen, while modifying its constant region to resemble the constant region of human IgM. The humanized anti-Cpn IgM possesses the advantages of uniformity and stable production of monoclonal antibodies, and can be accurately recognized by commercially available anti-human IgM secondary antibodies. Therefore, it mimics the real anti-Chlamydia pneumoniae IgM antibody in human serum and can replace human positive serum as a quality control material for Chlamydia pneumoniae (Cpn) IgM. This Cpn IgM quality control material can be applied to multiple platforms such as gold standard chromatography, chemiluminescence, and ELISA.

[0031] 3. This invention provides a method for preparing recombinant humanized anti-Cpn IgM monoclonal antibody, which allows for standardized production. Utilizing a mammalian cell expression system, it exhibits extremely high uniformity and stability, achieving highly consistent quality control across different batches and significantly improving the quality controllability of the reagent kit. It addresses the industry pain point of lacking reliable quality control materials for existing Chlamydia pneumoniae IgM detection, significantly improving the quality of related diagnostic reagent kits and the accuracy of clinical diagnosis, thus possessing extremely high commercial value and social benefits. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0033] Figure 1 The PCR amplification of the light and heavy chain variable regions in Example 1, wherein M: DL2000; 1, 2: light chain variable region amplification products; 3, 4: heavy chain variable region amplification products; Figure 2 The results are SDS-PAGE after purification in Example 1. M: Protein Marker; I: R-Anti-Cpn-IgM. Figure 3The results of the Kanghua Cpn IgM antibody test strip in Example 3 were used to detect the self-made anti-Cpn IgM quality control product. Figure 4 The results of testing three batches of self-made anti-Cpn IgM quality control products using the Kanghua Cpn IgM antibody test strip in Example 3 are shown. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0035] Example 1: Preparation of recombinant humanized anti-Cpn IgM monoclonal antibody 1.1 Method for preparing anti-Cpn hybridoma cells (1) Immunogen Cpn acquisition: Cpn-Ag (N0210) natural antigen product from Qingdao Shuojing Biotechnology Co., Ltd. was selected as the immunogen.

[0036] (2) Mouse immunization: Female balb / c mice aged 6-8 weeks were immunized with Cpn-Ag antigen; the immunoglobulin was mixed with an equal volume of Freund's complete adjuvant at a dose of 75 μg / mouse, emulsified completely, and administered subcutaneously to multiple sites on the back of the mice; three weeks later, a booster immunization was performed, with the immunoglobulin mixed with an equal volume of incomplete Freund's adjuvant at a dose of 35 μg / mouse, emulsified completely, and administered subcutaneously to multiple sites on the back of the mice; one week later, blood was collected from the tails of the mice to detect serum titers. When the serum titer reached 100,000 or higher, fusion was prepared; three days before fusion, the mice were immunized intraperitoneally by injection without adjuvant at a dose of 100 μg / mouse.

[0037] (3) Cell fusion: During fusion, the spleen of immunized mice was taken, the cells were ground and dispersed, and mouse B lymphocytes were collected after centrifugation. They were mixed at a ratio of 7:1 (B lymphocytes: SP2 / 0), centrifuged, and washed with serum-free DMEM medium to ensure that there was no fetal bovine serum in the mixed cells. Cell fusion was performed with PEG. After full fusion, the fusion was terminated with DMEM medium. Finally, the fused cells were resuspended with HAT medium, placed in a 96-well cell culture plate, and cultured in a carbon dioxide incubator.

[0038] (4) Hybridoma cell screening and subcloning: The medium was completely replaced with HAT medium on the 7th day after fusion, and the culture supernatant was detected by indirect ELISA on the 9th day. The specific testing methods are as follows: Coating: Dilute Cpn-Ag to 1 μg / mL with CBS buffer (pH 9.6), coat 100 μL / well into microplates, and incubate overnight at 4°C. Wash the plate 5 times with PBST buffer and blot dry. Block: Block the plate with PBST buffer containing 3% BSA, 200 μL / well, and incubate at 37°C for 2 h. Wash the plate 5 times with PBST buffer and blot dry. Detection: Add the cell supernatant to be tested to the plate, 100 μL / well, with mouse immune serum as a positive control and HAT medium as a blank control, and incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and blot dry. Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse antibody 20,000 times with PBST buffer and add 100 μL / well to the plate, incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and blot dry. Color development: Develop the color with TMB chromogenic solution in the dark, 100 μL / well, and incubate at 37°C for 15 min. Terminate with 2M sulfuric acid stop solution, 50 μL / well, and detect at 450 nm. Cells that react with Cpn-Ag and have high titers (OD450 ≥ 2.0) were selected for subcloning. Subcloning was performed using a serial dilution method. 200-500 cells were transferred to well A1 of a new 96-well cell culture plate and diluted 2-fold both longitudinally and laterally. After 7-9 days, the culture supernatant was tested using an indirect ELISA method. Wells with a single positive cell cluster were selected, and subcloning was continued until the positive rate of the 96-well plate reached 100%. Subcloning was repeated twice more. When the positive rate was 100% in both cases, subcloning was terminated to ensure the stability of the hybridoma cell line.

[0039] 1.2 Sequencing of anti-Cpn-Ag hybridoma cells (1) Hybridoma cell line culture and RNA extraction The prepared hybridoma cells were cultured in a large-scale manner, and the cultured cells were collected. RNA was extracted from the cells according to the instructions of Thermo's RNA extraction kit.

[0040] (2) Reverse transcription Following the instructions of Thermo's kit, the extracted RNA was immediately reverse transcribed to prepare cDNA.

[0041] (3) PCR amplification and recovery Using universal primers for the variable region of the mouse monoclonal antibody, and with the above-mentioned cDNA as a template, PCR amplification was performed on the variable regions of the light and heavy chains. The amplification products were identified by DNA gel extraction, and the PCR products were recovered by gel cutting using a TIANGEN kit. The light and heavy chain PCR products are shown below. Figure 1 .

[0042] (4) The above light chain and heavy chain PCR products were identified by vector construction and expression, positive clones were selected and sent to a sequencing company for sequencing.

[0043] 1.3 Variable region sequence analysis, humanization modification, and recombinant plasmid construction Sequencing results were analyzed using the IMGT antibody variable region analysis tool in the database. The amino acid sequence of the light chain variable region (VL) of the anti-Cpn-Ag monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.2. The sequencing results show that the light and heavy chain variable region sequences include three complementarity-determining regions CDR1, CDR2, and CDR3, and four backbone regions (FR1-FR4).

[0044] Analysis revealed that the light chain variable region comprises three complementarity-determining regions, CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.3, which is SVSY. The amino acid sequence of CDR2 is shown in SEQ ID NO.4, which is STS. The amino acid sequence of CDR3 is shown in SEQ ID NO.5, which is HQWSSYRT.

[0045] The heavy chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.6, which is GFTFSSYA. The amino acid sequence of CDR2 is shown in SEQ ID NO.7, which is ISSGGSYT. The amino acid sequence of CDR3 is shown in SEQ ID NO.8, which is ARHDYGSSYYAMDY.

[0046] The light chain variable region also includes the light chain variable region backbone regions of four FRs, FR1-FR4. The amino acid sequences of the light chain variable region backbone regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

[0047] The heavy chain variable region also includes the heavy chain variable region backbone regions of four FRs, FR1-FR4. The amino acid sequences of the heavy chain variable region backbone regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25.

[0048] The same signal peptide sequence was added to the N-terminus of the variable regions of the light and heavy chains, as shown in SEQ ID NO. 9. A human light chain constant region (hIgκC) sequence was added to the C-terminus of the light chain variable region: the sequence is referenced from Genbank: P01834.2, as shown in SEQ ID NO. 10. A human heavy chain constant region (hIgM-CH) was added to the C-terminus of the heavy chain variable region: the sequence is referenced from Genbank: AIC63051.1, as shown in SEQ ID NO. 11. Simultaneously, a 6*H linker was connected to the C-terminus via GGGGSGGGG. The amino acid sequence of the human J chain was referenced from Unprot: P01591, with the same signal peptide sequence added to its N-terminus. The full-length amino acid sequence of the J chain is shown in SEQ ID NO. 12. The full-length amino acid sequence of the humanized light chain was obtained, as shown in SEQ ID NO. 13. The full-length amino acid sequence of the humanized heavy chain was obtained, as shown in SEQ ID NO. 13. As shown in NO.14, the translation codons were optimized and synthesized by Beijing Qingke Biotechnology Co., Ltd. using mammalian cells as the host. The optimized full-length base sequence of the translation light chain is shown in SEQ ID NO.15; the full-length base sequence of the translation heavy chain is shown in SEQ ID NO.16; and the full-length base sequence of the J chain is shown in SEQ ID NO.17.

[0049] The light chain, heavy chain, and J chain base sequences were given to a gene synthesis company (Beijing Qingke Biotechnology Co., Ltd.) and synthesized into the cell expression vector pCDNA3.4. The light chain recombinant plasmid was named: pCDNA3.4-cpn-hL; the heavy chain recombinant plasmid was named: pCDNA3.4-cpn-hIgM; and the J chain recombinant plasmid was named: pCDNA3.4-hIgJ.

[0050] 1.4 Expression and purification of recombinant humanized anti-Cpn IgM monoclonal antibody (1) Cell resuscitation (a) Remove one HEK293 suspension cell from the liquid nitrogen tank. After removal, quickly place the cryovial into a water bath for rapid thawing, no more than 1 minute. The facility temperature is 37°C, ensuring the temperature remains stable at 37°C during thawing. When placing the cryovial into the water bath, ensure the liquid level does not exceed 2 / 3 of the tube's height to avoid water contact with the tube opening and causing contamination.

[0051] (b) In a clean bench, transfer HEK293 cells to a centrifuge tube containing 10 mL of culture medium, centrifuge at 1000 rpm for 10 min, collect the cell pellet, resuspend in culture medium, count the cells, and adjust the cell density to 0.4-0.6 × 10⁻⁶. 6 cells / mL.

[0052] (c) Transfer the resuspended cells to a shake flask. In principle, the culture volume should not exceed 25% of the total volume of the shake flask. Place the shake flask in a CO2 shaker and culture it under the following conditions: 37°C, 5% CO2, and 110 rpm. Culture for about 3 days, then observe and passage the cells.

[0053] (2) Cell passage (a) After about 3 days of cell resuscitation, the cells are in the logarithmic growth phase. Cells are then harvested for cell density (×10⁻⁶). 6 Cell counts (cells / mL) and viability (%) were measured, and cells were passaged after the tests.

[0054] (b) Calculate cell density ≥2×10 6 When the cell count / mL is above 90%, use 0.8-1.2 × 10⁻⁶ cells / mL. 6 The cells were passaged at a density of cells / mL and then transferred to shake flasks for culture.

[0055] (c) Continue passage 1-2 more times. Before transient transfection, cells need to be passaged stably at least 3 times. If the cell condition is poor or cell acclimatization is required during passage, the cells need to be passaged stably 3 times after the cell condition is stable or acclimatization is complete before transfection.

[0056] (3) Instantaneous transfection (a) Plasmid and PEI pretreatment: On the day of transfection, add light chain recombinant plasmid: heavy chain recombinant plasmid: J chain recombinant plasmid: transfection reagent (PEI) in a mass ratio of 2:1:0.5:3 (for example, to transfect 1L of suspension cells, add light chain recombinant plasmid: heavy chain recombinant plasmid: J chain recombinant plasmid: transfection reagent (PEI) in masses of 2mg, 1mg, 0.5mg, and 3mg respectively) to an appropriate amount of HEK293 suspension cell culture medium, mix thoroughly, and let stand for 10-15min.

[0057] (b) Transfection: Count the prepared cells and adjust the cell density to 3.0-3.5 × 10⁶ cells / year using the HEK293 suspension culture medium (model: 293 CD01) prepared in the above steps. 6 cells / mL.

[0058] (c) The transfected cells are loaded into sterile shake flasks (volume ≤30%) and the shake flasks are placed in a carbon dioxide shaker for incubation.

[0059] (4) Feeding 24 hours after transfection, add feed medium (model 293 F01) at 3-5% of the culture volume; add L-glutamine solution at 1% of the culture volume, and continue culturing. On days 3 and 5 post-transfection (day 0 is the day of transfection), cell viability is calculated (3-day cell viability ≥90%; 5-day cell viability ≥75%), and feed medium is added at 3-5% of the culture volume; L-glutamine solution is added at 1% of the culture volume, and culturing continues. On day 7, cell viability is measured (cell viability ≥50%), and the culture supernatant is collected. (HEK293 suspension cells, HEK293 suspension cell culture medium, and feed medium were all purchased from Hangzhou Peiding Biotechnology Co., Ltd.) (5) Collection and purification of expression supernatant (a) Collection of supernatant: The culture product was placed in a centrifuge bottle, balanced, centrifuged at 8°C and 9000 rpm for 30 min, and the supernatant was collected for purification.

[0060] (b) Ni column affinity chromatography purification Affinity chromatography was performed using an IMAC Bestarose-FF nickel ion affinity chromatography column (purchased from BorgLone Shanghai Biotechnology Co., Ltd.). The column was equilibrated with 10 column volumes of ultrapure water. The column was then equilibrated with 10 column volumes of equilibration buffer (20 mM PBS, pH 7.4) before adding the protein sample. After loading, unbound contaminants were washed away with 10 column volumes of equilibration buffer (20 mM PBS, pH 7.4). Contaminants were then washed away with 10 column volumes of washing buffer (20 mM PBS, 50 mM IM, pH 7.4). Finally, the target protein was eluted with elution buffer (20 mM PBS, 250 mM IM, pH 7.4).

[0061] Dialysis: Place the collected protein in a dialysis bag (specifications: MWCO=8-14kDa; diameter=8mm), using 20mM PBS (pH 7.4) as the dialysate, twice daily, changing the buffer twice a day (morning and evening), at 2-8℃ for 3 days (at least 6 times). After dialysis, remove the protein and place it in a 50 ml centrifuge tube. Centrifuge at 7500-9000 rpm, 4-15℃ for 30 min, collect the supernatant, and determine the concentration. The obtained protein sample is the recombinant humanized anti-Cpn IgM monoclonal antibody, named R-Anti-Cpn-IgM. After purification, R-Anti-Cpn-IgM was analyzed by SDS-PAGE gel chromatography. The results are shown below. Figure 2As shown, the SDS-PAGE gel image results correctly represent the light and heavy chains; their sizes are consistent with the theoretical values ​​of 23 kDa for light chains and 64 kDa for IgM heavy chains, and the purity is high.

[0062] Example 2: ELISA method for detecting the titer affinity of this R-Anti-Cpn-IgM The R-Anti-Cpn-IgM prepared in this invention can be detected by indirect ELISA using anti-human IgM and Cpn-Ag from different manufacturers. (1) Different manufacturers' Cpn natural antigens were diluted at 0.5 μg / mL in 0.01M pH9.6 carbonate buffer coating solution, 100 μL / well, and coated overnight at 4℃.

[0063] (2) Wash the plate three times with PBST, dry it, add 200 μL of blocking buffer (1% BSA) per well, and block for 2 hours.

[0064] (3) Wash the plate three times with PBST, dry it, and set aside.

[0065] (4) Sample addition: Add 1 mg / mL LR-Anti-Cpn-IgM at a ratio of 1:10 2 (10 μg / mL), 1:10 3 (1μg / mL), 1:10 4 (100ng / mL), 1:10 5 (10ng / mL), 1:10 6 (1 ng / mL), 1:10 7 (100 pg / mL), 1:10 8 Dilute to (10 pg / mL). Add 100 μL of each solution to a 96-well microplate and incubate at room temperature for 2 hours.

[0066] (5) Wash the plate three times with PBST and dry it. Add 0.1 mL of anti-human IgM-HRP antibody diluted 1:10000 with PBST from different manufacturers. Incubate at room temperature for 1 hour.

[0067] (6) Wash the plate five times with PBST and dry it. Add 100 μL of TMB colorimetric solution and react at room temperature in the dark for 10 minutes.

[0068] (7) Add 50 μL of stop solution (1M sulfuric acid) to terminate the reaction; measure its OD value at 450 nm. A P / N ≥ 2.1 is considered positive, otherwise it is considered negative. P is the OD450 value of the sample and N is the OD450 value of the blank control.

[0069] (8) The affinity was calculated by determining the half effective concentration (EC50) of R-Anti-Cpn-IgM using linear interpolation.

[0070] The results are shown in Table 1. The titer trend of R-Anti-Cpn-IgM against Cpn antigens from different manufacturers was consistent. The titer deviation (CV) for the same gradient test was ≤10%. The EC50 was calculated as EC50 = 1 ng / mL + (50% - 29.65%) / (53.25% - 29.65%) × (10 ng / mL - 1 ng / mL) = 8.76 ng / mL. The calculation shows that the EC50 is around 8.76 ng / mL, indicating high affinity. This indicates that the humanized monoclonal antibody still has strong titer affinity against the natural Cpn antigen.

[0071] The results are shown in Table 2. The titer of R-Anti-Cpn-IgM against different manufacturers of anti-human IgM showed a consistent trend at a dilution ratio of 1:10. 5 (10ng / mL), all detected values ​​were above 0.7; the humanized antibody titers against anti-human IgM from different manufacturers were normal, indicating that it can be used in combination with anti-human IgM antibodies from different manufacturers and has good compatibility.

[0072] In summary, the humanized anti-Cpn IgM monoclonal antibody in this patent can bind with high affinity to the Cpn antigen and can also be accurately recognized by commercially available anti-human IgM secondary antibodies. Therefore, it can be used as a quality control for anti-Cpn IgM to simulate real anti-Chlamydia pneumoniae IgM antibodies in human serum. In Table 1 of this embodiment, the secondary antibody used is anti-human IgM, derived from 9M of Qingdao Shuojing Biotechnology Co., Ltd. In Table 2 of this embodiment, the coating antigen is Cpn-Ag, derived from N0210 of Qingdao Shuojing Biotechnology Co., Ltd.

[0073] Table 1. ELISA assay of the titer and affinity of recombinant monoclonal antibody R-Anti-Cpn-IgM to Cpn antigens from different manufacturers.

[0074] Table 2. Affinity test of recombinant monoclonal antibody R-Anti-Cpn-IgM against anti-human IgM secondary antibodies from different manufacturers, as determined by ELISA.

[0075] Example 3: Preparation of recombinant humanized anti-Cpn IgM monoclonal antibody (R-Anti-Cpn-IgM) as a quality control sample for application testing. (1) Preparation of Cpn IgM quality control material Prepare a 20mM PBS buffer (pH 7.4) containing a certain concentration of BSA and Tween-20 to simulate serum matrix; perform serial dilutions of purified R-Anti-Cpn-IgM (2mg / mL) with the prepared simulated serum matrix at 1:100, 1:1000, 1:2000, 1:4000, and 1:10000; the concentrations of other components are: 0.1% BSA and 0.05% Tween-20; and add 0.1% ProClin 300 or 0.02% NaN3 to prevent microbial contamination.

[0076] (2) Kanghua Cpn IgM antibody test strip (gold standard chromatography) quality control product for detecting anti-Cpn IgM The anti-Cpn IgM quality control product prepared according to the embodiments of this invention was tested using the Kanghua Cpn IgM antibody test strip, and simultaneously tested with a purchased Yahuilong Cpn IgM-positive serum control. The test results are as follows: Figure 3 The results showed that the self-made anti-Cpn IgM quality control samples of this invention were all detectable, with deep color development, significantly superior to purchased Cpn IgM positive serum samples. This indicates that the recombinant monoclonal antibody R-Anti-Cpn-IgM prepared in this invention has high sensitivity.

[0077] (3) Inter-batch variability of anti-Cpn IgM quality control products detected by Kanghua Cpn IgM antibody test strip (gold standard chromatography). Following the above method for preparing anti-Cpn IgM quality control samples, three consecutive batches of purified R-Anti-Cpn-IgM monoclonal antibody were used to prepare anti-Cpn IgM quality control samples. The batch-to-batch differences were tested using Kanghua Cpn IgM antibody test strips. The test results are shown in [Figure number missing]. Figure 4 The three batches had the same dilution gradient, namely 1:100, 1:1000, and 1:10000. The quality control samples showed consistent color development and batch-to-batch stability.

[0078] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A recombinant humanized anti-Cpn IgM monoclonal antibody, characterized in that, The monoclonal antibody includes a light chain variable region and a heavy chain variable region; The light chain variable region includes three complementarity-determining regions CDR1, CDR2 and CDR3, whose amino acid sequences are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; The heavy chain variable region includes three complementarity-determining regions CDR1, CDR2 and CDR3, whose amino acid sequences are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody further includes a J chain; the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.13; the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.14; and the amino acid sequence of the J chain of the monoclonal antibody is shown in SEQ ID NO.

12.

4. A nucleic acid molecule, characterized in that, The monoclonal antibody encoding the recombinant humanized anti-Cpn IgM as described in claim 3.

5. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It contains the nucleic acid molecule as described in claim 4 or is transformed with the recombinant expression vector as described in claim 5.

7. A method for preparing the monoclonal antibody as described in claim 3, characterized in that, Includes the following steps: S1. Immunize mice with Cpn natural antigen and screen to obtain hybridoma cells; S2. Extract mRNA from hybridoma cells, reverse transcribe it into cDNA, amplify the variable regions of the light and heavy chains, and sequence it. S3. Ligate the light and heavy chain variable region sequences obtained from sequencing with the human constant region sequences to construct a recombinant expression vector; S4. Transfect HEK293 suspension cells with light chain, heavy chain and J chain recombinant plasmids in a certain proportion and culture them for secretion expression. S5. Purify to obtain the monoclonal antibody.

8. A quality control product against Cpn IgM, characterized in that, The quality control material contains the monoclonal antibody as described in claims 1-3.

9. The use of the monoclonal antibody as described in any one of claims 1-3 or the anti-Cpn IgM quality control material as described in claim 8 in a Cpn IgM antibody detection kit.

10. The application according to claim 9, characterized in that, The detection kit is an indirect method gold chromatography, ELISA, or chemiluminescence detection kit that relies on an anti-human IgM (μ chain) detection system.

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