Recombinant humanized anti-human Mp monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to a recombinant humanized anti-human Mp monoclonal antibody which comprises a light chain and a heavy chain, the monoclonal antibody is named as Mp21, and the amino acid sequence of the light chain is as shown in SEQ ID NO.1; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 2. The monoclonal antibody further comprises a J chain, and the amino acid sequence of the J chain is shown as SEQ ID NO: 3. The invention also provides an anti-Mp quality control product which comprises the recombinant humanized anti-human Mp monoclonal antibody, and discloses an application of the recombinant humanized anti-human Mp monoclonal antibody in preparation of an Mp antibody detection kit. The specific monoclonal antibody Mp21 aiming at the human mycoplasma pneumoniae (Mp) is successfully obtained, recombined and expressed, the immunogenicity is remarkably reduced through the humanized design of the monoclonal antibody Mp21, and the monoclonal antibody Mp21 is suitable for in-vitro diagnosis and potential treatment application. The antibody can be used as a positive quality control product of an Mp antibody detection kit, is clear in component, high in titer and good in linearity, and solves the problems that an imported quality control product in the current market is high in price, domestic raw materials are large in batch difference, and components are uncertain.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, immunology and in vitro diagnostics, and in particular to a recombinant humanized anti-human Mp monoclonal antibody, its preparation method and application. Background Technology

[0002] Mycoplasma pneumoniae (Mp) is a microorganism that lies between bacteria and viruses. It has a simple structure, but it can adhere to respiratory mucosal epithelial cells through a special structure at one end, causing infection. It has no cell wall and is resistant to antibiotics that act on the cell wall.

[0003] Mycoplasma pneumoniae causes respiratory infections in humans, especially pneumonia, primarily transmitted through droplets. Children and adolescents are the main susceptible populations. Clinical symptoms of mycoplasma pneumoniae pneumonia include fever, cough, and sore throat. Often, due to symptoms such as nasal congestion and runny nose, it is misdiagnosed as a common cold in the early stages, leading to a worsening of the condition and even development into pneumonia. Accurate detection and eradication of Mycoplasma pneumoniae in the early stages of infection can significantly reduce the outbreak rate of mycoplasma pneumoniae pneumonia. Therefore, the diagnosis of Mycoplasma pneumoniae is particularly important.

[0004] There are many clinical detection methods for Mycoplasma pneumoniae in humans, including serological testing, molecular diagnostic methods, and isolation and culture. Isolation and culture methods are complex, time-consuming, and have low positive rates, resulting in limited clinical applicability. Molecular biological diagnostic methods offer high sensitivity and rapid diagnosis, making them suitable for early diagnosis, but they require sophisticated equipment, advanced laboratory conditions, and skilled personnel, limiting their widespread application. Serological testing is simple, quick, and requires less stringent laboratory conditions; it can detect antigens, IgG antibodies, and IgM antibodies, and is suitable for both acute and convalescent phases.

[0005] Currently, there is a lack of standard positive control products for Mp antibody testing on the market. While imported positive control products have performance advantages, they are expensive, have unstable delivery times, and are unsuitable for product research and development and production. Domestic raw materials exhibit large batch-to-batch variations, uncertain composition, and insufficient independence, making it impossible to effectively monitor differences between different brands of reagent kits, and also suffer from significant batch-to-batch variability. Therefore, it is essential to develop positive control products with minimal batch-to-batch variation, strong applicability, and high potency. Summary of the Invention

[0006] The present invention aims to solve the above-mentioned problems. In a first aspect, it provides a recombinant humanized anti-human Mp monoclonal antibody, comprising a light chain and a heavy chain. The monoclonal antibody is named Mp21, and the amino acid sequence of its light chain is shown in SEQ ID NO.1; the amino acid sequence of its heavy chain is shown in SEQ ID NO.2.

[0007] Based on the above technical solution, the monoclonal antibody further includes a J chain, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0008] Furthermore, the light chain includes a light chain signal peptide, a light chain variable region, and a light chain humanized constant region; The amino acid sequence of the light chain signal peptide is shown in SEQ ID NO: 6; The light chain variable region includes four backbone regions and three complementary determinant regions. The backbone regions include FR1: DVVMTQTPLSLPVSLGDQASISCRSS, FR2: LHWYLQKPGQSPKLLIY, FR3: TRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC, and FR4: FGGGTKLEIK. The complementary determinant regions include CDR1: QSLVHSNGNTY, CDR2: KVS, and CDR3: SQNTHVPWT. The amino acid sequence of the humanized constant region of the light chain is shown in SEQ ID NO: 4.

[0009] Furthermore, the heavy chain includes a heavy chain signal peptide, a heavy chain variable region, and a heavy chain humanized constant region; The amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO: 7. The heavy chain variable region includes four backbone regions and three complementary determinant regions. The backbone regions include FR1: EVQLQESGPGLVKPFQSLSLTCTVT, FR2: WNWIRKFPGNKLEWMGY, FR3: YYTPSLRSRISITRDTSKNHFFLQLNSMTTEDTATYYC, and FR4: WGAGTTVTVSS. The complementary determinant regions include CDR1: GYSITSGY, CDR2: ITYSGSS, and CDR3: ALLRLPGDWSFDV. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 5.

[0010] Furthermore, the nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.8; and the nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO.9.

[0011] In a second aspect, the present invention provides a recombinant expression vector comprising the nucleotide sequence encoding the light or heavy chain of a monoclonal antibody as described in the first aspect, wherein the vector is pCDNA3.4.

[0012] Thirdly, the present invention provides a host cell comprising a recombinant expression vector as described in the third aspect, wherein the host cell is HEK293 suspension cells.

[0013] Fourthly, based on the same invention, the present invention also provides an anti-Mp quality control product, wherein the quality control product comprises a recombinant humanized anti-human Mp monoclonal antibody as described in the first aspect.

[0014] Furthermore, the present invention also provides the application of the above-mentioned recombinant humanized anti-human Mp monoclonal antibody or anti-Mp quality control in the preparation of Mp antibody detection kits.

[0015] More preferably, the Mp antibody detection kit is a colloidal gold immunochromatographic detection kit.

[0016] The present invention has the following beneficial effects: 1. This invention successfully obtained and recombinantly expressed a specific monoclonal antibody Mp21 against human Mycoplasma pneumoniae (Mp). Its humanized design significantly reduced immunogenicity, making it suitable for in vitro diagnostics and potential therapeutic applications.

[0017] 2. Antibodies prepared by recombinant expression systems (such as HEK293 suspension cells) have a purity of over 95% as determined by SDS-PAGE, with consistent performance between batches and small batch-to-batch differences, making them suitable for use as standardized reagents.

[0018] 3. This antibody can be used as a positive control for Mp antibody detection kits. It has a clear composition, high potency, and good linearity, which solves the problems of high price of imported quality control products, large batch-to-batch differences of domestic raw materials, and uncertain composition in the current market.

[0019] 4. This antibody exhibits excellent detection performance in various serological detection methods, including colloidal gold immunochromatography platform, with high titer and high sensitivity, making it suitable for developing rapid and accurate Mp antibody diagnostic kits.

[0020] 5. By adopting a mature eukaryotic expression system and codon optimization strategy, combined with standardized transfection and purification processes, efficient and stable large-scale antibody production can be achieved, which has good industrialization prospects. Attached Figure Description

[0021] 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.

[0022] Figure 1Antibody SDS-PAGE detection results; Figure 2 Antibody titer test results; Figure 3 : Colloidal gold platform test results. Detailed Implementation

[0023] 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.

[0024] Example 1: Preparation of hybridoma cell lines secreting monoclonal antibodies against human Mycoplasma pneumoniae 1.1 Animal Immunization Mycoplasma pneumoniae was inoculated into broth culture medium and cultured in a shaker incubator at 37°C. The bacterial pellet was collected by high-speed centrifugation, washed with PBS and centrifuged 2-3 times. The collected bacterial pellet was then sonicated and concentrated to obtain the immunogenic antigen.

[0025] Six-week-old female balb / c mice were immunized with the human Mp antigen prepared above. For the primary immunization, the antigen was emulsified with an equal volume of Freund's complete adjuvant and administered subcutaneously to multiple sites on the back of the mice at a dose of 100 μg / mouse. Booster immunizations were performed every 15 days, with the antigen emulsified with an equal volume of incomplete Freund's adjuvant and administered subcutaneously to multiple sites on the back of the mice at a dose of 50 μg / mouse. On day 7 after each booster immunization, blood was collected from the tail of the mice to determine serum titers. Fusion was only performed when the serum titer reached 50,000 or higher. The results are shown in Table 1. Three days before fusion, mice were immunized intraperitoneally without adjuvant at a dose of 100 μg / mouse.

[0026] Table 1. Results of serum titer detection in immunized mice

[0027] 1.2 Cell Fusion Under aseptic conditions, spleens of immunized mice were harvested, thoroughly ground and dispersed. The homogenate was passed through a 200-mesh sieve to remove undispersed tissue fragments. The cells were centrifuged at 1000 rpm for 10 min to collect mouse lymphocytes. These lymphocytes were then mixed with mouse myeloma cells (sp2 / 0) at a ratio of 10:1. After centrifugation and washing, the cells were ensured to be free of fetal bovine serum. Cell fusion was initiated by adding 1 mL of PEG (molecular weight 1450) dropwise over 1 min at a 37°C water bath, gradually increasing the speed. The mixture was gently mixed and allowed to stand for 1 min. The fusion was terminated with 25 mL of serum-free DMEM, added gradually at a rate of 1 mL, 1 mL, 2 mL, 4 mL, 6 mL, and 12 mL per minute, stopping the reaction within 6 min. The fused cells were incubated at 37°C in a 5% CO2 incubator for 15 min, centrifuged at 800 rpm for 10 min, resuspended in HAT medium, aliquoted into 96-well cell culture plates, and cultured at 37°C in a 5% CO2 incubator.

[0028] 1.3 Hybridoma cell screening and subcloning The fused cells were completely replaced with HAT medium on day 7, and the culture supernatant was detected by conventional indirect ELISA on day 9.

[0029] The specific testing methods are as follows: Dilute Mp antigen to 1 μg / mL with coating buffer, add 100 μL / well to an ELISA plate, and incubate overnight at 4°C. Wash the plate 3 times. Add 150 μL of blocking buffer to each well of the ELISA plate and incubate at 37°C for 2 hours. Wash the plate 3 times. Add 100 μL / well of the cell supernatant to be tested to the ELISA plate, using mouse immune serum as a positive control and HAT medium as a blank control, and incubate at 37°C for 1 hour. Wash the plate 3 times. Dilute HRP-labeled goat anti-mouse antibody 10,000 times and add 100 μL / well to the ELISA plate, incubating at 37°C for 1 hour. Wash the plate 5 times. Add 100 μL of TMB chromogenic solution to each well of the ELISA plate and incubate at 37°C for 10 minutes; stop with 2M sulfuric acid, 50 μL / well, and read the data at 450 nm.

[0030] Subcloning is performed on cell wells that show a strong positive reaction. Generally, this process is repeated three times. Once the positive rate of a single-clone cell line reaches 100%, it is considered a stable cell line, and a hybridoma cell line is obtained.

[0031] Example 2: Hybridoma cell sequencing 2.1 Cell Culture The hybridoma cell line obtained in Example 1 was revived, expanded, and the culture was centrifuged at 1000 rpm for 10 min to collect the cell pellet.

[0032] 2.2 RNA extraction RNA was extracted from the cells according to the instructions of Thermo's RNA extraction kit.

[0033] 2.3 Reverse transcription Following the instructions of Thermo's kit, the RNA was immediately reverse transcribed to prepare cDNA.

[0034] 2.4 PCR amplification and recovery Using universal primers for the variable region of the IgG subtype mouse monoclonal antibody, and with the above 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, and the PCR products were recovered by gel cutting using a kit from TIANGEN.

[0035] 2.5 Sequencing The light chain and heavy chain PCR products were ligated into the pCDNA3.4 vector, respectively, and transformed into DH5α. The mixture was then plated, cultured, and cloned. Five to ten positive clones were selected and sent to a sequencing company for sequencing.

[0036] Example 3: Preparation of recombinant humanized anti-human mycoplasma pneumoniae monoclonal antibody 3.1 Sequence Analysis and Modification After sequencing, the antibody sequence was analyzed using the IMGT antibody variable region analysis tool in the database. The constant regions of the light and heavy chains were humanized, and the amino acid sequence after codon optimization is as follows: The amino acid sequence of the light chain of the recombinant humanized anti-human mycoplasma pneumoniae monoclonal antibody is shown in SEQ ID NO: 1. The light chain includes a light chain signal peptide, a light chain variable region, and a light chain humanized constant region (M subtype). The amino acid sequence of the light chain signal peptide is shown in SEQ ID NO: 6. The light chain variable region contains four backbone regions and three complementarity-determining regions. The backbone regions include FR1: DVVMTQTPLSLPVSLGDQASISCRSS, FR2: LHWYLQKPGQSPKLLIY, FR3: TRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC, and FR4: FGGGTKLEIK. The complementarity-determining regions include CDR1: QSLVHSNGNTY, CDR2: KVS, and CDR3: SQNTHVPWT. The amino acid sequence of the light chain humanized constant region is shown in SEQ ID NO: 4.

[0037] The amino acid sequence of the heavy chain of the recombinant humanized anti-human mycoplasma pneumoniae monoclonal antibody is shown in SEQ ID NO: 2. The heavy chain includes a heavy chain signal peptide, a heavy chain variable region, and a heavy chain constant region (M isotype). The amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO: 7. The heavy chain variable region contains four backbone regions and three complementarity-determining regions. The backbone regions include FR1: EVQLQESGPGLVKPFQSLSLTCTVT, FR2: WNWIRKFPGNKLEWMGY, FR3: YYTPSLRSRISITRDTSKNHFFLQLNSMTTEDTATYYC, and FR4: WGAGTTVTVSS. The complementarity-determining regions include CDR1: GYSITSGY, CDR2: ITYSGSS, and CDR3: ALLRLPGDWSFDV. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 5.

[0038] The recombinant humanized anti-human mycoplasma pneumoniae monoclonal antibody consists of two polypeptide chains (light chain and heavy chain) and also contains a J chain, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0039] In summary, the full-length amino acid sequences of the humanized light and heavy chains were obtained, as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The translation codons were then optimized using the online codon optimization website Jcat. The optimized full-length base sequence of the light chain is shown in SEQ ID NO: 8, and the full-length base sequence of the heavy chain is shown in SEQ ID NO: 9.

[0040] The detailed sequence is shown in Table 2.

[0041] Table 2 Sequence List

[0042] 3.2 Plasmid Construction The light chain, heavy chain, and J chain sequences were given to Qingke Biotechnology Co., Ltd. for gene synthesis and were constructed into the expression vector pCDNA3.4, ultimately yielding light chain plasmid L21, heavy chain plasmid H21, and J chain plasmid J21.

[0043] 3.3 Transfection and Expression Resuscitate HEK293 suspension cells and passage them more than 3 times to ensure that the cell viability remains stable at over 95%.

[0044] During transfection, adjust the cell density to 2.5-3.5 × 10⁶ cells / year. 6cells / mL, cell viability over 95%.

[0045] Mix the light chain plasmid, heavy chain plasmid, J chain plasmid, and transfection reagent thoroughly in a specific ratio. After standing for 10-20 minutes, add the mixture to the prepared cells and mix well. Incubate the cells in a CO2 shaker at 37°C, 120 rpm, and 5% CO2.

[0046] Feeding was performed on days 1, 3, and 5 after transfection, and cell expression supernatant was collected on day 7 (at which point the cell viability was over 60%).

[0047] 3.4 Antibody purification The cell culture supernatant was precipitated with ammonium sulfate, and the precipitated protein was collected. The protein was purified according to the BorgLone heparin column purification manual. The eluted protein was collected, filtered through a 0.22 μm filter membrane, aliquoted, named Mp21, and stored in a -80℃ freezer for subsequent detection and validation.

[0048] Example 4: Performance Detection of Recombinant Humanized Anti-human Mycoplasma pneumoniae Monoclonal Antibody 4.1 Antibody purity detection Mp21 was subjected to reducing SDS-PAGE analysis, and the purity was above 95%. The test results are as follows... Figure 1 As shown.

[0049] 4.2 Antibody titer detection The conventional indirect ELISA method was used. Mp antigen (5 μ / mL) was coated, and Mp21 antibody was detected at 8 gradients, starting from 1000 ng / mL and diluted 2-fold. The results are as follows: Figure 2 As shown, the titer was higher than the control (commercially available Mp antibody).

[0050] 4.3 Antibody batch-to-batch variation detection Following the method in Example 3, three batches of protein were transfected and purified. The batch-to-batch difference was tested using an indirect ELISA method. The test results are shown in Table 3. The antibody batch-to-batch difference was small.

[0051] Table 3 Results of antibody batch-to-batch difference detection

[0052] 4.4 Antibody Detection on Colloidal Gold Platform (1) Preparation of colloidal gold test strips Mouse anti-human IgM monoclonal antibody was labeled onto colloidal gold, blocked with blocking buffer, centrifuged, and the supernatant was collected. The precipitate was resuspended in colloidal gold diluent, evenly spread onto glass fiber, and dried to serve as the gold-labeled pad. Mp antigen and goat anti-mouse antibody were diluted separately with coating diluent, evenly coated onto an NC membrane, and dried to serve as the detection line and control line, respectively. The sample pad, gold-labeled pad, NC membrane, and absorbent paper were sequentially fixed onto a PVC plate to prepare the colloidal gold test strip.

[0053] (2) Quality control product test results Mp21 was diluted with diluent at dilution gradients of 1:1000, 1:500, 1:200, 1:100, 1:50, and 1:10, and used as test samples. The test results are as follows: Figure 3 As shown, it has high valence and good linearity.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant humanized anti-human Mp monoclonal antibody, characterized in that, The monoclonal antibody, named Mp21, comprises a light chain and a heavy chain. The amino acid sequence of its light chain is shown in SEQ ID NO.1, and the amino acid sequence of its heavy chain is shown in SEQ ID NO.

2.

2. The recombinant humanized anti-human Mp monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody further includes a J chain, the amino acid sequence of which is shown in SEQ ID NO:

3.

3. The recombinant humanized anti-human Mp monoclonal antibody according to claim 1, characterized in that, The light chain includes a light chain signal peptide, a light chain variable region, and a light chain humanized constant region; The amino acid sequence of the light chain signal peptide is shown in SEQ ID NO: 6; The light chain variable region includes four backbone regions and three complementary determinant regions. The backbone regions include FR1: DVVMTQTPLSLPVSLGDQASISCRSS, FR2: LHWYLQKPGQSPKLLIY, FR3: TRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC, and FR4: FGGGTKLEIK. The complementary determinant regions include CDR1: QSLVHSNGNTY, CDR2: KVS, and CDR3: SQNTHVPWT. The amino acid sequence of the humanized constant region of the light chain is shown in SEQ ID NO:

4.

4. The recombinant humanized anti-human Mp monoclonal antibody according to claim 1, characterized in that, The heavy chain includes a heavy chain signal peptide, a heavy chain variable region, and a heavy chain humanized constant region; The amino acid sequence of the heavy chain signal peptide is shown in SEQ ID NO:

7. The heavy chain variable region includes four backbone regions and three complementary determinant regions. The backbone regions include FR1: EVQLQESGPGLVKPFQSLSLTCTVT, FR2: WNWIRKFPGNKLEWMGY, FR3: YYTPSLRSRISITRDTSKNHFFLQLNSMTTEDTATYYC, and FR4: WGAGTTVTVSS. The complementary determinant regions include CDR1: GYSITSGY, CDR2: ITYSGSS, and CDR3: ALLRLPGDWSFDV. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:

5.

5. The recombinant humanized anti-human Mp monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.8; the nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO.

9.

6. A recombinant expression vector, characterized in that, The vector comprises the nucleotide sequence encoding the light or heavy chain of a monoclonal antibody as described in claim 5, wherein the vector is pCDNA3.

4.

7. A host cell, characterized in that, The invention comprises a recombinant expression vector as described in claim 6, wherein the host cell is HEK293 suspension cells.

8. A quality control product resistant to Mp, characterized in that, The quality control product comprises a recombinant humanized anti-human Mp monoclonal antibody as described in any one of claims 1-5.

9. The use of the recombinant humanized anti-human Mp monoclonal antibody as described in any one of claims 1-5 or the anti-Mp quality control product as described in claim 8 in the preparation of an Mp antibody detection kit.

10. The application according to claim 1, characterized in that, The Mp antibody detection kit is a colloidal gold immunochromatographic detection kit.

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