Anti-human Mycoplasma pneumoniae CARDS TX monoclonal antibody MM03H

Abstract

This invention discloses a monoclonal antibody MM03H against human Mycoplasma pneumoniae CARDS TX, prepared from a hybridoma cell line deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21420. Currently, there are no commercially available anti-CARDS toxin antibodies, and even fewer ELISA kits targeting the CARDS TX antigen. This invention, for the first time, discloses a monoclonal antibody MM03H against human Mycoplasma pneumoniae CARDS TX protein. Upon binding to the human Mycoplasma pneumoniae CARDS TX protein, it can inhibit the entry of the human Mycoplasma pneumoniae CARDS TX protein across the cell membrane into the cytoplasm.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody, specifically to an anti-human mycoplasma pneumoniae CARDS TX monoclonal antibody MM03H. Background Technology

[0002] Mycoplasma pneumoniae pneumonia (MPP) is a common type of community-acquired pneumonia, most frequently seen in school-aged children and adolescents. The exact pathogenic mechanism of Mycoplasma pneumoniae (MP) remains to be elucidated. In 2005, Kannan et al., while studying the interaction between MP and human surfactant protein A, discovered a protein approximately 68 kDa in size that could bind to surfactant protein A. This protein was later confirmed to have vacuolating toxin activity and, as a virulence factor directly related to MP pathogenesis, was named MP community-acquired respiratory distress syndrome toxin (CARDS TX).

[0003] The main functions of CARDS TX currently identified are: adenosine diphosphate ribosyltransferase activity and cellular vacuolating toxin activity. Adenosine diphosphate (ADP) ribosylation includes mono-ADP ribosylation and poly-ADP ribosylation, and CARDS TX participates in the mono-ADP ribosylation process. Kannan et al. used NAD tracing to treat CHO cells with rCARDS TX and observed that some proteins in the cells were modified by ADP ribosylation, but how these proteins further affect host cells and participate in the specific mechanisms of MP pathogenesis still needs further investigation. Co-incubation of CARDS TX with CHO cells can induce cytoplasmic vacuolization. When CARDS TX acts on the respiratory tract, it can cause significant slowing, asynchrony, or even cessation of ciliary movement, ciliary structure destruction, edema of airway epithelial cells and submucosal cells, cytoplasmic vacuolization, and nuclear fragmentation, ultimately leading to cell death.

[0004] Animal experiments have confirmed that CARDS-TX can induce an increase in pro-inflammatory cytokines, as well as the infiltration of peribronchial and perivascular inflammatory cells and vacuolization of airway epithelial cells. As a virulence factor of mycoplasma pneumoniae (MP), CARDS-TX not only induces inflammatory responses in the respiratory system but also mediates weakened airway ciliary movement, epithelial cell damage, and even apoptosis. Therefore, CARDS-TX plays an important role in the pathogenic factors of MP. Studies have found a positive correlation between CARDS-TX concentration in bronchoalveolar lavage fluid (BALF) and the degree of lung injury; the higher the CARDS-TX concentration in BALF, the more severe the infiltration of peribronchial and perivascular lymphocytes. Simultaneously, inflammatory factors such as IL-12 and IFN-γ are significantly elevated in BALF. Furthermore, the proportion of children infected with drug-resistant strains of Mycoplasma pneumoniae increases, and some children may develop refractory Mycoplasma pneumoniae pneumonia, in which case macrolide antibiotics often have poor therapeutic effects. As one of the virulence components of Mycoplasma pneumoniae (MP), CARDS-TX possesses strong immunogenicity. Therefore, the development of anti-CARDS-TX antibodies is of great value for the prevention and treatment of Mycoplasma pneumoniae pneumonia. However, there are currently no commercially available anti-CARDS-TX antibodies on the market, and even fewer ELISA detection kits targeting CARDS-TX antigens. Therefore, the development of functional monoclonal antibodies against human Mycoplasma pneumoniae CARDS-TX is of great value for further research on the biological function of CARDS-TX and provides new possibilities for the clinical treatment of Mycoplasma pneumoniae pneumonia. Summary of the Invention

[0005] The objective of this invention is to provide an anti-human mycoplasma pneumoniae CARDS TX monoclonal antibody and a hybridoma cell line capable of producing the said anti-CARDS TX monoclonal antibody.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is: a hybridoma cell line, wherein the hybridoma cell line is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 21420.

[0007] The method for preparing the hybridoma cell line disclosed in this invention includes the following steps:

[0008] (1) Expression of full-length CARDS TX protein: The full-length sequence of CARDS TX was cloned into the pFastBac insect system expression vector; recombinant baculovirus P1 was obtained by transfecting insect cells with recombinant baculovirus DNA extracted from Ecoli strain containing recombinant baculoviruses and high-titer recombinant virus was used to infect HighFive cells in the logarithmic growth phase to express the target protein and purified by nickel column, eluted and desalted to the final protein buffer; the purified protein was used to immunize BALB / C mice;

[0009] (2) Obtaining fusion cell growth clones: Spleen cells were aseptically removed from immunized mice as antigen-sensitized B cells. The B cells were fused with myeloma cell line AG8 using conventional methods. Then, the conventional fusion cell HAT screening method was used to screen the cells and obtain fusion cell growth clones.

[0010] (3) After screening and identification using biochemical and immunological techniques such as Western blotting and flow cytometry, a hybridoma cell line with a high level of antibody secretion was selected. The hybridoma cell line was deposited in the China General Microbiological Culture Collection Center and classified as: Mouse anti-human mycoplasma pneumoniae CARDS TX monoclonal antibody hybridoma cell line MM03H.

[0011] The preservation information of the hybridoma cell line described in this invention is as follows: Preservation unit: China General Microbiological Culture Collection Center (CGMCC); Preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: January 7, 2021; Preservation number: CGMCC No. 21420; Classification and name: Mouse anti-human mycoplasma pneumoniae CARDS TX monoclonal antibody hybridoma cell line MM03H.

[0012] In the above technical solution, the full-length CARDS TX protein expressed in step (1) has strong immunogenicity, and the spatial configuration of the expressed antigen molecule can be exposed on the cell membrane surface in a natural state, thereby more effectively stimulating the body's immune response.

[0013] In the above technical solutions, the method for preparing anti-human Mycoplasma pneumoniae CARDS TX monoclonal antibody can be carried out according to conventional methods known in the art (Kohler and Milstein, Nature 265:495-497, 1975). Alternatively, the corresponding humanized form of anti-monoclonal antibody can be prepared according to the method described in US Patent 5,585,089.

[0014] There are two methods for preparing monoclonal antibodies using the above-mentioned hybridoma cell lines:

[0015] 1) Inoculate the above hybridoma cells into the hybridoma culture medium, and then isolate and purify the required monoclonal antibody from the culture medium after culture;

[0016] 2) Inoculate the above-mentioned hybridoma cells into the peritoneal cavity of the animal, and isolate and purify the required monoclonal antibody from the ascites fluid of the animal.

[0017] The present invention also provides the heavy chain variable region amino acid sequence of the monoclonal antibody MM03H: SEQ.ID.NO:1; and the light chain variable region amino acid sequence: SEQ.ID.NO:2.

[0018] SEQ.ID.NO:1:QVQLKESGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSNYYGPMDYWGQGTSVTVSS

[0019] Corresponding to:

[0020] Gln Val Gln Leu Lys Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala SerVal Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Met His TrpVal Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr AsnAsp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Asp LysSer Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala ValTyr Tyr Cys Ala Arg Ser Asn Tyr Tyr Gly Pro Met Asp Tyr Trp Gly Gln Gly ThrSer Val Thr Val Ser Ser

[0021] SEQ.ID.NO:2:

[0022] DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQLGNTLTWTFGGGTKLEIK

[0023] Corresponding to:

[0024] Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly AspArg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp TyrGln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu HisSer Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu ThrIle Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Leu Gly Asn ThrLeu Thr Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

[0025] The present invention also provides the DNA molecular sequence encoding the heavy chain variable region of the monoclonal antibody MM03H: SEQ.ID.NO:3; and the DNA molecular sequence encoding the light chain variable region: SEQ.ID.NO:4.

[0026] SEQ.ID.NO:3:

[0027] caggtgcagctgaaggagtctggacctgagctggtaaagcctggggcttcagtgaagatgtcctgcaaggcttctggatacacattcactagctatgttatgcactgggtgaagcagaagcctgggcagggccttgagtggattggatatattaatccttacaatgatggtactaag tacaatgagaagttcaaaggcaaggccacactgacttcagacaaatcctccagcacagcctacatggagctcagcagcctgacctctgaggactctgcggtctattactgtgcaagatctaattattatggccctatggactactggggtcaaggaacctcagtcaccgtctcctca

[0028] SEQ.ID.NO:4:

[0029] gatattcagatgacacagactacatcctccctgtctgcctctctggggagacagtcaccatcagttgcagggcaagtcaggacattaggaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagat tacattcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaactgggtaatacgcttacgtggacgttcggtggaggcaccaagctggaaataaaa

[0030] This invention discloses the application of the above-mentioned monoclonal antibody MM03H in blocking human Mycoplasma pneumoniae CARDS TX protein; or its application in preparing reagents for detecting the expression level of Mycoplasma pneumoniae CARDS TX. The ELISA detection kit prepared by the MM03H monoclonal antibody of this invention, combined with conventional components, has significantly improved accuracy compared to existing human Mycoplasma pneumoniae in vitro toxin (CARDS-TX) ELISA kits.

[0031] This invention discloses the application of the above-mentioned monoclonal antibody MM03H in inhibiting human Mycoplasma pneumoniae CARDS TX protein, especially inhibiting human Mycoplasma pneumoniae CARDS TX protein from crossing the cell membrane into the cytoplasm.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] This invention discloses for the first time an anti-human Mycoplasma pneumoniae CARDS TX protein monoclonal antibody, MM03H. Upon binding to the human Mycoplasma pneumoniae CARDS TX protein, it can inhibit the entry of the human Mycoplasma pneumoniae CARDS TX protein across the cell membrane into the cytoplasm. The inhibitory effect is positively correlated with the antibody concentration used. Currently, there are no commercially available anti-human Mycoplasma pneumoniae CARDS TX protein monoclonal antibodies on the market, nor are there any literature reports on the preparation of mouse monoclonal antibodies that inhibit the entry of human Mycoplasma pneumoniae CARDS TX protein across the cell membrane into the cytoplasm. Attached Figure Description

[0034] Figure 1 Karyotype analysis of chromosomes from the hybridoma cell line in Example 1 (magnified 1000 times).

[0035] Figure 2 This is a graph showing the results of Western Blot analysis of the recognition of antigen by the monoclonal antibody MM03H in Example 1;

[0036] Figure 3 This is a diagram showing the results of Ig subclass identification in Example 1;

[0037] Figure 4 In Example 2, flow cytometry was used to analyze the effect of the anti-human mycoplasma pneumoniae CARDS TX antibody MM03H on inhibiting the entry of human mycoplasma pneumoniae CARDS TX protein into cells. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. The raw materials, specific operating methods, and testing methods of this invention are conventional techniques.

[0039] Example 1: Preparation of anti-human Mycoplasma pneumoniae CARDS TX monoclonal antibody

[0040] (1) Preparation of full-length human Mycoplasma pneumoniae CARDS TX protein:

[0041] a) Construction of human Mycoplasma pneumoniae CARDS TX gene expression vector

[0042] The full-length CDS sequence of the *Mycoplasma pneumoniae* CARDS TX gene was obtained from the NCBI database. The codon TGA encoding tryptophan was mutated to TGG, without altering the translated amino acid sequence. The mutated gene sequence is SEQ.ID.NO:5. The mutated sequence was directly synthesized by a conventional gene synthesis company. The synthesized DNA fragment was then inserted into the insect system expression vector pFastBac after being treated with restriction endonucleases and T4 ligase, and transformed into competent *E. pneumoniae* strain DH10Bac.

[0043] SEQ.ID.NO:5:

[0044]

[0045] b) Expression and purification of human Mycoplasma pneumoniae CARDS TX protein:

[0046] Recombinant baculotropic DNA was extracted from Ecoli bacteria containing recombinant baculotropic DNA. This DNA was then used to infect insect cells to obtain P1 recombinant baculovirus, which was further amplified to obtain high-titer recombinant virus. The high-titer recombinant virus was used to infect insect cells in the logarithmic growth phase to express the target protein. High Five cells were used as the insect cells. The nickel column was equilibrated with equilibration buffer, and the culture supernatant after centrifugation and filtration was loaded onto the nickel column. The column was then washed with equilibration buffer, and finally, the target protein was eluted with imidazole at different concentrations, followed by desalting to the final protein buffer.

[0047] (2) Preparation of hybridoma cell lines secreting specific mouse anti-human mycoplasma pneumoniae CARDS TX antibodies

[0048] Using the human Mycoplasma pneumoniae CARDS TX protein obtained above as an immunogen, Balb / c mice were immunized three times (10... 7 / 500ul / mouse (3-week interval). On the fourth day after the last immunization, mouse spleen cells were fused with P3X63Ag8 mouse myeloma cell line (10 96-well plates). Hybridoma culture supernatant was used for preliminary screening to identify positive clones whose antibodies could bind to the human Mycoplasma pneumoniae CARDS TX protein. After rescreening and subcloning, several hybridoma cell lines stably secreting specific mouse anti-human Mycoplasma pneumoniae CARDS TX antibodies were obtained. One of these lines was retained and named MM03H.

[0049] The preservation information for the above hybridoma cell line is as follows: Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: January 7, 2021; Accession number: CGMCC No. 21420; Classification and name: Mouse anti-human mycoplasma pneumoniae CARDS TX monoclonal antibody hybridoma cell line MM03H.

[0050] Hybridoma cells, after continuous in vitro passage, can still stably secrete specific antibodies; chromosome analysis of the hybridoma cell lines shows (see...). Figure 1 The chromosome number of hybridoma cells is 80-110.

[0051] (3) Production and characterization of anti-human mycoplasma pneumoniae CARDS TX monoclonal antibody

[0052] a) Production of monoclonal antibodies using in vivo ascites induction method

[0053] Female Balb / c mice aged 6-8 weeks were injected intraperitoneally with Pristane (0.5 ml / mouse). One week later, hybridoma cells (1 × 10⁻⁶) were inoculated intraperitoneally. 6 / ani), and simultaneously inject an equal volume mixture of Pristane and Freund's incomplete adjuvant (0.2 ml / ani). Ascites fluid was harvested 8-10 days later, and the supernatant was collected by centrifugation and stored at -80°C.

[0054] Ascites fluid, after fibrin removal and salting out, was purified by protein G affinity chromatography. The protein peak eluate was collected, dialyzed against phosphate-buffered saline (PBS), and the antibody protein concentration was determined using a 751 UV spectrophotometer to be 1.0–1.6 mg / ml. SDS-PAGE results showed that the mouse anti-human Mycoplasma pneumoniae CARDS TX antibody secreted by MM03H could recognize the recombinant human Mycoplasma pneumoniae CARDS TX protein (see [link to study]). Figure 2 ).

[0055] b) Ig subclass identification

[0056] Ig subclasses were identified using a rapid test strip assay. The results showed that MM03H was a mouse IgG2b antibody (see [link to test strip assay]). Figure 3 ).

[0057] Example 2: In vitro biological effects of monoclonal antibodies on cells

[0058] This embodiment describes the effect of the anti-human mycoplasma pneumoniae CARDS TX antibody MM03H of the present invention on inhibiting the entry of human mycoplasma pneumoniae CARDS TX protein into cells.

[0059] 293T cells were seeded into 6-well culture plates and cultured in RPMI-1640 medium containing 10% FBS until the cell confluence reached more than 50%.

[0060] The concentration of FITC-labeled human Mycoplasma pneumoniae CARDS TX protein was adjusted to 1 mg / ml with PBS. 10 μl of human Mycoplasma pneumoniae CARDS TX protein was added to each group of EP tubes. The concentration of anti-human Mycoplasma pneumoniae CARDS TX antibody MM03H was adjusted to 1 mg / ml with PBS. The antibody was added to each group of EP tubes at antibody:protein ratios of 1:1, 1:5, and 1:20, respectively. After gently mixing with a pipette, the tubes were incubated in a CO2 incubator for 1 h.

[0061] The culture medium was aspirated from the 6-well culture plate. The incubated mixture of human Mycoplasma pneumoniae CARDS TX protein and antibody was then added to the 6-well culture plate with PBS to bring the volume to 1 ml. Parallel experiments were conducted with a negative control group (without fluorescent protein) and a positive control group (with fluorescent protein only).

[0062] After incubation, the cells were digested and pipetted to prepare a single-cell suspension. After washing twice with PBS, the cells were resuspended in 400 μL of flow cytometry loading buffer (PBS containing 2% FBS) and then analyzed by flow cytometry to detect cell fluorescence intensity. Flow cytometry results showed that, compared with the positive control group, antibody MM03H could inhibit the entry of fluorescent human Mycoplasma pneumoniae CARD TX protein into the cell cytoplasm, significantly reducing cell fluorescence intensity. The degree of fluorescence reduction was correlated with the antibody concentration used (see [link to relevant documentation]). Figure 4 This indicates that the monoclonal antibody MM03H inhibits the entry of human Mycoplasma pneumoniae CARDS TX protein across the cell membrane into the cytoplasm, and the inhibitory effect is positively correlated with the antibody concentration used.

Claims

1. The application of monoclonal antibodies in the preparation of reagents for treating Mycoplasma pneumoniae pneumonia, characterized in that, The monoclonal antibody is a monoclonal antibody prepared from a hybridoma cell line, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 21420.

2. The application of monoclonal antibodies in the preparation of reagents for blocking Mycoplasma pneumoniae CARDS TX protein, characterized in that, The monoclonal antibody is a monoclonal antibody prepared from a hybridoma cell line, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 21420.

3. The use of monoclonal antibodies in the preparation of drugs for treating Mycoplasma pneumoniae CARDS TX or inhibiting the entry of human Mycoplasma pneumoniae CARDS TX protein across the cell membrane into the cytoplasm, characterized in that, The monoclonal antibody is a monoclonal antibody prepared from a hybridoma cell line, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 21420.

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