CN113150139B - PBP2a monoclonal antibody and preparation method and application thereof - Google Patents
PBP2a monoclonal antibody and preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a PBP2a monoclonal antibody and a preparation method and application thereof, wherein the preparation process of the PBP2a monoclonal antibody comprises the following steps: the method comprises the steps of synthesizing a staphylococcus aureus PBP2a gene, expressing and purifying a PBP2a protein, immunizing a Balb/c mouse by taking recombinant PBP2a protein as immunogen, fusing splenocytes of the immunized mouse with myeloma NS-1 cells of the mouse, screening hybridoma cells secreting specific McAb by an indirect ELISA method, inducing the mouse to generate ascites by hybridoma cell strains, purifying an antibody by protein G affinity chromatography, and finally obtaining the PBP2a protein monoclonal antibody. The monoclonal antibodies of PBP2a obtained by the application can be combined with PBP2 a; the titer of the obtained PBP2a monoclonal antibody is more than 200 ten thousand, and the PBP2a protein monoclonal antibody has high purity, strong specificity and high sensitivity, and can be used for preparing a diagnostic reagent for detecting PBP2a genes.
Description
Technical Field
The invention relates to the field of immunotherapy biomedicine, and particularly relates to a PBP2a monoclonal antibody and a preparation method and application thereof.
Background
Due to the widespread use of broad spectrum antimicrobial agents, the increasing proportion of methicillin-resistant staphylococcus aureus (MRSA) in clinically isolated staphylococci has developed into one of the major pathogenic bacteria of serious nosocomial infections. Since the first discovery of MRSA in 1961, MRSA infection is spreading and prevalent worldwide at a staggering rate. Therefore, rapid detection of MRSA in clinical specimens is of great significance. Current studies have shown that the major cause of MRSA production is the production of the penicillin binding protein PBP2a by staphylococcus aureus. The protein exists on the cell surface by a drug resistance gene and has extremely low affinity to beta-lactam antibacterial agents.
Because PBP2a has a low affinity for β -lactams, PBP2a is not inhibited when other PBPs are inhibited and cannot perform normal physiological functions in the presence of β -lactams, and can act in place of other inhibited PBPs, allowing bacterial cells to survive and develop tolerance.
The existing PBP2a detection method has poor specificity and long time consumption, and delays the diagnosis and treatment of patients; the monoclonal antibody of PBP2a protein in the prior art has low purity titer and poor specificity. Therefore, the establishment of a sensitive, rapid and specific PBP2a detection diagnosis method and the preparation of the PBP2a protein monoclonal antibody with high purity titer and strong specificity have important clinical application value.
Disclosure of Invention
2. The invention provides a PBP2a monoclonal antibody and a preparation method and application thereof aiming at various defects of the prior art, the 6-strain PBP2a monoclonal antibody is prepared, and the prepared monoclonal antibody has high purity titer, strong specificity and important clinical application value.
3. In a first aspect, the present application provides a PBP2a monoclonal antibody, comprising a 1B4, 4F10 and 5G4 monoclonal antibodies, the protein sequence of which comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are as follows (1) to (3):
the heavy chain variable region of the 1B4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO.2, and the light chain variable region of the 1B4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 3;
(2) the heavy chain variable region of the 4F10 monoclonal antibody has an amino acid sequence shown as SEQ ID No.4, and the light chain variable region of the 4F10 monoclonal antibody has an amino acid sequence shown as SEQ ID No. 5;
(3) the heavy chain variable region of the 5G4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO.6, and the light chain variable region of the 5G4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 7.
7. In a second aspect of the present application, there is provided a method for preparing the above PBP2a monoclonal antibody, comprising the following steps: the method comprises the steps of synthesizing a staphylococcus aureus PBP2a gene, expressing and purifying a PBP2a protein, immunizing a Balb/c mouse by taking recombinant PBP2a protein as immunogen, fusing splenocytes of the immunized mouse with myeloma NS-1 cells of the mouse, screening hybridoma cells secreting specific McAb by an indirect ELISA method, inducing the mouse to generate ascites by hybridoma cell strains, purifying the antibody by a protein G affinity chromatography method, and finally obtaining the PBP2a protein monoclonal antibody.
As a further illustration of the present application, the synthetic Staphylococcus aureus PBP2a gene has an amino acid sequence as shown in SEQ ID No. 1.
As a further explanation of the present application, the method specifically comprises the steps of:
step 1): synthesis of PBP2a Gene
Obtaining an amino acid sequence of a staphylococcus aureus PBP2a gene from a GenBank sequence database; carrying out codon optimization of the base sequence of the PBP2a gene according to the codon preference of escherichia coli, and synthesizing a gene sequence after the codon optimization;
step 2): expression and purification of PBP2a Gene
The PBP2a gene is connected into a pET-28a vector, and the plasmid is transformed into escherichia coli BL21(DE3) and is induced to express by IPTG; collecting thalli, crushing the thalli under high pressure, centrifuging, collecting supernatant, and purifying protein by using Ni column affinity chromatography; carrying out fine purification on the purified protein again by using a Superdex-200 column to obtain a PBP2a recombinant protein with high purity, and collecting the PBP2a protein as an antigen for later use;
step 3): immunization of mice
Immunizing 3 female BALB/c mice of 6-8 weeks old by using the purified PBP2a protein as an antigen, collecting blood at the tail 10 days after the conventional immunization, and detecting the antibody titer generated by the mice by using indirect ELISA; injecting recombinant antigen into tail vein at last time 3 days before fusion, wherein the dose is 60 mug/mouse, and performing boosting immunization once;
and step 4): preparation of NS-1 myeloma cells
One week before fusion, NS-1 myeloma cells stored in liquid nitrogen were revived and cultured at 25cm 2 Subculturing in cell culture flask with 15% fetal calf serum DMEM for one week, and adjusting cell concentration to 10 6 Per mL; during fusion, myeloma cells in a logarithmic growth phase are selected, the original culture medium in a bottle is discarded, a proper amount of serum-free DMEM culture solution is added, the cells are gently blown down and transferred into a 50mL centrifuge tube, 400g of the centrifuge tube is centrifuged for 5 minutes, the cells are washed for 3 times, the supernatant is discarded, cell precipitates are re-suspended by the serum-free culture solution, and the cell precipitates are counted for standby;
and step 5): preparation of immune spleen cells
Taking one BALB/c mouse with the best immune effect, killing the mouse by orbital bleeding, and soaking the mouse in 75% alcohol for 5min for disinfection; fixing the sterilized mice in a super clean bench, taking out spleen, and removing adipose tissues and connective tissues adhered with cells by scissors; washing spleen with serum-free culture solution, transferring into 40 μm cell filter screen, gently grinding with inner core of syringe, gently washing filter screen with serum-free culture solution, and collecting spleen cell suspension; centrifuging at 400g for 5min, washing cells for 3 times, discarding supernatant, suspending cell precipitates by serum-free DMEM culture solution, and counting for later use;
step 6): cell fusion
Uniformly mixing NS-1 myeloma cells and immune spleen cells in a 50mL centrifuge tube in a ratio of 1:10, centrifuging for 10min at 400g, sucking out supernatant, and slightly flicking the bottom of the centrifuge tube to slightly loosen cell precipitates; slowly dropping 1mL of 50% PEG solution preheated to 37 ℃ into the lmin; gently stirring the cells with a pipette for 1 min; then adding 10mL of DMEM culture medium into the cell mixture, dropwise adding lmL into the cell mixture in the first minute, adding lmL into the cell mixture in the second minute, adding 3mL into the cell mixture in the 3min-4min, and adding the rest 5mL into the cell mixture after 5 min; incubating the cell mixture in a water bath at 37 ℃ for 15 min; then 400g centrifugation for 5min, removing the supernatant, using 20ml 15% fetal bovine serum DMEM medium heavy suspension cell precipitation, and transfer to 75cm 2 Cell culturePlacing the bottle in a cell culture box for incubation for 16-24 h; transferring the fused cell suspension to a 50ml centrifuge tube, and centrifuging for 10min at 400 g; the supernatant was removed, the cell pellet was resuspended in 2.5mL of 15% fetal bovine serum DMEM medium, 22.5mL of semisolid medium was added and mixed well and poured into 3.5cm diameter dishes of approximately 2mL each at 37 deg.C with 5% CO 2 Culturing; ten days later, the surface of the culture medium of the visible plate is provided with visible white cell clone clusters;
step 7): positive hybridoma cell selection
Sucking up the cell mass in the plate under the aseptic condition, putting the cell mass into a 96-well plate culture solution, and continuously culturing for 4 days; carrying out indirect ELISA detection 4 days later; meanwhile, the serum of a normal mouse before immunization is used as a negative control, the serum of the mouse after immunization is used as a positive control, and the positive judgment is carried out when the light absorption value is 2.1 times larger than that of the negative control group; detecting hybridoma cell holes which have specific antibodies and grow in a single clone and have good shapes, and then cloning; after at least 3 times, transferring the hybridoma cells in the positive hole to a 24-hole culture plate, and freezing and storing the hybridoma cells when the hybridoma cells in the 24-hole culture plate grow well;
step 8): preparation and purification of monoclonal antibodies
Injecting 0.5ml of liquid paraffin into the abdominal cavity of a BALB/c mouse aged 8 weeks; hybridoma cells were cultured at 1X 10 5 Inoculating at a concentration of 25cm 2 Culturing in a culture flask until the cell activity is optimal, and adjusting the cell number to about 1 × 10 6 Inoculating to the abdominal cavity of a mouse injected with liquid paraffin; collecting ascites after 7-10 days; the ascites was diluted 3-fold or more with 20mM PBS pH7.4, the supernatant was centrifuged and purified by HiTrap protein G affinity chromatography to obtain the PBP2a protein monoclonal antibody.
As further illustrated in the present application, in step 3), the method for immunizing the mouse is: 60 mu g of the original emulsified mice each, 350 mu l of the total volume of antigen +1 multiplied by PBS, 3500 times of mixed emulsification with the equal volume of Freund's adjuvant and 200 times/min; first immunization: injecting 0.2ml of emulsion containing 60 mu g of antigen into groin of each mouse by using a 2ml injector subcutaneously, and recording the injection time and the injection part; and (3) second immunization: two weeks apart, each mouse was injected intraperitoneally with 0.2ml of emulsion containing 60 μ g of antigen using a 2ml syringe, and the time and location of injection was recorded; and (3) third immunization: two weeks apart, each mouse was injected subcutaneously in the groin with 0.2ml of emulsion containing 60 μ g of antigen using a 2ml syringe, and the time and location of injection was recorded.
As a further illustration of the present application, in step 7), the ELISA detection method: coating the antigen on an enzyme label plate by 100 ng/hole, standing overnight at 4 ℃, washing for three times, adding 200mL of confining liquid, incubating for 2h at 37 ℃, washing for three times, then adding hybridoma cell culture supernatant, incubating for 2h at 37 ℃, washing for three times by using the washing liquid, adding goat anti-mouse IgG-HRP diluted by 1:10000, incubating for 1h at 37 ℃, washing for three times, developing for 10min at 37 ℃ in a dark place, adding 2M H after developing sufficiently, and performing color development 2 SO 4 The reaction was stopped and the absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader.
As a further explanation of the present application, in step 8), Protein G column was used for purification, and the new column was passed through the column with 5ml of ultrapure water, and then 5ml of 0.4M PB buffer (pH 7.0) was used to equilibrate the purification cartridge; the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the binding site; the column was equilibrated with 10ml of 0.4M PB buffer pH 7.0; 5ml of 0.1M glycine-hydrochloric acid buffer pH 2.7 to remove the antibody on the binding site, and adding 1M Tris-HCl pH 8.0 to neutralize the glycine and maintain the pH at neutrality suitable for antibody preservation; and (4) carrying out antibody sequence determination on the hybridoma cells of the purified antibody.
In a third aspect, the present application provides a pharmaceutical composition, which comprises the monoclonal antibody and a pharmaceutically acceptable excipient.
In a fourth aspect, the present application provides a detection reagent or kit comprising the monoclonal antibody described above.
The application in the fifth aspect provides the application of the monoclonal antibody in preparing a diagnostic reagent for detecting PBP2a gene.
Compared with the prior art, the invention has the following beneficial technical effects:
the monoclonal antibodies of PBP2a obtained by the application can be combined with PBP2 a; the titer of the obtained PBP2a monoclonal antibody is more than 200 ten thousand, and the PBP2a protein monoclonal antibody has high purity, strong specificity and high sensitivity, and can be used for preparing a diagnostic reagent for detecting PBP2a genes.
Drawings
FIG. 1 is a diagram showing the result of purification assay of recombinant PBP2a protein of the present invention.
FIG. 2 is a graph showing the results of purity measurement of PBP2a monoclonal antibody of the present invention.
FIG. 3 shows the results of Ig classes and subclasses identification of the 6 monoclonal antibodies of the present invention.
FIG. 4 shows the result of the measurement of the titer of the PBP2a monoclonal antibody of the present invention.
FIG. 5 is a diagram of immunoblot analysis of PBP2a monoclonal antibody of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, a detailed description of the present invention will be given below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
Example (b):
the experimental procedure and method:
obtaining of PBP2a Gene
The amino acid sequence of the PBP2a gene is obtained from a GenBank database, the codon optimization of the base sequence of the PBP2a gene is carried out according to the codon preference of escherichia coli, and the gene sequence after the codon optimization is synthesized by the Committee biological engineering (Shanghai) corporation. PBP2a encodes 668 amino acids.
PBP2a amino acid sequence:
MKKIKIVPLILIVVVVGFGIYFYASKDKEINNTIDAIEDKNFKQVYKDSSYISKSDNGEVEMTERPIKIYNSLGVKDINIQDRKIKKVSKNKKRVDAQYKIKTNYGNIDRNVQFNFVKEDGMWKLDWDHSVIIPGMQKDQSIHIENLKSERGKILDRNNVELANTGTAYEIGIVPKNVSKKDYKAIAKELSISEDYIKQQMDQNWVQDDTFVPLKTVKKMDEYLSDFAKKFHLTTNETESRNYPLGKATSHLLGYVGPINSEELKQKEYKGYKDDAVIGKKGLEKLYDKKLQHEDGYRVTIVDDNSNTIAHTLIEKKKKDGKDIQLTIDAKVQKSIYNNMKNDYGSGTAIHPQTGELLALVSTPSYDVYPFMYGMSNEEYNKLTEDKKEPLLNKFQITTSPGSTQKILTAMIGLNNKTLDDKTSYKIDGKGWQKDKSWGGYNVTRYEVVNGNIDLKQAIESSDNIFFARVALELGSKKFEKGMKKLGVGEDIPSDYPFYNAQISNKNLDNEILLADSGYGQGEILINPVQILSIYSALENNGNINAPHLLKDTKNKVWKKNIISKENINLLTDGMQQVVNKTHKEDIYRSYANLIGKSGTAELKMKQGETGRQIGWFISYDKDNPNMMMAINVKDVQDKGMASYNAKISGKVYDELYENGNKKYDIDE。
PBP2a nucleic acid sequence:
ATGAAAAAGATCAAAATCGTTCCTCTGATCCTGATCGTGGTTGTCGTGGGCTTCGGTATCTATTTCTACGCTTCTAAAGACAAAGAAATCAACAACACTATTGACGCTATCGAAGACAAAAACTTCAAACAGGTTTACAAAGACAGCTCCTACATCTCTAAATCTGACAACGGTGAAGTTGAAATGACCGAACGTCCAATTAAAATCTACAACAGCCTGGGTGTGAAAGACATCAACATTCAGGACCGTAAAATTAAAAAGGTTTCTAAAAACAAAAAACGTGTGGACGCCCAGTACAAAATCAAAACCAACTACGGTAACATCGATCGTAACGTTCAGTTCAACTTCGTCAAAGAAGACGGCATGTGGAAACTGGACTGGGACCACAGCGTGATTATCCCAGGTATGCAGAAAGATCAGTCTATTCACATCGAAAACCTGAAATCCGAACGTGGCAAAATCCTGGATCGCAACAACGTTGAACTGGCCAACACCGGCACCGCTTATGAAATCGGTATTGTTCCTAAAAACGTCTCTAAAAAAGACTATAAAGCTATCGCCAAAGAACTGAGCATTTCCGAAGACTATATTAAACAGCAGATGGATCAGAACTGGGTTCAGGACGACACCTTCGTTCCACTGAAAACCGTTAAAAAGATGGACGAGTATCTGTCCGACTTCGCTAAAAAATTCCATCTGACCACTAACGAAACCGAAAGCCGTAACTACCCACTGGGCAAAGCTACCTCCCATCTGCTCGGTTACGTTGGTCCTATCAACAGCGAAGAACTGAAACAGAAAGAATACAAAGGTTACAAAGACGACGCTGTTATCGGCAAAAAAGGTCTGGAAAAACTGTATGACAAGAAACTGCAGCACGAAGACGGCTACCGCGTTACCATTGTTGACGACAACTCCAACACCATCGCTCACACCCTGATCGAAAAGAAGAAAAAGGATGGCAAAGACATCCAGCTGACCATTGACGCCAAAGTTCAGAAATCCATTTACAACAACATGAAAAACGACTACGGTTCTGGCACTGCTATTCACCCACAGACCGGCGAACTTCTGGCTCTGGTTTCCACTCCTAGCTATGATGTCTACCCTTTCATGTATGGTATGTCCAATGAAGAATACAACAAACTGACTGAAGATAAAAAAGAACCATTGCTGAACAAGTTCCAGATCACTACCTCTCCTGGTTCCACCCAGAAAATCCTGACCGCCATGATCGGCCTGAACAACAAAACCCTGGACGATAAAACTTCTTACAAAATTGATGGTAAAGGCTGGCAGAAAGATAAATCCTGGGGTGGCTACAACGTGACCCGTTACGAAGTTGTGAACGGCAACATTGATCTGAAACAGGCTATCGAATCCAGCGATAACATCTTCTTCGCTCGTGTTGCTCTGGAACTGGGCTCTAAAAAATTCGAAAAAGGTATGAAAAAACTGGGTGTGGGCGAAGACATTCCATCTGATTACCCTTTCTACAACGCCCAGATCTCTAACAAAAACCTGGATAACGAAATTCTGCTTGCTGACTCTGGCTACGGTCAGGGCGAAATCCTGATTAACCCAGTTCAGATCCTGTCTATCTACTCTGCCCTGGAAAACAACGGTAACATCAACGCTCCACACCTGCTGAAGGACACCAAAAACAAAGTTTGGAAGAAAAACATCATTTCCAAAGAAAACATCAACCTGCTGACTGATGGCATGCAGCAGGTGGTGAACAAAACTCACAAGGAAGATATCTACCGTTCTTACGCTAACTTGATCGGTAAATCGGGTACCGCTGAACTGAAAATGAAACAGGGTGAAACTGGTCGCCAGATCGGTTGGTTCATCTCTTACGATAAAGACAACCCTAACATGATGATGGCCATCAACGTTAAAGACGTTCAGGATAAAGGTATGGCTAGCTATAACGCTAAAATCTCTGGTAAAGTTTACGATGAACTGTATGAAAACGGTAACAAAAAATACGACATCGATGAAtaa。
expression and purification of PBP2a Gene
The PBP2a gene was ligated into pET-28a vector and the plasmid was transformed into E.coli BL21(DE3) and expression was induced with IPTG. Collecting thallus, crushing thallus under high pressure, centrifuging, collecting supernatant, and purifying protein by Ni column affinity chromatography. The purified protein is refined again by using Superdex-200 column to obtain PBP2a recombinant protein with high purity, and PBP2a protein is collected as antigen for later use (see figure 1 for details).
3. Immunization of mice
3 female BALB/c mice 6-8 weeks old were immunized with purified PBP2a protein as antigen, the protocol is shown in Table 1, 10 days after the third conventional immunization, tail blood was collected, and the antibody titer produced by the mice was measured by indirect ELISA. The final injection of recombinant antigen at a dose of 60 μ g/mouse was performed in tail vein one time on day 3 before fusion.
Table 1: immunization protocol for mice
Preparation of NS-1 myeloma cells
One week before fusion, NS-1 myeloma cells stored in liquid nitrogen were revived and cultured at 25cm 2 In a cell culture flask. Subculturing with 15% fetal calf serum DMEM for one week, and adjusting cell concentration to 10 6 One per mL. During fusion, myeloma cells in a logarithmic growth phase are selected, the original culture medium in a bottle is discarded, a proper amount of serum-free DMEM culture solution is added, the cells are gently blown down and transferred into a 50mL centrifuge tube, 400g of the centrifuge tube is centrifuged for 5 minutes, the cells are washed for 3 times, the supernatant is discarded, the cell sediment is re-suspended by the serum-free culture solution, and the cell sediment is counted for standby.
5. Preparation of immune spleen cells
One BALB/c mouse with the best immune effect is taken, orbital bleeding is killed, and the mouse is soaked in 75% alcohol for 5min for disinfection. Fixing the sterilized mice in a super clean bench, taking out spleen, and removing adipose tissues and connective tissues adhered with cells by scissors; washing spleen with serum-free culture solution, transferring into 40 μm cell filter screen, lightly grinding with inner core of syringe, gently washing filter screen with serum-free culture solution, and collecting spleen cell suspension; centrifuging at 400g for 5min, washing the cells for 3 times, discarding the supernatant, suspending the cell precipitate by serum-free DMEM culture solution, and counting for later use.
6. Cell fusion
NS-1 myeloma cells and immune spleen cells were mixed in a 1:10 ratio in a 50mL centrifuge tube and centrifuged at 400g for 10 min. Sucking out supernatant, and flicking the bottom of the centrifuge tube to slightly loosen cell sediment; slowly dropping 1mL of 50% PEG solution preheated to 37 ℃ into the lmin; gently stirring the cells with a pipette for 1 min; adding 10mL of DMEM medium into the cell mixture, dropwise adding lmL in the first minute, lmL in the second minute, 3mL in the 3min-4min, and the rest 5mL after 5 min; incubating the cell mixture in a water bath at 37 ℃ for 15 min; then 400g centrifugation for 5min, removing the supernatant, using 20ml 15% fetal bovine serum DMEM medium heavy suspension cell precipitation, and transfer to 75cm 2 And putting the cell culture bottle in a cell culture box for incubation for 16-24 h. Transferring the fused cell suspension to a 50ml centrifuge tube, and centrifuging for 10min at 400 g; removing supernatant, resuspending the cell pellet in 2.5mL of 15% DMEM medium, adding 22.5mL of semisolid medium, mixing, and pouring into plates with a diameter of 3.5cm, each plate containing about 2mL of semisolid medium, 37 deg.C, and 5% CO 2 And (5) culturing. After ten days, the culture medium surface of the plate was visible with a macroscopic white colony of cells.
7. Positive hybridoma cell selection
The cell pellet was aseptically pipetted up into a 96-well plate and cultured for 4 days. Indirect ELISA detection was performed after 4 days. Coating the antigen on an enzyme label plate at 100 ng/well, standing overnight at 4 ℃, washing three times, adding 200mL of blocking solution, incubating for 2h at 37 ℃, and washing three timesAdding hybridoma cell culture supernatant, incubating at 37 deg.C for 2h, washing with washing solution for three times, adding 1:10000 diluted goat anti-mouse IgG-HRP, incubating at 37 deg.C for 1h, washing for three times, developing at 37 deg.C in dark for 10min, adding 2M H 2 SO 4 The reaction was stopped and the absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader. And meanwhile, the serum of a normal mouse before immunization is used as a negative control, and the serum of the mouse after immunization is used as a positive control, so that the positive judgment is carried out when the light absorption value is 2.1 times greater than that of the negative control group. Detecting hybridoma cell holes which have specific antibodies and grow in a single clone and have good shapes, and then cloning; and (4) after at least 3 times, transferring the hybridoma cells in the positive hole to a 24-hole culture plate, and freezing and storing the hybridoma cells when the hybridoma cells in the 24-hole culture plate grow well.
8. Preparation and purification of monoclonal antibodies
0.5ml of liquid paraffin was injected into the abdominal cavity of BALB/c mice aged 8 weeks. Hybridoma cells were cultured at 1X 10 5 Inoculating at a concentration of 25cm 2 Culturing in a culture flask until the cell activity is optimal, and adjusting the cell number to about 1 × 10 6 The mice were inoculated into the abdominal cavity of the mice injected with liquid paraffin. Ascites was collected 7-10 days later.
9. Purification of monoclonal antibodies
The ascites fluid was diluted 3-fold or more with 20mM PBS pH7.4, centrifuged to take the supernatant, and purified by HiTrap protein G affinity chromatography. Purifying by using a Protein G column, wherein 5ml of ultrapure water is firstly used for passing through a new column, and then 5ml of 0.4M PB buffer solution (pH 7.0) is used for balancing a purifying small column; the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the binding site; the column was equilibrated with 10ml of 0.4M PB buffer (pH 7.0); the antibody at the binding site was eluted with 5ml of 0.1M glycine-hydrochloric acid buffer (pH 2.7) and glycine was neutralized by adding 1M Tris-HCl (pH 8.0) to maintain pH at neutrality suitable for antibody preservation. Protein electrophoresis was performed on 6 purified PBP2a monoclonal antibodies, and the loading amount was 1. mu.g, which showed that the antibodies 1A12, 1B4, 2A7, 3A5, 4F10 and 5G4 had high purity, and the heavy and light chains of the antibodies could be clearly seen (see FIG. 2 for details).
10. Subclass identification of monoclonal antibodies
The monoclonal antibody subclass was identified with reference to the instructions of the kit for identifying monoclonal antibody subclasses from Sigma (cat # ISO2-1 KT). The results show that: 1A12, 2A7, 3A5 and 4F10 are IgG1 immunoglobulin, and 1B4 and 5G4 are IgG2a immunoglobulin (see figure 3 for details). The light chains of the 6-strain antibody were all Kappa chains.
7. Sequencing of monoclonal antibodies
Hybridoma cells of the three strains of antibodies 1B4, 4F10 and 5G4 were sent to tsingri biotechnology limited of south kyo for antibody sequencing. The method comprises the following steps: total RNA was isolated from hybridoma cells according to the technical manual of RNeasy Plus Micro Kit. The total RNA is then reverse transcribed into cDNA using isotype specific antisense primers or universal primers according to the SMARTScribe reverse transcriptase technical Manual. The antibody fragments of the heavy and light chains were amplified according to the standard protocol for rapid amplification of the GenScript cDNA ends (RACE). The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen clones with the correct size insert.
The sequencing results were as follows:
the amino acid sequence of the heavy chain variable region of monoclonal antibody 1B4 is:
QIQLVQSRPELKKTGETVKISCKASGYNFTNYGMNWVKQAPGKGLKWMGWINT NTGEPTYAEEFKGRFAFSLKTSASTAYLQINNLKNEDTATYFCFYYGNNWGQGTLVTVS A。
the amino acid sequence of the light chain variable region of monoclonal antibody 1B4 is:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKV SDRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIK。
the amino acid sequence of the heavy chain variable region of monoclonal antibody 4F10 is:
EVQLQQSGPELVRPGASVKMSCKASGYTFPGYVMHWVKQRPGQGLEWIGHINP YYDDIKYNENFKGKATLTSDKSSSTAFLEFSSLTSEDSAVYYCTISAYWGQGTLVTVSA。
the amino acid sequence of the light chain variable region of monoclonal antibody 4F10 is:
DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSAGKTYLNWLLQRPGQSPKRLIYLVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK。
the amino acid sequence of the heavy chain variable region of monoclonal antibody 5G4 is:
EVQLQQSGPELVKPGASVKMSCKVSGYTFTSYVMHWVRQKPGQGLEWIGHISPS NDGIDYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCTISAYWGQGTLVTVSA。
the amino acid sequence of the light chain variable region of monoclonal antibody 5G4 is:
DVVMTQTPLTLAVTFGQPASISCKSSQSLIDRAGKTYLNWLLQRPGQSPKRLIYLV SKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK。
12. potency assay for monoclonal antibodies
PBP2a protein was coated at 100 ng/well, single antibody as 1: 10000. 1: 20000. 1: 40000. 1: 80000. 1: 160000, 1: 320000, 1: 640000, 1: 1280000, 1: 2560000, the concentrations (ng/ml) of the antibodies used are 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0.390625 respectively. The A450nm value was determined by indirect ELISA. The maximum dilution of the monoclonal antibody reacting with the target antigen of the immunity protein is the titer, and the ratio of the reading of the measuring hole to the negative control value is more than 2.1, and the result is positive. The specific operation steps are as follows: antigen was coated and coated overnight at 4 ℃. Blocking with 3% BSA blocking solution at 37 ℃ for 2 h; PBP2a monoclonal antibody is used as a primary antibody, and incubation is carried out for 1h at 37 ℃; HRP-goat anti-mouse IgG was used as a secondary antibody, and incubated at 37 ℃ for 1 h. Developing for 5min by TMB developing solution; stop with 2mM sulfuric acid. The results show that the 6-strain antibody titer is over 200 ten thousand, wherein the 1B4 titer is the highest (see the detailed picture in figure 4).
13. And (5) identifying the specificity of the monoclonal antibody.
The specificity of the antibody was detected by Western-Blot using PBP2a protein as an antigen. The specific experimental steps are as follows: the loading amount of PBP2a protein was 1. mu.g, and after protein electrophoresis and electrotransfer to PVDF membrane, the membrane was removed, washed with TBS and 5min X3. Blocking with 3% BSA4 ℃ overnight. The purified monoclonal antibody was added at a ratio of 1:50000 (antibody use concentration 0.02. mu.g/ml) and incubated for 1 h. TBST washing membrane, 5min x 3. HRP-labeled goat anti-mouse IgG antibody (secondary antibody) diluted at 1:10000 was added and incubated for 1 h. TBST washing membrane, 5min x 3. ECL developing solution is prepared according to the proportion of 1:1 of A/B solution, and is exposed on a luminoscope. Western-blotting results show that all the antibodies of the 6 strains can be combined with the PBP2a protein (see the attached figure 5 for details).
The foregoing is illustrative of embodiments of the present invention and is not to be construed as limiting thereof in any way. The scope of the present invention is defined by the claims and is not limited by the embodiments described above, and any simple modifications or equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Sequence listing
<110> Shenzhen Shenhua disease prevention and control center (Shenzhen Shenhua region sanitation inspection center and Shenzhen Shenhua region occupational disease prevention and control center)
<120> PBP2a monoclonal antibody, preparation method and application thereof
<141> 2021-02-01
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Thr Ile Asp Ala Ile Glu Asp Lys Asn Phe Lys Gln Val Tyr Lys Asp
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Ser Ser Tyr Ile Ser Lys Ser Asp Asn Gly Glu Val Glu Met Thr Glu
50 55 60
Arg Pro Ile Lys Ile Tyr Asn Ser Leu Gly Val Lys Asp Ile Asn Ile
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Gln Asp Arg Lys Ile Lys Lys Val Ser Lys Asn Lys Lys Arg Val Asp
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Ala Gln Tyr Lys Ile Lys Thr Asn Tyr Gly Asn Ile Asp Arg Asn Val
100 105 110
Gln Phe Asn Phe Val Lys Glu Asp Gly Met Trp Lys Leu Asp Trp Asp
115 120 125
His Ser Val Ile Ile Pro Gly Met Gln Lys Asp Gln Ser Ile His Ile
130 135 140
Glu Asn Leu Lys Ser Glu Arg Gly Lys Ile Leu Asp Arg Asn Asn Val
145 150 155 160
Glu Leu Ala Asn Thr Gly Thr Ala Tyr Glu Ile Gly Ile Val Pro Lys
165 170 175
Asn Val Ser Lys Lys Asp Tyr Lys Ala Ile Ala Lys Glu Leu Ser Ile
180 185 190
Ser Glu Asp Tyr Ile Lys Gln Gln Met Asp Gln Asn Trp Val Gln Asp
195 200 205
Asp Thr Phe Val Pro Leu Lys Thr Val Lys Lys Met Asp Glu Tyr Leu
210 215 220
Ser Asp Phe Ala Lys Lys Phe His Leu Thr Thr Asn Glu Thr Glu Ser
225 230 235 240
Arg Asn Tyr Pro Leu Gly Lys Ala Thr Ser His Leu Leu Gly Tyr Val
245 250 255
Gly Pro Ile Asn Ser Glu Glu Leu Lys Gln Lys Glu Tyr Lys Gly Tyr
260 265 270
Lys Asp Asp Ala Val Ile Gly Lys Lys Gly Leu Glu Lys Leu Tyr Asp
275 280 285
Lys Lys Leu Gln His Glu Asp Gly Tyr Arg Val Thr Ile Val Asp Asp
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Gly Lys Asp Ile Gln Leu Thr Ile Asp Ala Lys Val Gln Lys Ser Ile
325 330 335
Tyr Asn Asn Met Lys Asn Asp Tyr Gly Ser Gly Thr Ala Ile His Pro
340 345 350
Gln Thr Gly Glu Leu Leu Ala Leu Val Ser Thr Pro Ser Tyr Asp Val
355 360 365
Tyr Pro Phe Met Tyr Gly Met Ser Asn Glu Glu Tyr Asn Lys Leu Thr
370 375 380
Glu Asp Lys Lys Glu Pro Leu Leu Asn Lys Phe Gln Ile Thr Thr Ser
385 390 395 400
Pro Gly Ser Thr Gln Lys Ile Leu Thr Ala Met Ile Gly Leu Asn Asn
405 410 415
Lys Thr Leu Asp Asp Lys Thr Ser Tyr Lys Ile Asp Gly Lys Gly Trp
420 425 430
Gln Lys Asp Lys Ser Trp Gly Gly Tyr Asn Val Thr Arg Tyr Glu Val
435 440 445
Val Asn Gly Asn Ile Asp Leu Lys Gln Ala Ile Glu Ser Ser Asp Asn
450 455 460
Ile Phe Phe Ala Arg Val Ala Leu Glu Leu Gly Ser Lys Lys Phe Glu
465 470 475 480
Lys Gly Met Lys Lys Leu Gly Val Gly Glu Asp Ile Pro Ser Asp Tyr
485 490 495
Pro Phe Tyr Asn Ala Gln Ile Ser Asn Lys Asn Leu Asp Asn Glu Ile
500 505 510
Leu Leu Ala Asp Ser Gly Tyr Gly Gln Gly Glu Ile Leu Ile Asn Pro
515 520 525
Val Gln Ile Leu Ser Ile Tyr Ser Ala Leu Glu Asn Asn Gly Asn Ile
530 535 540
Asn Ala Pro His Leu Leu Lys Asp Thr Lys Asn Lys Val Trp Lys Lys
545 550 555 560
Asn Ile Ile Ser Lys Glu Asn Ile Asn Leu Leu Thr Asp Gly Met Gln
565 570 575
Gln Val Val Asn Lys Thr His Lys Glu Asp Ile Tyr Arg Ser Tyr Ala
580 585 590
Asn Leu Ile Gly Lys Ser Gly Thr Ala Glu Leu Lys Met Lys Gln Gly
595 600 605
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610 615 620
Pro Asn Met Met Met Ala Ile Asn Val Lys Asp Val Gln Asp Lys Gly
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Ala
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Pro Lys Leu Leu Ile Tyr Lys Val Ser Asp Arg Phe Ser Gly Val Pro
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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
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50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Phe
65 70 75 80
Leu Glu Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Thr Ile Ser Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
100 105 110
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1 5 10 15
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20 25 30
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35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
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35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
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Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly
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Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Claims (4)
1. A PBP2a monoclonal antibody, comprising monoclonal antibodies 1B4, 4F10 and 5G4, wherein: the protein sequence of the monoclonal antibody contains a heavy chain variable region and a light chain variable region; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are as follows (1) to (3):
(1) the heavy chain variable region of the 1B4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO.2, and the light chain variable region of the 1B4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 3;
(2) the heavy chain variable region of the 4F10 monoclonal antibody has an amino acid sequence shown as SEQ ID NO.4, and the light chain variable region of the 4F10 monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 5;
(3) the heavy chain variable region of the 5G4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO.6, and the light chain variable region of the 5G4 monoclonal antibody has an amino acid sequence shown as SEQ ID NO. 7.
2. A pharmaceutical composition comprising the monoclonal antibody of claim 1 and a pharmaceutically acceptable excipient.
3. A detection reagent or kit comprising the monoclonal antibody of claim 1.
4. Use of the monoclonal antibody according to any one of claims 1 to 3 for the preparation of a diagnostic reagent for the detection of the PBP2a gene.
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