Recombinant protein for screening and specifically recognizing anti-CK-MB antibody and preparation method thereof
By obtaining the N-terminal domain of the M subunit of CK-MB and the B subunit of CK-MB fuses with the IgG1 Fc sequence, the recombinant protein M/B-NTD-Fc is formed, which solves the problem of difficult to obtain specific recognition of CK-MB antibodies in the prior art, and achieves high specificity and accuracy of CK-MB detection.
Patent Information
- Application Number
- CN202510230465.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-30
AI Technical Summary
It is difficult to obtain antibodies specifically recognize the spatial conformation of CK-MB heterodimers in the prior art, resulting in the problem of pseudo-elevation in CK-MB detection.
By obtaining the N-terminal domains of the M and B subunits of CK-MB and fusing with the IgG1 Fc sequence, the recombinant protein M/B-NTD-Fc was formed, which was used to screen for specific recognition of anti-CK-MB antibodies.
This method can effectively exclude the recognition of CK-MM and CK-BB or monoclonal antibodies with high cross-reactions, improving the specificity and accuracy of CK-MB detection.
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Abstract
Description
Technical Field
[0001] The present invention relates to a recombinant protein for screening specific recognition anti-CK-MB antibodies and a preparation method thereof, belonging to the technical field of antibodies. Background Art
[0002] Creatine kinase isoenzymes (CK) are widely distributed in cells and tissues that rapidly consume ATP, such as muscle and brain, catalyzing the conversion of creatine to phosphocreatine using ATP. CK exists in four isoenzymes: muscle-type (CK-MM), brain-type (CK-BB), hybrid-type (CK-MB), and mitochondrial-type (CK-Mt). CK-MM is primarily found in various muscle cells, CK-BB is primarily found in brain cells, CK-MB is primarily found in cardiomyocytes, and CK-Mt is primarily found in the mitochondria of cardiac and skeletal muscles. In normal human serum, CK-MM predominates, with minimal levels of CK-BB and even less of CK-MB. Myocardial cell damage triggers the release of CK-MB, leading to a rapid increase in serum CK-MB levels within a short period of time. This increase typically occurs within 6 hours of onset, peaks within 24 hours, and gradually decreases to normal levels after 72 hours. This suggests that CK-MB can be used for the diagnosis of early myocardial damage.
[0003] In the "Guidelines for the Diagnosis and Treatment of Acute ST-Segment Elevation Myocardial Infarction (2019)" and the "Guidelines for the Diagnosis and Treatment of Non-ST-Segment Elevation Acute Coronary Syndrome (2016)", CK-MB is listed as an auxiliary diagnostic tool for acute myocardial infarction (AMI).
[0004] CK-MM, CK-BB, and CK-MB are homodimers or heterodimers composed of M and B subunits. CK-Mt includes the sarcomeric type (CK-Mt S) and the universal type (CK-Mt U), which exist as homodimers or octamers. The amino acid sequence homology between the M and B subunits is as high as 80.58%, with a large number of conserved sequences. Even compared with the two mitochondrial CKs, the amino acid sequences of the four CK subunits share 74.52% homology, also with a large number of conserved segments. The isoenzyme-specific box region in the sequence determines the isoenzyme type and polymerization form, while the six broadly homologous CK framework regions form the enzyme framework and determine its catalytic function. The seven regions on both sides of the framework vary significantly and determine the isoenzyme type and species specificity (SM Muhlebach et al., 1994). CK-MM, CK-BB, and CK-MB are all three isoenzymes with identical physiological activities. Both the M and B subunits are composed of an N-terminal domain (NTD) and a C-terminal domain (CTD). The structures of the CK-MM and CK-BB homodimers have been determined, showing high similarity in spatial conformation. Regarding the CK-MB heterodimer, Dar-Fu Tai et al. proposed that during heterodimer formation, the CTD conformation remains unchanged, while the NTD conformation changes, forming the heterodimer interface.
[0005] At present, the mainstream methods for clinical determination of CK-MB include immunosuppression method, latex immunoturbidimetry and chemiluminescence immunoassay. These methods all rely on specific antibodies for CK. Mainstream reagents use antibodies that specifically recognize the M subunit to capture CK-MM and CK-MB in the blood, and use antibodies that specifically recognize the B subunit to detect CK-MB in blood samples (the content of CK-BB in serum is relatively low), thereby determining the content of CK-MB in serum. However, the difficulty of CK-MB immunoassay technology lies in the difficulty in obtaining antibodies that specifically recognize CK-MB, and it is impossible to effectively eliminate the interference of CK-MM, CK-BB and giant CK (complexes formed by CK and immunoglobulins and mitochondrial CK, which can lead to false increases in CK-MB detection) in serum. The reasons why it is difficult to obtain antibodies that specifically recognize CK-MB include: 1. CK-MB, CK-MM, and CK-BB have high sequence homology, with little difference in spatial conformation, which only exists at the interaction interface between the two subunits; 2. The two subunits of CK-MB easily dissociate and rearrange into CK-MM and CK-BB during storage, resulting in the body primarily forming antibodies that recognize CK-MM and CK-BB after immunization of animals, while antibodies that recognize the spatial conformation of the CK-MB subunit interaction interface are very rare; 3. The CTD portion of CK-MM, CK-BB, and CK-MB has a stable conformation, which can easily interfere with the spatial conformation of the heterodimer interaction interface during the screening process. In summary, in the process of developing CK-specific antibodies, it is difficult to obtain antibodies that specifically recognize the spatial conformation of the CK-MB heterodimer. Most antibodies recognize the M and B subunits, or antibodies with significant cross-recognition properties. Summary of the Invention
[0006] The present invention provides a recombinant protein for screening specific anti-CK-MB antibodies and a preparation method thereof, which can effectively solve the above problems.
[0007] The present invention is achieved in that:
[0008] A method for preparing a recombinant protein for screening for specific recognition of anti-CK-MB antibodies comprises the following steps:
[0009] S1, obtain the N-terminal sequence M-NTD of the M subunit of CK-MB; obtain the N-terminal sequence B-NTD of the B subunit of CK-MB;
[0010] S2, M-NTD is fused to the first human IgG1 Fc sequence Fc1 to obtain M-NTD-Fc1; B-NTD is fused to the second human IgG1 Fc sequence Fc2 to obtain B-NTD-Fc2;
[0011] S3, M-NTD-Fc1 and B-NTD-Fc2 are simultaneously inserted into the expression vector for recombinant expression to obtain the heterodimer M / B-NTD-Fc.
[0012] In some embodiments, the sequence of the M-NTD is shown as SEQ.ID No.1.
[0013] In some embodiments, the sequence of the B-NTD is shown as SEQ.ID No.2.
[0014] In some embodiments, the sequence of Fc1 is shown as SEQ.ID No.3.
[0015] In some embodiments, the sequence of Fc2 is shown as SEQ.ID No.4.
[0016] In some embodiments, the sequence of the M-NTD-Fc1 is shown as SEQ.ID No.5.
[0017] In some embodiments, the sequence of the M-NTD-Fc2 is shown as SEQ.ID No.6.
[0018] A recombinant protein prepared by the method described above.
[0019] A method for preparing CK-MB monoclonal antibody is provided, which is obtained by immunizing animals with the recombinant protein.
[0020] A CK-MB monoclonal antibody prepared by the method.
[0021] The beneficial effects of the present invention are:
[0022] The recombinant protein provided by the present invention is unique in that it only contains the N-terminal structural portion of the two CK-MB subunits, whose spatial conformation changes significantly during the formation of heterodimers, and the key interactive interface structure between the heterodimers. This design deliberately excludes the C-terminal structural portion, which has high homology and maintains unchanged spatial conformation during the formation of heterodimers. Such a recombinant protein design gives it high specificity and can be widely used in the immune response and screening process for CK-MB monoclonal antibodies, effectively excluding those monoclonal antibodies that may recognize CK-MM and CK-BB or have high cross-reactivity, thereby ensuring the accuracy and specificity of the detection. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments is not intended to limit the scope of the invention for which protection is sought, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0024] The present invention provides a method for preparing a recombinant protein for screening a specific anti-CK-MB antibody, the method comprising the following detailed steps:
[0025] First, in the first step (S1), the N-terminal sequence of the M subunit of creatine kinase isoenzyme MB (CK-MB) is obtained, recorded as M-NTD; at the same time, the N-terminal sequence of the B subunit of CK-MB is obtained, recorded as B-NTD.
[0026] Secondly, in the second step (S2), the obtained M-NTD sequence is fused with the Fc sequence of the first human immunoglobulin G1 (IgG1), denoted as Fc1, to obtain the fusion protein sequence M-NTD-Fc1; similarly, the B-NTD sequence is fused with the Fc sequence of the second human IgG1, denoted as Fc2, to obtain the fusion protein sequence B-NTD-Fc2.
[0027] Then, in the third step (S3), the M-NTD-Fc1 sequence and B-NTD-Fc2 sequence prepared above are simultaneously inserted into an appropriate expression vector for recombinant expression, and finally the heterodimer M / B-NTD-Fc is obtained. The above-mentioned recombinant expression technologies are well known to those skilled in the art, and commonly used expression systems include but are not limited to HEK293 cell expression systems and CHO cell expression systems. During the cell expression culture process, the supernatant of the cell culture is collected, and the above-mentioned recombinant protein is enriched from the supernatant by protein A affinity chromatography technology. Subsequently, the heterodimer is further separated and purified using metal chelate chromatography and avidin affinity chromatography techniques, and these purification techniques are also well known to those skilled in the art.
[0028] The recombinant protein provided by the present invention has unique structural characteristics, namely, it only contains the N-terminal structure of the two CK-MB subunits, which changes their spatial conformation during heterodimer formation, and the interactive interface structure of the heterodimer. This recombinant protein does not contain the C-terminal structure, which has high homology and does not change its spatial conformation during heterodimer formation. Therefore, this recombinant protein can be specifically used in the immunization and screening process of recognizing CK-MB monoclonal antibodies, effectively excluding those that recognize CK-MM and CK-BB or have high cross-reactivity, thereby improving the specificity and accuracy of the screening.
[0029] In some embodiments, to reduce the production of homodimers during recombinant expression, the present invention adds positively and negatively charged amino acid residue sequences to the N-terminus of the Fc sequence. To isolate and purify heterodimers, the present invention adds a 6×His Tag and a Strep Tag to the C-terminus of the Fc sequence.
[0030] In some embodiments, the sequence of the M-NTD is shown as MPFGNTHNKFKLNYKPEEEYPDLSKHNNHMAKVLTLELYKKLRDKETPSGF TVDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKELFDPIISDRHGGYKPT DK (SEQ. ID No. 1).
[0031] In some embodiments, the sequence of the B-NTD is as shown in MPFSNSHNALKLRFPAEDEFPDLSAHNNHMAKVLTPELYAELRAKSTPSGFT LDDVIQTGVDNPGHPYIMTVGCVAGDEESYEVFKDLFDPIIEDRHGGYKPSDEHKTDLNPDNLQGGDDLDPN (SEQ. ID No. 2).
[0032] In some embodiments, the sequence of Fc1 is shown as GGGSKKKKKEPKSCDKTHTCPPCPAELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSReEMTKNQVSLTClVKGFYPSDIAVEWESNGQPENNYdTTPPVLDSDGSFFLYSdLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ. ID No. 3).
[0033] In some embodiments, the sequence of Fc2 is shown as GGGSDDDDDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRkEMTKNQVSLTClVKGFYPSDIAVEWESNGQPENNYKTTPPVLkSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWSHPQFEK (SEQ. ID No. 4).
[0034] In some embodiments, the sequence of M-NTD-Fc1 is as shown in MPFGNTHNKFKLNYKPEEEYPDLSKHNNHMAKVLTLELYKKLRDKETPSGFTVDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKELFDPIISDRHGGYKPTDKGGGSKKKKKEPKSCDKTHTCPPCPAELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSReEMTKNQVSLTClVKGFYPSDIAVEWESNGQPENNYdTTPPVLDSDGSFFLYSdLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(SEQ.ID No.5).
[0035] In some embodiments, the sequence of M-NTD-Fc2 is as shown in MPFSNSHNALKLRFPAEDEFPDLSAHNNHMAKVLTPELYAELRAKSTPSGFTLDDVIQTGVDNPGHPYIMTVGCVAGDEESYEVFKDLFDPIIEDRHGGYKPSDEHKTDLNPDNLQGGDDLDPNGGGSDDDDDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRkEMTKNQVSLTClVKGFYPSDIAVEWESNGQPENNYKTTPPVLkSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWSHPQFEK(SEQ.ID No.6).
[0036] The embodiment of the present invention provides a recombinant protein prepared by a specific method. The recombinant protein has unique structural characteristics. Specifically, it only contains the N-terminal structure of the two CK-MB subunits whose spatial conformation changes significantly during the formation of heterodimers, and the interactive interface structure between the heterodimers. It is worth noting that the recombinant protein does not contain those C-terminal structural parts whose homology is high and whose spatial conformation remains unchanged during the formation of heterodimers. Because of this, the recombinant protein can be specifically used in the immune response and screening process of identifying CK-MB monoclonal antibodies, and can effectively exclude those monoclonal antibodies that recognize CK-MM and CK-BB or have high cross-reactivity, thereby significantly improving the specificity and accuracy of the screening process and ensuring the reliability and validity of the experimental results.
[0037] The present invention provides a method for preparing a CK-MB monoclonal antibody. This method utilizes recombinant technology to produce a recombinant protein with a specific antigenic epitope. This recombinant protein is then injected into the animal as an immunogen, stimulating the animal's immune system to produce antibodies specific to CK-MB. Through a series of screening and purification steps, a highly pure and specific CK-MB monoclonal antibody is ultimately obtained.
[0038] The present invention also provides a CK-MB monoclonal antibody obtained using the above-described preparation method. This monoclonal antibody not only has high specificity, enabling accurate recognition and binding to the CK-MB antigen, but also exhibits excellent stability and affinity in practical applications. The CK-MB monoclonal antibody obtained using this preparation method can be widely used in clinical diagnosis, scientific research, and other fields, providing strong technical support for the further development of related fields.
[0039] Example 1 Preparation of recombinant proteins M-NTD-Fc1 and B-NTD-Fc2
[0040] 1. Sequence synthesis and expression
[0041] Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the recombinant protein sequence and insert it into the pTT5 vector. Following the instructions in the Thermo Fisher FreeStyle 293 Expression System User Manual, the vector containing the recombinant protein sequence was transfected into HEK293-F cells, cultured, and the cell culture supernatant was collected.
[0042] 2. Separation and purification
[0043] Supernatant from HEK293-F cells was loaded onto a Protein A affinity chromatography column equilibrated with 20 mM phosphate buffer, pH 7.4, at a linear flow rate of 200-400 cm / h to enrich the target protein. After loading, the column was eluted with 20 mM acetate buffer, pH 3.5, and the eluted fractions were collected.
[0044] The pH of the Protein A affinity chromatography elution fraction was adjusted to 7.4 and loaded onto a nickel ion chelate affinity chromatography column equilibrated with 20 mM phosphate buffer, pH 7.4. His-tag dimer components were isolated and purified at a linear flow rate of 200-400 cm / h. After loading, the column was eluted with 20 mM phosphate buffer, pH 7.4, containing 200 mM imidazole, and the eluted fractions were collected.
[0045] The eluted fraction from the nickel chelate affinity chromatography was loaded onto an avidin affinity chromatography column equilibrated with 20 mM phosphate buffer, pH 7.4, at a linear flow rate of 200-400 cm / h to isolate and purify the Strep Tag-containing dimer. After loading, the column was eluted with 20 mM phosphate buffer, pH 7.4, containing 2.5 mM desthiobiotin, and the eluted fractions were collected. This eluted fraction is the heterodimer M / B-NTD-Fc.
[0046] The eluted fractions from the avidin affinity chromatography were concentrated by ultrafiltration using a 30 kD ultrafiltration tube and buffer exchanged, and the recombinant protein was stored in 20 mM phosphate buffer at pH 7.4 containing 100 mM sodium chloride.
[0047] Example 2
[0048] Application of M / B-NTD-Fc in the screening of CK-MB monoclonal antibodies
[0049] 1. Animal immunization
[0050] Recombinant human CK-MB antigen (Cat. No. DRA12, Suzhou Jinan Protein); Freund's complete adjuvant (Cat. No. 77140, Thermo Fisher); Freund's incomplete adjuvant (Cat. No. 77145, Thermo Fisher); HAT media supplement (50×) (Cat. No. 21060017, Thermo Fisher); HT media supplement (50×) (Cat. No. H0111067030, Thermo Fisher); PEG (Cat. No. P7181, Sigma); RPMI 1640 (Cat. No. L210KJ, Shanghai Yuanpei Biotechnology); fetal bovine serum (FBS) (Cat. No. C04001-500, Shanghai Xiaopeng Biotechnology); DMEM (Cat. No. L310KJ, Shanghai Yuanpei Biotechnology); Penicillin Streptomycin (Cat. No. 15140122, Gibco); HRP-labeled goat anti-mouse antibody (Cat. No. D110087, Shanghai Sangon Biotechnology Co., Ltd.); Protein A Resin (Cat. No. SA023010, Changzhou Tiandirenhe Biotechnology Co., Ltd.).
[0051] The recombinant human CK-MB antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points into 6-8 week old SPF grade Balb / c mice (Fuzhou Wu's Animal Experimental Center). 200 μg / mouse was injected subcutaneously at multiple points. 2 weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points at 100 μg / mouse. Boost the immunization twice and perform a shock injection into the abdomen 3 days before fusion.
[0052] 2. Preparation of Feeder Cells
[0053] BALB / c mouse peritoneal macrophages were used as feeder cells. One day prior to fusion, BALB / c mice were sacrificed by cervical dislocation and the entire body was immersed in 75% alcohol. Under aseptic conditions in a clean hood, the abdominal skin was incised to expose the peritoneum. 5 mL of RPMI 1640 basal medium supplemented with 1% penicillin-streptomycin was injected intraperitoneally with a syringe. The cells were repeatedly rinsed, the rinse fluid was collected, and the cells were centrifuged at 1000 rpm for 5 minutes. The pellet was then resuspended in RPMI 1640 complete medium supplemented with 1% HAT. The cells were adjusted to a concentration of 1 × 10^5 cells / mL and plated in a 96-well plate at 150 μL / well. The cells were cultured overnight at 37°C in 5% CO2.
[0054] 3. Preparation of Immune Splenocytes
[0055] Three days after the last immunization, remove the spleen under sterile conditions, place it on a plate, rinse once with RPMI 1640 basal medium, grind and filter on a nylon mesh in a small beaker to prepare a cell suspension. Centrifuge, discard the supernatant, and resuspend in RPMI 1640 basal medium. Repeat this process three times and count the cells.
[0056] 4. Cell Fusion
[0057] (1) Preheat 40 mL of HAT culture medium, 15 mL of serum-free DMEM culture medium, and 1 mL of 50% PEG (M12000) in a 37°C water bath.
[0058] (2) Take a suspension of mouse myeloma Sp2 / 0 cells (2.5×10^7 cells) and the above-mentioned immune spleen cells (10^8 cells) and add them to a 50 mL centrifuge tube, mix well, and add DMEM serum-free culture medium to 40 mL. Centrifuge for 10 minutes, pour off the supernatant, and mix well.
[0059] (3) Place the centrifuge tube in 37°C preheated water, take 0.7 mL of preheated 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of serum-free culture medium preheated at 37°C;
[0060] (4) Add serum-free DMEM to 40 mL, centrifuge for 10 minutes, and pour off the supernatant. Add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum, mix thoroughly with a pipette, and add 2 drops to each well of four 96-well cell culture plates containing feeder cells. Incubate in a 37°C, 7% CO2 incubator.
[0061] 5. Selection and Culture of Hybridoma Cells
[0062] On the 1st, 3rd, 5th and 7th day after cell fusion, the HAT culture medium is used to replace the culture medium. The surviving cells are hybridoma cells, while non-hybridoma cells die, and true hybrid cells are selected.
[0063] 6. Detection of specific antibodies
[0064] Hybridoma cells were divided into two groups of 20 plates each. Indirect ELISA was performed using recombinant human CK-MB antigen (Cat. No. DRA12, Suzhou Nearshore Protein) and recombinant protein M / B-NTD-Fc as coating antigens, respectively. Positive cell lines were selected with supernatant OD490 values greater than 1.5. A total of 113 positive cell lines were obtained using recombinant human CK-MB antigen as the screening antigen, and 92 positive cell lines were obtained using recombinant protein M / B-NTD-Fc as the screening antigen.
[0065] Recombinant human CK-MB antigen, recombinant protein M / B-NTD-Fc, recombinant human CK-MM antigen (Cat. No. DCC96, Suzhou Nearshore Protein) and recombinant human CK-BB antigen (Cat. No. DRA02, Suzhou Nearshore Protein) were used as coating antigens, and the culture supernatants of the selected positive cell lines were retested by indirect ELISA. The results are shown in Table 1.
[0066] Table 1
[0067]
[0068] From the data shown in Table 1, it can be clearly observed that when we used the recombinant protein M / B-NTD-Fc as the screening antigen, the positive cell lines obtained showed superior specificity in recognizing CK-MB compared to the case of using the recombinant protein CK-MB as the screening antigen. By using M / B-NTD-Fc as the screening antigen, we successfully avoided the adverse effects that the C-terminal conserved region may have during the screening process. Specifically, these positive cell lines showed significantly lower cross-reactivity when cross-reacting with CK-MM and CK-BB, which means that they can more accurately recognize the target antigen CK-MB without being interfered with by other similar proteins. This improvement effectively improves the accuracy and reliability of the screening process. Ultimately, the implementation of this strategy greatly improved the overall efficiency of hybridoma cell screening, making the screening process more efficient and accurate, and laying a solid foundation for subsequent research and application.
[0069] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.
Claims
1. A method for preparing a recombinant protein for screening a recombinant protein that specifically recognizes an anti-CK-MB antibody, characterized in that: The following steps are involved: S1, obtain the N-terminal sequence M-NTD of the M subunit of CK-MB; obtain the N-terminal sequence B-NTD of the B subunit of CK-MB; S2, fusing M-NTD to the first human IgG1 Fc sequence Fc1 to obtain M-NTD-Fc1; fusing B-NTD to the second human IgG1 Fc sequence Fc2 to obtain B-NTD-Fc2; S3, inserting M-NTD-Fc1 and B-NTD-Fc2 into the expression vector at the same time, and performing recombinant expression to obtain the heterodimer M / B-NTD-Fc.
2. The preparation method according to claim 1, characterized in that: The sequence of the M-NTD is shown in SEQ.ID No.
1.
3. The preparation method according to claim 1, characterized in that: The sequence of the B-NTD is shown in SEQ.ID No.
2.
4. The preparation method according to claim 1, characterized in that: The sequence of Fc1 is shown in SEQ.ID No.
3.
5. The preparation method according to claim 1, characterized in that: The sequence of Fc2 is shown in SEQ.ID No.
4.
6. The preparation method according to claim 1, characterized in that: The sequence of the M-NTD-Fc1 is shown as SEQ.ID No.
5.
7. The preparation method according to claim 1, characterized in that: The sequence of the M-NTD-Fc2 is shown in SEQ.ID No.
6.
8. A recombinant protein prepared by the method according to any one of claims 1 to 7.
9. A method for preparing CK-MB monoclonal antibody, characterized in that: The method is obtained by immunizing an animal with the recombinant protein according to claim 8.
10. A CK-MB monoclonal antibody prepared by the method of claim 9.