An antibody against a cell surface protein of Clostridium butyricum

Abstract

The present invention provides an antibody to a cell surface protein of Clostridium butyricum, and belongs to the technical field of Clostridium butyricum screening. The antibody to the cell surface protein of Clostridium butyricum is a first monoclonal antibody or a second monoclonal antibody. The present invention is based on a magnetic bead sorting method, and uses an antibody magnetic strain that couples and recognizes Clostridium butyricum choline binding protein A and cell wall anchor protein to capture Clostridium butyricum, thereby achieving the purpose of efficient and rapid separation of Clostridium butyricum, being convenient and low in cost, and the separated Clostridium butyricum can be used for culture, sequencing and other aspects.

Description

Technical Field

[0001] The invention belongs to the technical field of screening Clostridium butyricum, and in particular relates to an antibody against Clostridium butyricum cell surface protein. Background Art

[0002] The intestinal microbiota is one of the important areas of human health research. Among them, Clostridium butyricum (butyric acid bacteria) as a probiotic plays a key role in maintaining intestinal health. As a type of Gram-positive bacteria, Clostridium butyricum is widely present in the soil, human and animal intestines. It plays an important role in inhibiting the growth of intestinal pathogens and maintaining the balance of intestinal microorganisms by producing substances such as short-chain fatty acids. In addition, Clostridium butyricum can also reduce the adhesion and colonization of pathogens to the intestinal mucosa through the mechanism of competitive inhibition of adhesion, thus playing an important role in maintaining the intestinal health of the host.

[0003] Currently, the screening and research of Clostridium butyricum mainly relies on traditional microbial culture techniques, which include the use of selective culture media to isolate specific strains through plate screening. This method is effective in some cases, especially for those microorganisms that can grow under laboratory conditions. However, there are many types of microorganisms in the human intestine, and many intestinal microorganisms are difficult to grow under laboratory conditions, which makes it particularly difficult to screen specific microorganisms using traditional methods.

[0004] Existing technologies have obvious defects in screening Clostridium butyricum. First, the traditional plate screening method is inefficient and it is difficult to quickly and accurately isolate the target bacteria from complex intestinal samples. Secondly, this method is costly, requires a large amount of culture medium and manual operation, and traditional methods are almost powerless against those microorganisms that are difficult to grow under laboratory conditions. In addition, even if the separation is successful, there is the problem of weak strains being eliminated during the culture process, which further limits the in-depth research on probiotics such as Clostridium butyricum.

[0005] In summary, although Clostridium butyricum is important for maintaining intestinal health, the existing traditional screening methods have obvious shortcomings in efficiency, cost and applicability. These defects limit researchers' in-depth understanding of the functions and mechanisms of action of Clostridium butyricum, and also affect the application and promotion of probiotics in clinical and health fields. Therefore, the development of more efficient, economical and more widely applicable Clostridium butyricum screening technology is an important need in current microbiology and medical research. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for screening antibodies against Clostridium butyricum cell surface proteins, comprising:

[0007] Expression and purification of cbpA-CLP protein;

[0008] Immunizing and cell fusion the cbpA-CLP protein to screen out hybridoma cell lines that can recognize cbpA and CLP;

[0009] Cultivating the hybridoma cell line and purifying antibodies against cbpA and CLP;

[0010] Sequencing the cbpA and CLP antibodies to determine the variable region sequences of the antibodies;

[0011] The antibodies of cbpA and CLP were sorted by carboxyl magnetic beads, and Clostridium butyricum was enriched by the carboxyl magnetic beads.

[0012] Preferably, the expression and purification of cbpA-CLP protein comprises:

[0013] The N-terminus of cbpA expressed by Escherichia coli and the fusion protein of the cell wall anchor protein CLP are fused and connected through a connecting peptide, so that cbpA and CLP are connected in series, and the expressed cbpA-CLP protein is obtained after purification.

[0014] Preferably, the cbpA-CLP protein is subjected to immunization and cell fusion to screen out hybridoma cell lines capable of recognizing cbpA and CLP, comprising:

[0015] Immunizing experimental animals with the cbpA-CLP protein;

[0016] Taking the spleen of the experimental animal, preparing a spleen cell suspension, and mixing it with a myeloma cell line to obtain a mixed cell fluid;

[0017] performing a fusion treatment on the mixed cell fluid to obtain fused cells;

[0018] The fused cells are screened to obtain hybridoma cell lines that recognize cbpA and CLP, respectively.

[0019] Preferably, the screening of the fusion cells to obtain hybridoma cell lines that recognize cbpA and CLP respectively comprises:

[0020] Based on the limiting dilution method, the fused cells are cultured on a culture plate using HT culture medium;

[0021] detecting the cell supernatant in each well of the culture plate to determine the cells producing the antibody;

[0022] Subclone the cells in the positive wells until a stable monoclonal cell line is obtained;

[0023] The specificity of the antibodies produced by each cell line was determined by detecting the reactions of the antibodies in the supernatant with cbpA-CLP protein, cbpA protein, and CLP protein, and the monoclonal cell lines producing the specificity were designated as hybridoma cell lines recognizing cbpA and hybridoma cell lines recognizing cbpA.

[0024] Preferably, after immunizing the experimental animal with the cbpA-CLP protein, the method further comprises:

[0025] After immunization, blood is collected from the experimental animals, and the antibody titer and titer in the serum are detected.

[0026] Preferably, the culturing of the hybridoma cell line and purification of antibodies to cbpA and CLP comprises:

[0027] Resuscitating the hybridoma cell line in the presence of serum, culturing the hybridoma cell line in the absence of serum, and collecting the target antibody corresponding to the hybridoma cell line;

[0028] The target antibodies are purified and their purity is identified to obtain the antibodies against cbpA and CLP.

[0029] Preferably, the purification of the target antibody comprises:

[0030] Centrifuging and filtering the target antibody to obtain an antibody supernatant;

[0031] The antibody supernatant is purified by using a Protein A agarose affinity chromatography column to capture the antibody, and the captured antibody is eluted by an eluent to obtain the purified target antibody.

[0032] Preferably, the cbpA and CLP antibodies are sorted by carboxyl magnetic beads, and Clostridium butyricum is enriched by the carboxyl magnetic beads, comprising:

[0033] Pre-treating the cbpA and CLP antibodies, and the carboxyl magnetic beads, respectively;

[0034] Carrying out antibody coupling reaction between the pretreated antibody and the carboxyl magnetic beads to obtain coupled magnetic beads;

[0035] adding the coupled magnetic beads to a stool suspension to enrich Clostridium butyricum that specifically binds to the coupled magnetic beads, thereby obtaining enriched strain magnetic beads;

[0036] Releasing the enriched strain magnetic beads and separating the magnetic beads to obtain Clostridium butyricum;

[0037] Preferably, the pretreatment comprises:

[0038] The buffer of the cbpA antibody and the buffer of the CLP antibody are replaced with MES buffer; and the carboxyl magnetic beads are washed with MES buffer, and EDC and NHS are added after resuspending to activate the carboxyl magnetic beads.

[0039] Preferably, the step of adding the coupled magnetic beads to a stool suspension to enrich Clostridium butyricum that specifically binds to the coupled magnetic beads to obtain enriched strain magnetic beads comprises:

[0040] adding the coupled magnetic beads to the fecal suspension, and incubating at 37° C. for 2 hours to allow the coupled magnetic beads to specifically bind to Clostridium butyricum;

[0041] The magnetic beads and the unbound part are separated by a magnetic frame to obtain the enriched strain magnetic beads.

[0042] Preferably, after the cbpA and CLP antibodies are sorted by carboxyl magnetic beads and Clostridium butyricum is enriched by the carboxyl magnetic beads, the method further comprises:

[0043] The enriched Clostridium butyricum was cultured under anaerobic conditions for 48 hours;

[0044] According to the morphological characteristics of the cultured Clostridium butyricum, a single colony is selected as a colony sample;

[0045] The colony sample is used as a template to perform PCR amplification, and the expected DNA fragment is identified by PCR technology;

[0046] Recovering the DNA bands and sequencing the recovered DNA to obtain sequence information;

[0047] The homology with the Clostridium butyricum sequence was determined based on the sequence information.

[0048] The invention provides a method for screening antibodies against cell surface proteins of Clostridium butyricum, comprising: expressing and purifying cbpA-CLP proteins; immunizing and cell fusing the cbpA-CLP proteins to screen hybridoma cell lines capable of recognizing cbpA and CLP; culturing the hybridoma cell lines and purifying antibodies against cbpA and CLP; sequencing the antibodies against cbpA and CLP to determine variable region sequences of the antibodies; and sorting the antibodies against cbpA and CLP by carboxyl magnetic beads, and enriching the cbpA and CLP cells to obtain Clostridium butyricum.

[0049] The method for screening antibodies to Clostridium butyricum cell surface proteins provided by the present invention has the following beneficial effects: 1. In the present invention, two cell wall surface protein gene fragments are connected in series and then recombinantly expressed by using Escherichia coli, and the obtained fusion protein can screen antibodies to two different targets in one cell fusion; 2. The screened LPXTG antibody and anchor protein antibody are respectively coupled to magnetic beads and mixed to obtain mixed magnetic beads, and the two magnetic beads form a synergistic effect; 3. The magnetic bead method is used to enrich Clostridium butyricum in feces, which can be used for strain identification and sequencing, and can also be used for the cultivation of the bacteria.

[0050] In summary, the present invention is based on the magnetic bead sorting method, and uses an antibody magnetic strain that couples and recognizes choline binding protein A of Clostridium butyricum and cell wall anchor protein to capture Clostridium butyricum, so as to achieve the purpose of efficiently and quickly separating Clostridium butyricum from fecal samples. In addition, there is no need to use expensive instruments such as flow cytometers, and separation is performed using a magnetic stand, which is convenient and low-cost. The separated Clostridium butyricum can be used for culture, sequencing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the process of screening antibodies against Clostridium butyricum cell surface proteins of the present invention;

[0052] Figure 2 This is the electrophoresis diagram of the purified antibody in Example 3 of the method for screening antibodies against cell surface proteins of Clostridium butyricum of the present invention;

[0053] Figure 3 This is the DNA electrophoresis diagram in Example 6 of the method for screening antibodies against Clostridium butyricum cell surface proteins of the present invention.

[0054] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. 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 creative work are within the scope of protection of the present invention.

[0056] refer to Figure 1 The present invention provides a method for screening antibodies against Clostridium butyricum cell surface proteins, comprising:

[0057] Step S1, expressing and purifying cbpA-CLP protein;

[0058] Step S2, immunizing and cell fusion the cbpA-CLP protein to screen out hybridoma cell lines that can recognize cbpA and CLP;

[0059] Step S3, culturing the hybridoma cell line and purifying antibodies to obtain cbpA and CLP;

[0060] Step S4, sequencing the cbpA and CLP antibodies to determine the variable region sequences of the antibodies;

[0061] Step S5, sorting the antibodies of cbpA and CLP by carboxyl magnetic beads, and enriching Clostridium butyricum by the carboxyl magnetic beads.

[0062] As mentioned above, in the present invention, a method for separating specific microorganisms by coupling antibodies to magnetic beads is used. In this method, antibodies that can recognize surface proteins of specific microorganisms are first coupled to magnetic beads, and then these coupled magnetic beads are added to a sample containing target microorganisms. Through the action of the magnetic field, microorganisms that specifically bind to the antibodies can be quickly adsorbed and separated. Afterwards, the antigen-antibody complex is dissociated by an appropriate method to obtain purified target microorganisms.

[0063] This magnetic bead-based microbial separation method has the following advantages: Compared with the traditional plate culture method, the magnetic bead method can quickly capture the target microorganisms directly from complex samples, greatly shortening the time required for the separation process. Since the separation is achieved by combining specific antibodies with antigens on the surface of microorganisms, this method has high specificity and can accurately separate the target microorganisms. Many intestinal microorganisms are difficult to culture under laboratory conditions, but by coupling magnetic beads to antibodies, these microorganisms can be directly separated from the sample without relying on their growth and reproduction. Since the separation process is carried out in a closed system and is easy to operate, the risk of external contamination of the sample during processing can be effectively reduced.

[0064] In summary, the magnetic bead-coupled antibody method provides a new method with high efficiency, strong specificity and wide applicability in the field of microbial separation. It is particularly suitable for those microbial species that are difficult to separate by traditional culture methods, and has important significance for microbiological research and related applications.

[0065] The cell surface proteins of Gram-positive bacteria are involved in many important biological functions, promoting activities such as host cell invasion, immune response escape and adhesion to infection sites. Choline binding protein A (cbpA) is a cell surface protein of Clostridium butyricum that can specifically bind to the polymeric immunoglobulin receptor. It consists of a signal peptide, an N-terminal coiled coil and a C-terminal cell wall binding domain, of which the N-terminal part is exposed outside the cell wall and the C-terminal part is anchored to the cell wall surface. Cell wall anchor protein (CLP_3241) is a protein anchored to the cell wall surface, containing 8 NEAT domains, most of which exist outside the cell and participate in ion transport. These two proteins are located on the cell wall surface and are easily bound to the corresponding antibodies.

[0066] In the present invention, based on the above characteristics, CLP (cell wall anchored protein) and cbpA (choline binding protein A) are used as target proteins for separation and identification of Clostridium butyricum, which is based on the unique presence and biological functions of these two proteins on the surface of Clostridium butyricum:

[0067] (1) Surface exposure and identifiability: CLP and cbpA are both proteins on the surface of the cell wall of C. butyricum. Their exposed position on the bacterial surface makes them ideal targets for antibody recognition and binding. Due to their location on the cell surface, antibodies can easily bind to them specifically without penetrating the cell wall or cell membrane. (2) Specificity: CLP and cbpA are specifically expressed in C. butyricum, which means that they can be used as biomarkers to distinguish C. butyricum from other microorganisms. Using antibodies against these specific proteins for separation can improve the specificity of the screening process and reduce the interference of non-target microorganisms. (3) Biological function: These proteins play an important role in the biological functions of C. butyricum, such as participating in the adhesion process of bacteria, immune evasion, etc. Studying these proteins not only helps to isolate and identify bacteria, but also helps to further understand their role in the physiological and pathological processes of C. butyricum. (4) Convenience of isolation and identification: Due to these characteristics of CLP and cbpA, they have become powerful tools for bacterial isolation and identification. Through antibodies against these proteins, rapid and specific capture and separation of Clostridium butyricum can be achieved, providing purified bacterial samples for subsequent biological research or applications.

[0068] In summary, CLP and cbpA were selected based on their specificity in C. butyricum, surface exposure, and importance in bacterial biological functions, making them ideal target proteins for C. butyricum-specific recognition and isolation.

[0069] The main principle of the present invention to enrich and screen Clostridium butyricum using CLP and cbpA is to use the affinity of specific antibodies to the surface proteins of target bacteria to achieve rapid and specific separation of microorganisms. Specifically, this method involves the following key steps and principles:

[0070] Preparation of fusion protein: Through genetic engineering technology, the gene fragments of choline binding protein A (cbpA) and cell wall anchor protein (CLP_3241) of Clostridium butyricum are connected in series, and Escherichia coli is used as a host to express the desired fusion protein. The principle of this step is based on molecular cloning technology and recombinant protein expression technology, and the purpose is to obtain a fusion protein that can simultaneously recognize two different targets. Antibody production and screening: By immunizing mice, the above fusion protein is used as an antigen to stimulate mice to produce specific antibodies. Subsequently, monoclonal antibodies that can specifically recognize cbpA and CLP are screened by the fusion method of spleen cells and myeloma cells. The principle of this step is based on immunology and cell fusion technology, aiming to obtain highly specific antibodies against specific protein targets. Antibody-coupled magnetic beads and capture of microorganisms: The screened monoclonal antibodies are coupled to carboxyl magnetic beads respectively. These coupled magnetic beads can specifically bind to cbpA and CLP proteins on the surface of Clostridium butyricum through antibodies. Using the magnetic field, bacteria bound to antibodies are quickly separated. The principle of this step is based on the antigen-antibody specific affinity in immunology and magnetic separation technology. Release and culture of bacteria: The magnetic beads and bacteria are dissociated by enzyme cleavage and other methods to release the captured Clostridium butyricum, which is then counted and cultured. The principle of this step is based on the enzyme cleavage reaction in biochemistry to achieve the dissociation of antibodies and bacteria, as well as the bacterial culture technology in microbiology.

[0071] The present invention realizes the rapid and specific separation and subsequent cultivation and research of Clostridium butyricum by combining molecular biology, immunology and magnetic separation technology, and provides an efficient tool for microbiological research and application.

[0072] In step S4, cell line sequencing is performed, which is a key link in this process. Its role is to understand the binding characteristics of the antibody and its antigen by determining the variable region sequence of the antibody. The variable region of the antibody is the key part that determines its specificity and affinity. Through sequence analysis, the ability of the antibody to recognize and bind to the antigen can be predicted. Secondly, knowing the precise sequence of the variable region of the antibody, antibody engineering, such as humanization, affinity maturation, etc., can be performed through molecular biology techniques to improve its application value in clinical or research. Thirdly, once the sequence information of the variable region of the antibody is obtained, these sequences can be cloned into expression vectors through genetic engineering methods, and a large number of antibodies can be recombinantly expressed in vitro. This is crucial for the large-scale production and application of antibodies.

[0073] In summary, the present invention obtains a fusion protein that can simultaneously recognize two different targets by connecting two cell wall surface protein gene fragments of Clostridium butyricum in series and expressing them recombinantly using Escherichia coli. The LPXTG antibody and anchor protein antibody produced by this fusion protein are coupled to magnetic beads respectively, and the two magnetic beads are mixed and used to form a synergistic effect to enhance the specific capture efficiency. Through the magnetic bead sorting method, the present invention can efficiently and quickly enrich Clostridium butyricum from fecal samples without relying on expensive instruments such as flow cytometers. Only magnetic frames can be used for separation. This process is not only convenient but also low in cost. The isolated Clostridium butyricum can be used for further strain identification, sequencing and cultivation, providing an efficient separation tool for microbiological research and application.

[0074] Furthermore, the step S1, expressing and purifying the cbpA-CLP protein, comprises:

[0075] Step S11, the N-terminus of cbpA expressed by Escherichia coli and the fusion protein of the cell wall anchor protein CLP are fused and connected through a connecting peptide to connect cbpA and CLP in series, and the expressed cbpA-CLP protein is obtained after purification.

[0076] The above steps mainly involve the use of molecular biology techniques to construct a fusion protein, specifically, the N-terminus of the cbpA protein expressed by Escherichia coli is fused to the cell wall anchor protein CLP through a connecting peptide to form a cbpA-CLP fusion protein. This operation has several key purposes and advantages: Enhanced specificity: By connecting cbpA and CLP, two proteins with different biological functions in series, the fusion protein can have the biological characteristics of both proteins at the same time. This design can improve the specific recognition and binding of Clostridium butyricum in subsequent experiments, because the fusion protein can interact with two different targets on the surface of Clostridium butyricum at the same time. Simplified purification process: By constructing a fusion protein, a single protein containing two functional domains can be obtained in an expression system, which simplifies the subsequent protein purification and processing steps. Traditionally, if the two proteins are expressed and purified separately, it may take more time and resources, and the fusion protein strategy provides a more efficient solution. Enhanced functionality: The design of the connecting peptide ensures that the spatial structure of cbpA and CLP is not disturbed and their respective biological activities are maintained. This structural consideration ensures that the fusion protein can play a synergistic role in the combination of the two proteins in subsequent applications. Convenient for subsequent applications: The obtained cbpA-CLP fusion protein can be used in a variety of biomedical research, such as being used as a specific probe for the detection and separation of Clostridium butyricum, or playing a role in studying the mechanism of interaction between Clostridium butyricum and the host.

[0077] In summary, by constructing the cbpA-CLP fusion protein, this step provides significant advantages in achieving comprehensive protein functions, simplifying experimental procedures, and improving experimental efficiency. This not only helps to study the biological characteristics of Clostridium butyricum in more depth, but also provides an effective strategy for microbial research and application based on protein engineering.

[0078] Furthermore, the step S2, immunizing and cell fusion the cbpA-CLP protein to screen out hybridoma cell lines capable of recognizing cbpA and CLP, comprises:

[0079] Step S21, immunizing experimental animals with the cbpA-CLP protein; wherein the experimental animals may be mice.

[0080] Step S22, taking the spleen of the experimental animal, preparing a spleen cell suspension, and mixing it with a myeloma cell line to obtain a mixed cell fluid;

[0081] Step S23, performing fusion treatment on the mixed cell fluid to obtain fused cells;

[0082] Step S24, screening the fused cells to obtain hybridoma cell lines that recognize cbpA and CLP respectively.

[0083] The above method describes the process of producing antibodies specific to LPXTG-CLP protein by immunizing mice, and then using fusion technology to produce specific monoclonal antibodies. In this process, animal spleen cells and myeloma cell lines (such as SP2 / 0) are used for fusion, mainly based on the following reasons: Immune response produces antibodies: The spleen cells of animals (such as mice) are important sites for immune response and can produce antibodies against specific antigens (LPXTG-CLP protein in this case). After immunizing mice, their spleen cells will be rich in B cells that are specific to the injected antigen. Obtaining immortal cell lines: Myeloma cell lines (such as SP2 / 0) are a type of immune cell cancerous line that can proliferate indefinitely but do not produce antibodies themselves. When these cells are fused with immunized spleen cells, hybridoma cells that can proliferate indefinitely and produce specific antibodies can be obtained. Producing monoclonal antibodies: By fusing spleen cells and myeloma cells, the hybridoma cells formed can proliferate indefinitely, and each hybridoma cell can produce a specific antibody, which is called a monoclonal antibody. By screening these hybridoma cells, cell lines that produce monoclonal antibodies against specific antigen sites can be found. Improve screening efficiency: HT culture medium and limiting dilution method can be used to effectively screen and clone hybridoma cells that produce the desired antibodies. This method can screen out single cells that specifically produce the desired antibodies from millions of cells, greatly improving the efficiency and accuracy of screening.

[0084] In short, by fusing animal spleen cells and myeloma cells, we can combine the advantages of both, namely the specific antibody production ability of spleen cells and the immortality of myeloma cells, thereby efficiently producing monoclonal antibodies against specific antigens.

[0085] Furthermore, the step S24, screening the fusion cells to obtain hybridoma cell lines that recognize cbpA and CLP, respectively, comprises:

[0086] Step S241, culturing the fused cells on a culture plate using HT culture medium based on a limiting dilution method;

[0087] Step S242, detecting the cell supernatant in each well of the culture plate to determine the cells producing the antibody;

[0088] Step S243, performing subclone screening on the cells in the positive wells until a stable monoclonal cell line is obtained;

[0089] Step S244, by detecting the reaction of the antibody in the supernatant with the cbpA-CLP protein, cbpA protein, and CLP protein, the specificity of the antibody produced by each cell line is determined, and the monoclonal cell line that produces the specificity is used as the hybridoma cell line that recognizes cbpA and the hybridoma cell line that recognizes cbpA.

[0090] As mentioned above, limiting dilution assay is an experimental method for obtaining clonal cell populations from a single cell source, which is used in cell biology and immunology research. This method mainly dilutes cells to a sufficiently low concentration to ensure that each culture container (such as each well of a 96-well plate) receives only one cell, so that the clonal population generated by the proliferation of cells in the well is derived from only a single cell.

[0091] As mentioned above, HT medium is a special medium for culturing hybridoma cells, which is usually used in the selection and maintenance of hybridoma cells. HT refers to "Hypoxanthine-Thymidine", and these two components play a role in a specific cell selection system.

[0092] The above step S241 is to culture the fused cells, using HT medium and limiting dilution method to culture the fused cells on the culture plate. This method ensures that there are only a single or very few cells in each well through gradual dilution, so that the clones finally formed by the cells in each well are derived from a single fused cell. Through this step, multiple monoclonal cell populations that may produce the desired antibodies can be obtained.

[0093] Then the antibody is tested, and the culture supernatant of each well is tested to determine which cells are producing antibodies. This is usually achieved by ELISA or other immunoassay methods. In this step, the cell wells that produce antibodies are identified, i.e., positive wells. This step directly detects antibodies and quickly and effectively identifies valuable fusion cells.

[0094] The third step is subclone screening: further subclone screening is performed on the cells in the positive wells, and the limiting dilution method is repeated until a stable monoclonal cell line is obtained. In this step, a monoclonal cell line that is purified and stably produces the target antibody can be obtained. This ensures the consistency and stability of the screened cell line in biological characteristics and antibody production.

[0095] The fourth step is to confirm the specificity. By detecting the reaction between the antibody in the culture supernatant and the cbpA-CLP fusion protein, cbpA protein and CLP protein, the specificity of the antibody produced by the cell line is determined. The monoclonal antibody production cell line that specifically recognizes cbpA and CLP is confirmed and selected. This step ensures that the final selected cell line can produce highly specific antibodies against specific proteins, providing important biological materials for subsequent application research.

[0096] In short, this series of steps combines cell biology and immunology methods, from the culture of fusion cells to the screening and confirmation of monoclonal antibodies, and each step is to ensure the acquisition of high-quality and highly specific monoclonal antibodies.

[0097] Furthermore, the step S21, after immunizing the experimental animal with the cbpA-CLP protein, further comprises:

[0098] Step S25, after immunization, blood is collected from the experimental animal, and the antibody titer and titer in the serum are detected.

[0099] It should be noted that the reason for testing the antibody titer and titer in the serum after blood collection is to evaluate the effectiveness of the immune response and determine whether the production of specific antibodies has been successfully stimulated.

[0100] Specifically, in this process, by detecting the titer of antibodies in the serum, the intensity of the mouse's response to the LPXTG-CLP protein can be evaluated. If the antibody titer is high, it means that the immune response is good and the mouse has successfully produced antibodies against the protein. By detecting the antibody titer after different immunization stages, it can be determined whether the immunization needs to continue or whether a sufficient antibody level has been reached. If the titer reaches the expected level, the immunization can be stopped and the next experimental operation can be carried out. If multiple mice are immunized at the same time, by comparing the antibody titers in their serum, the individuals with the strongest antibody response can be selected for subsequent spleen cell collection and hybridoma cell preparation, which helps to improve the success rate of hybridoma cells producing specific antibodies. In addition, it can also provide a basis for the screening of hybridoma cells: knowing the antibody titer in mouse serum helps to set reasonable screening criteria in the subsequent hybridoma cell screening process, so as to more specifically screen out cell lines that can produce high-titer antibodies.

[0101] In short, detecting the titer and titer of antibodies in serum is a key step, which is directly related to the success rate and efficiency of subsequent experiments, ensures the effectiveness of the immunization program, and provides important preliminary information for obtaining hybridoma cell lines with high specificity antibodies.

[0102] Furthermore, the step S3, culturing the hybridoma cell line and purifying antibodies to obtain cbpA and CLP, comprises:

[0103] Step S31, resuscitating the hybridoma cell line in the presence of serum, culturing the hybridoma cell line in the absence of serum, and collecting the target antibody corresponding to the hybridoma cell line;

[0104] Step S32, purifying and identifying the purity of the target antibody to obtain the cbpA and CLP antibodies.

[0105] The above methods describe the process of culturing and purifying specific antibodies from hybridoma cell lines.

[0106] First, the hybridoma cell line is revived under serum conditions to provide the necessary growth factors and nutrients. The cells are then cultured under serum-free conditions, which helps with the subsequent purification of antibodies because the interference of other proteins in the serum is reduced under serum-free conditions. Then, the target antibody is collected from the supernatant of the hybridoma cells cultured under serum-free conditions, which usually involves centrifugation and filtration steps to remove cell debris and larger impurities. In this way, a relatively pure antibody supernatant can be obtained, laying the foundation for subsequent purification work. The third step is to purify the antibody supernatant using a Protein A agarose affinity chromatography column. Protein A has a high affinity to bind to the Fc region of most mammalian IgG antibodies, and the antibody is eluted from the Protein A column by the eluent. This purification method has strong specificity and can effectively purify the target antibody from complex samples. The purification process is simple to operate and has good reproducibility. Finally, purity identification is performed, and the purified antibody is usually identified by SDS-PAGE or other protein electrophoresis methods to ensure that the obtained antibody has sufficient purity for subsequent research or application. Purity identification is a key step in ensuring antibody quality. High-purity antibodies are essential for the reliability and reproducibility of experimental results.

[0107] Through the above steps, specific cbpA and CLP antibodies can be effectively cultured and purified from hybridoma cell lines, providing high-quality antibody resources for further experiments.

[0108] Furthermore, in step S32, the target antibody is purified, including:

[0109] Step S321, centrifuging and filtering the target antibody to obtain an antibody supernatant;

[0110] Step S322, purifying the antibody supernatant using a Protein A agarose affinity chromatography column to capture the antibody, and eluting the captured antibody with an eluent to obtain the purified target antibody.

[0111] First, the target antibody was centrifuged and filtered. In this step, the culture supernatant containing the target antibody was centrifuged and filtered. Centrifugation is used to remove cell debris and larger impurities, while filtration further removes smaller particles and undissolved materials to ensure that the supernatant obtained contains only dissolved proteins and other small molecules. In this step, a clear antibody supernatant containing the target antibody to be purified can be obtained.

[0112] Then, Protein A affinity chromatography purification is performed, and the antibody is purified using a Protein A affinity chromatography column. Protein A is a protein with high affinity to the Fc region of most mammalian IgGs, which can specifically bind to and capture antibodies. The target antibody is captured by passing the antibody supernatant through a chromatography column filled with Protein A. Subsequently, an appropriate eluent is used to detach and elute the antibody bound to the column by changing conditions such as pH or ionic strength, thereby obtaining the purified target antibody.

[0113] This purification method has high specificity, and only specific antibodies will be captured and eluted by Protein A, greatly improving the purity of the target antibody. In addition, Protein A affinity chromatography is a mature technology with standardized operation and good reproducibility, which is suitable for large-scale antibody purification.

[0114] Furthermore, in step S5, the antibodies of cbpA and CLP are sorted by carboxyl magnetic beads, and Clostridium butyricum is enriched by the carboxyl magnetic beads, comprising:

[0115] Step S51, pre-treating the cbpA and CLP antibodies, and the carboxyl magnetic beads respectively;

[0116] As mentioned above, this step includes preparing the antibodies of cbpA and CLP and performing necessary pretreatment on the carboxyl magnetic beads. The pretreatment may include washing the magnetic beads to remove impurities and activating the carboxyl groups so that they can be effectively coupled with the antibodies. Through pretreatment, the carboxyl groups on the surface of the magnetic beads are ensured to be in an activated state and can efficiently undergo coupling reactions with the antibodies. At the same time, it also ensures that the antibodies maintain their biological activity, laying a good foundation for the subsequent coupling and enrichment processes.

[0117] Wherein, the preprocessing includes:

[0118] The buffer of the cbpA antibody and the buffer of the CLP antibody are replaced with MES buffer; and the carboxyl magnetic beads are washed with MES buffer, and EDC and NHS are added after resuspending to activate the carboxyl magnetic beads.

[0119] As mentioned above, MES buffer (2-(N-morpholinoethyl) succinic acid) is a biological buffer with a pH range of 5.5 to 6.7, which is suitable for most biological reactions. Here, MES buffer is used to adjust the reaction environment to ensure that the antibody and magnetic bead coupling reaction is carried out under appropriate pH conditions.

[0120] As mentioned above, EDC is a carboxyl activating reagent that can convert carboxyl groups (-COOH) into more reactive intermediates, which is helpful for reacting with amino groups (-NH 2 ) molecules (such as antibodies) to form stable amide bonds.

[0121] As mentioned above, the synergistic use of NHS and EDC can increase the activation efficiency of the carboxyl group, making the carboxyl group more likely to react with the amino group. The addition of NHS helps to form stable NHS esters, which can improve the coupling efficiency and help the antibody to be stably fixed on the magnetic beads for a long time.

[0122] As mentioned above, the activation of magnetic beads refers to the reaction of the carboxyl groups on the surface of the magnetic beads with EDC and NHS through chemical reactions to form activated carboxyl groups, which can form covalent bonds with the amino groups in the antibody. This process is necessary to ensure that the antibody is stably coupled to the magnetic beads. The activated magnetic beads can react efficiently with the amino groups in the antibody to form stable covalent bonds, which is crucial for the subsequent specific capture of target substances (such as Clostridium butyricum).

[0123] Step S52, performing antibody coupling reaction on the pretreated antibody and the carboxyl magnetic beads to obtain coupled magnetic beads;

[0124] In this step, the pretreated antibody reacts with the carboxyl magnetic beads, and the antibody is usually fixed on the surface of the magnetic beads by forming a stable covalent bond to obtain coupled magnetic beads, that is, the surface of the magnetic beads carries specific antibodies. Through antibody coupling, the magnetic beads can specifically recognize and capture Clostridium butyricum containing the corresponding antigen (cbpA and CLP in this case), thereby improving the specificity and efficiency of the enrichment process.

[0125] Step S53, adding the coupled magnetic beads to the feces suspension to enrich Clostridium butyricum that specifically binds to the coupled magnetic beads to obtain enriched strain magnetic beads;

[0126] As mentioned above, the coupled magnetic beads are added to the fecal suspension containing Clostridium butyricum. The magnetic beads will bind to the cbpA and CLP on its surface, and the strains bound to the magnetic beads are enriched by magnetic separation to obtain magnetic beads enriched with specific Clostridium butyricum. This step can quickly and specifically enrich the target strains from complex samples, greatly reducing the interference of non-target microorganisms.

[0127] Step S54, releasing the enriched strain magnetic beads and separating the magnetic beads to obtain Clostridium butyricum.

[0128] As described above, the enriched strain magnetic beads are treated to release and separate the bound Clostridium butyricum, which involves changing the buffer conditions or using enzyme treatment to obtain purified Clostridium butyricum. This step allows Clostridium butyricum to be effectively separated from the coupled magnetic beads, providing a pure strain for further research or application.

[0129] Furthermore, the step S53, adding the coupled magnetic beads to the feces suspension to enrich Clostridium butyricum that specifically binds to the coupled magnetic beads to obtain enriched strain magnetic beads, comprises:

[0130] Step S531, adding the coupled magnetic beads to the feces suspension, and incubating at 37° C. for 2 hours to allow the coupled magnetic beads to specifically bind to Clostridium butyricum;

[0131] As described above, the magnetic beads coupled with antibodies are added to the fecal suspension and incubated at 37°C for 2 hours to allow the antibodies on the magnetic beads to specifically bind to Clostridium butyricum. This step ensures the specific antibody-antigen (i.e., magnetic beads-Clostridium butyricum) binding, providing a basis for subsequent enrichment and separation.

[0132] Step S532, using a magnetic rack to separate the magnetic beads from the unbound portion to obtain the enriched strain magnetic beads.

[0133] The magnetic beads bound to Clostridium butyricum and the unbound sample fraction were separated using a magnetic stand to obtain magnetic beads enriched with Clostridium butyricum. Magnetic separation is a fast, efficient and easy-to-operate process that can effectively separate target bacteria from complex samples.

[0134] Furthermore, in step S5, after the antibodies of cbpA and CLP are sorted by carboxyl magnetic beads and Clostridium butyricum is obtained by enrichment using the carboxyl magnetic beads, the method further comprises:

[0135] Step S6, culturing the enriched Clostridium butyricum under anaerobic conditions for 48 hours;

[0136] In the above, the enriched Clostridium butyricum was cultured under anaerobic conditions for 48 hours. The anaerobic culture simulates the natural growth environment of Clostridium butyricum, which helps to maintain its physiological state and activity.

[0137] Step S7, screening individual colonies of the cultured Clostridium butyricum according to their morphological characteristics as colony samples;

[0138] In the above, according to the morphological characteristics of the cultured Clostridium butyricum, a single colony is screened from the culture plate. Selecting a single colony is conducive to obtaining pure Clostridium butyricum and avoiding cross contamination.

[0139] Step S8, using the colony sample as a template to perform PCR amplification, and identifying it as the expected DNA fragment through PCR technology;

[0140] In the above, the selected colony samples are used as templates for PCR amplification to identify their DNA. PCR technology can quickly and accurately amplify target DNA fragments, providing materials for subsequent sequence analysis.

[0141] Step S9, recovering the DNA bands and sequencing the recovered DNA to obtain sequence information;

[0142] Step S10, determining the homology with the Clostridium butyricum sequence according to the sequence information.

[0143] In the above, the PCR product is subjected to electrophoresis, the target DNA band is recovered, and sequencing is performed. This step can obtain accurate DNA sequence information, providing a basis for further analysis.

[0144] In the above, the DNA sequence obtained by sequencing is compared to determine its homology with the known Clostridium butyricum sequence. Through sequence comparison, it can be confirmed whether the enriched bacteria are Clostridium butyricum, ensuring the accuracy of the experimental results.

[0145] Overall, this series of steps not only enriched and isolated the target Clostridium butyricum from complex fecal samples by combining molecular biology and microbiology methods, but also performed detailed molecular identification and analysis, ensuring the accuracy and reliability of the results.

[0146] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0147] Example 1: Protein recombinant expression purity and concentration detection

[0148] In this example, the purity and concentration of protein recombinant expression were tested. Escherichia coli was used to express the N-terminal 36-380aa of cbpA (NCBI accession number GEQ15565.1), connected with the peptide GGGGS, and then connected with the 152-281aa fusion protein of the cell wall anchor protein CLP_3241 (NCBI accession number WP_003413819.1), and cbpA-CLP was purified, and the purity and concentration of the antigen were characterized.

[0149] Experimental methods:

[0150] (1) cbpA-CLP was expressed in E. coli, and the nucleotide sequence of the cbpA-CLP fusion protein after codon optimization of cbpA and CLP was shown in SEQ ID NO.1; the amino acid sequence of cbpA-CLP was shown in SEQ ID NO.2;

[0151] (2) The nucleotide sequence of the cbpA protein synthesized by full gene synthesis is shown in SEQ ID NO.3; the amino acid sequence of cbpA is shown in SEQ ID NO.4.

[0152] (3) The CLP protein was fully gene-synthesized, and its nucleotide sequence was shown in SEQ ID NO.5, and its amino acid sequence was shown in SEQ ID NO.6, and then transformed into Escherichia coli BL21;

[0153] (4) After ultrasonic disruption, the supernatant after centrifugation was purified by nickel column, the protein A280 concentration was measured by ultra-micro spectrophotometer, and the protein purity was analyzed by SDS-PAGE.

[0154] Table 1. Parameters of recombinant expression protein in Example 1

[0155] Protein name Purification tags A280 concentration SDS-PAGE protein purity cbpA-CLP His 2.2mg / mL 95％ cbpA His 1.5 mg / mL 93％ CLP His 2.1mg / mL 92％

[0156] Example 2: Mouse immunization, antiserum titer detection and hybridoma cell fusion

[0157] In this example, mouse immunization, antiserum titer detection, and hybridoma cell fusion were performed. The recombinant protein cbpA-CLP was used to immunize mice, and after immunization, the mouse blood was collected to detect the titer and titer of the serum, and the spleen was taken and fused with myeloma cells to screen out cell lines that recognize cbpA and CLP.

[0158] Experimental methods:

[0159] (1) LPXTG-CLP protein was mixed with Freund's complete adjuvant and then placed in a mixer for emulsification. The first immunization dose was 50 μg protein, and the second to fourth immunization doses were 50 μg protein. Immunization was performed every two weeks. After 3-4 immunizations, 2 mL of blood from the mouse ear vein was collected to detect the serum antibody titer.

[0160] (2) Prepare spleen cell suspension and wash with PBS, then mix with SP2 / 0 cells at a ratio of spleen cells: SP2 / 0 = 10:1. Centrifuge at 1 000 rpm for 5 min, drip the mixed cells, and tap to loosen the cell clumps. Add 1 mL of PEG1450 in a 37°C water bath, react in a 37°C water bath for 2 min, and slowly add 20 mL of RPMI 1640 stop solution along the tube wall. After the fusion of cells is terminated, centrifuge at 800 rpm for 5 min, and aspirate the residual liquid.

[0161] (3) The screened cells were diluted to 1 cell / well by limiting dilution method using HT medium, and the cells were plated on 96-well cell culture plates. When the monoclonal cells grew to a medium size and the density was about 104 cells or more, the titer could be detected, and then the positive cells were taken again, and the subclone screening was repeated. After the supernatant of cells in all microwells was detected to be positive, the same monoclonal screening was performed again until all positive results were obtained again after limiting dilution method. It can be determined that positive cell lines were screened at this time, and hybridoma cells were obtained. The cells were numbered 16 and 19, and the recognition sites of cbpA-CLP, cbpA, and CLP proteins were detected for 16 and 19, respectively. According to the results, the two antibodies recognized cbpA and CLP proteins, respectively. The test results are shown in Table 2.

[0162] Table 2. Detection results of cells No. 16 and 19 recognizing cbpA and CLP proteins

[0163] Protein name Antibody No. 16 Antibody 19 cbpA-CLP + + cbpA - + CLP + -

[0164] Among them, "+" means that the antigen and antibody react, and "-" means that the antigen and antibody do not react.

[0165] Example 3: Serum-free culture of hybridoma cells and purification of antibodies

[0166] In this example, serum-free culture of hybridoma cells and purification of antibodies were performed. Hybridoma cells were acclimated in serum-free culture medium, and a large amount of culture supernatant was subjected to affinity chromatography to obtain antibodies, and the purity was identified by SDS-PAGE.

[0167] Experimental methods:

[0168] (1) Resuscitate hybridoma cells in serum-free medium. During several consecutive cell passages, gradually reduce the serum ratio (e.g., from 20% serum to 15% serum to 10% serum and then to serum-free medium), and gradually increase the substitute additives to allow the hybridoma cells to adapt and continue to grow. When preparing monoclonal antibodies, resuscitate hybridoma cells in serum-free medium.

[0169] (2) When the cells are in good condition, increase the amount of culture medium and the container for culturing cells. When a large number of cells die, collect the liquid, centrifuge to obtain the supernatant, filter and collect, and obtain the sample to be purified;

[0170] (3) Loading the purified sample onto a Protein A agarose affinity chromatography column at a flow rate of 0.5 mL / min to allow the antibody to bind to Protein A;

[0171] (4) Elute with elution buffer to obtain the antibody, and identify its purity by SDS PAGE. The electrophoresis results are referenced Figure 2 .

[0172] Example 4: Cell line sequencing

[0173] In this example, RNA was extracted from the cell line and reverse transcribed by RT PCR to obtain cDNA, and DNA sequencing was performed using universal primers for the light chain and heavy chain variable regions of mouse IgG antibodies to determine the variable region sequence of the antibody.

[0174] Experimental methods:

[0175] (1) culturing hybridoma cells No. 16 and No. 19, lysing them, and extracting total RNA and mRNA from the lysates;

[0176] (2) cDNA was synthesized by reverse transcription of mRNA using random hexamer primers (5'-Pd(NNNNNN)-3'N=G, A, T or C), followed by two rounds of nested PCR:

[0177] (3) The first-strand cDNA was used as a template for amplification. The forward primer was complementary to the corresponding heavy and light chain leader sequences, and the reverse primer was a sequence within the constant region of the heavy and light chains.

[0178] Heavy chain forward primer: as shown in SEQ ID NO.7; heavy chain reverse primer: as shown in SEQ ID NO.8; light chain forward primer: as shown in SEQ ID NO.9; light chain reverse primer: as shown in SEQ ID NO.10.

[0179] The PCR amplification program was as follows: denaturation at 94°C for 2 min; denaturation at 94°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 60 s, and 40 PCR cycle extensions; final extension at 72°C for 5 min.

[0180] (4) The gene products with restriction enzyme cutting sites were amplified in the second round and connected to the cloning vector. Then, through sequencing and analysis, the light chain and heavy chain variable region sequences of antibodies No. 16 and No. 19 were obtained. The sequencing results are as follows:

[0181] The nucleotide sequence of the light chain of the first monoclonal antibody is shown in SEQ ID NO.11;

[0182] The amino acid sequences of the light chain CDR and FR regions of the first monoclonal antibody are as follows: FR-L1: as shown in SEQ ID NO.12; CDR-L1: as shown in SEQ ID NO.13; FR-L2: as shown in SEQ ID NO.14; CDR-L2: as shown in SEQ ID NO.15; FR-L3: as shown in SEQ ID NO.16; CDR-L3: as shown in SEQ ID NO.17; FR-L4: as shown in SEQ ID NO.18;

[0183] The nucleotide sequence of the first monoclonal antibody heavy chain is shown in SEQ ID NO.19;

[0184] The amino acid sequences of the CDR and FR regions of the heavy chain of the first monoclonal antibody are as follows: FR-H1: as shown in SEQ ID NO.20; CDR-H1: as shown in SEQ ID NO.21; FR-H2: as shown in SEQ ID NO.22; CDR-H2: as shown in SEQ ID NO.23; FR-H3: as shown in SEQ ID NO.24; CDR-H3: as shown in SEQ ID NO.25; FR-H4: as shown in SEQ ID NO.26.

[0185] The nucleotide sequence of the light chain of the second monoclonal antibody is shown in SEQ ID NO.27;

[0186] The amino acid sequences of the light chain CDR and FR regions of the second monoclonal antibody are as follows: FR-L1: as shown in SEQ ID NO.28; CDR-L1: as shown in SEQ ID NO.29; FR-L2: as shown in SEQ ID NO.30; CDR-L2: as shown in SEQ ID NO.31; FR-L3: as shown in SEQ ID NO.32; CDR-L3: as shown in SEQ ID NO.33; FR-L4: as shown in SEQ ID NO.34;

[0187] The nucleotide sequence of the second monoclonal antibody heavy chain is shown in SEQ ID NO.35;

[0188] The amino acid sequences of the CDR and FR regions of the heavy chain of the second monoclonal antibody are as follows: FR-H1: as shown in SEQ ID NO.36; CDR-H1: as shown in SEQ ID NO.37; FR-H2: as shown in SEQ ID NO.38; CDR-H2: as shown in SEQ ID NO.39; FR-H3: as shown in SEQ ID NO.40; CDR-H3: as shown in SEQ ID NO.41; FR-H4: as shown in SEQ ID NO.42.

[0189] Example 5: Magnetic bead sorting and enrichment of Clostridium butyricum

[0190] In this embodiment, antibodies to cbpA and CLP are coupled to carboxyl magnetic beads, respectively, Clostridium butyricum is separated from a fecal suspension, the purity and yield of the separated Clostridium butyricum are compared, the optimal separation conditions are found, the magnetic beads are attracted by a magnetic frame, and after washing, magnetic beads containing Clostridium butyricum are obtained, and then the antibodies and magnetic beads are dissociated by papain cleavage, and the magnetic beads are collected by a magnetic frame, and the supernatant is the Clostridium butyricum suspension.

[0191] Experimental methods:

[0192] (1) Replace the buffer of antibodies 16 and 19 with 15 mM MES buffer, pH 6.0, dilute the antibody to 2 mg / mL with MES buffer, take 1 mg of carboxyl magnetic beads, wash them three times with MES buffer, and resuspend them to 10 mg / mL with 0.1 mL of MES buffer;

[0193] (2) Weigh EDC and NHS and dissolve them in MES buffer. The concentration of EDC is 20 mg / mL and the concentration of NHS is 24 mg / mL.

[0194] (3) Take 50uL EDC and 50uL NHS respectively and add them to the washed magnetic beads for activation at 25℃ for 30min. Take 100ug and 200ug of antibody and 1mg of activated carboxyl magnetic beads (diameter 50nm, 200nm and 1000nm), react at 25℃ for 16h, magnetic separation, aspirate the supernatant and detect the remaining antibody content in the supernatant at the same time, calculate the amount and concentration of magnetic bead-coupled antibody, continue to wash the magnetic beads two to three times, and resuspend them with physiological saline.

[0195] (4) Take 5 mL of fecal suspension, add 1 mg of coupled magnetic beads, mix and incubate at 37°C for 2 h, separate the magnetic beads using a magnetic rack, and remove the unbound microorganisms and supernatant.

[0196] (5) Then, the magnetic beads bound to Clostridium butyricum were resuspended in physiological saline, and an antibody label removal reagent, i.e., 0.05% papain (S10011, Yuanye Biotechnology), was mixed with the labeled magnetic beads and incubated at 37°C for 3 h to cut the Fc and Fab of the mouse monoclonal antibody to separate the magnetic beads from Clostridium butyricum. The magnetic beads were then collected using a magnetic stand, and the supernatant was the Clostridium butyricum suspension. After the Clostridium butyricum was diluted, it was dropped into a blood cell counting plate and counted under a microscope. After the yield was calculated, 200 nm and the condition of magnetic beads to antigen = 5:1 was used to enrich Clostridium butyricum.

[0197] Table 3. Antigen mass ratio and yield corresponding to different carboxyl magnetic beads

[0198]

[0199] Example 6: Cultivation and sequencing of butyrate shuttle

[0200] In this example, a hemocytometer was used for counting, and an equal amount of Clostridium butyricum was taken for plating culture, colony count, and sequencing.

[0201] Experimental methods:

[0202] (1) Dilute the isolated Clostridium butyricum to 10 3 -10 4 / mL, and then spread on TSN solid culture medium, placed in a 37℃ constant temperature incubator for anaerobic culture for 48h, and observed the morphological characteristics of the colonies.

[0203] (2) Pick 10 single colonies and use them as templates for PCR amplification of 16S rDNA. The amplified products are separated by agarose gel electrophoresis. Figure 3 , a specific band of about 1500 bp in size was observed, which was consistent with the expected band size.

[0204] (3) The target band was purified by gel recovery and nucleotide sequencing, and the sequencing results were compared with the NCBI database by BLAST. The results showed that the 16S rDNA gene sequences of the 10 colonies were 100% homologous to Clostridium butyricum (NCBI accession number: NR_042144.1), so the isolate was determined to be Clostridium butyricum of the genus Clostridium.

[0205] In summary, the antibody screening method for Clostridium butyricum cell surface protein provided by the present invention is based on the strategy of tandem expression of cell surface proteins. Two proteins are expressed in tandem with a flexible connecting peptide, and antibodies with different recognition targets can be obtained in one cell fusion. Two antibodies are screened to recognize different target proteins, and the cell lines are sequenced to protect their light chain and heavy chain variable region sequences; the method for enriching Clostridium butyricum from feces established by the present invention uses two antibodies to capture Clostridium butyricum, which can increase coverage and reduce off-target, and the magnetic bead enrichment method can achieve high-throughput screening with higher specificity, which is more efficient than selective culture medium screening.

[0206] The above are preferred implementation modes and corresponding embodiments of the present invention. It should be pointed out that, for those skilled in the art, without departing from the creative concept of the present invention, several modifications and improvements may be made, including but not limited to the adjustment of proportions, processes, dosages and reaction vessels, which all fall within the scope of protection of the present invention.

Claims

1. An antibody against a Clostridium butyricum cell surface protein, characterized in that: is a first monoclonal antibody or a second monoclonal antibody; The light chain nucleotide sequence of the first monoclonal antibody is shown in SEQ ID NO.11; The amino acid sequences of the light chain CDR and FR regions of the first monoclonal antibody are as follows: FR-L1: as shown in SEQ ID NO.12; CDRL1: as shown in SEQ ID NO.13; FR-L2: as shown in SEQ ID NO.14; CDR-L2: as shown in SEQ ID NO.15; FR-L3: as shown in SEQ ID NO.16; CDR-L3: as shown in SEQ ID NO.17; FR-L4: as shown in SEQ ID NO.18; The heavy chain nucleotide sequence of the first monoclonal antibody is shown in SEQ ID NO.19; The amino acid sequences of the light chain CDR and FR regions of the first monoclonal antibody are as follows: FR-H1: as shown in SEQ ID NO.20; CDR-H1: as shown in SEQ ID NO.21; FR-H2: as shown in SEQ ID NO.22; CDR-H2: as shown in SEQ ID NO.23; FR-H3: as shown in SEQ ID NO.24; CDR-H3: as shown in SEQ ID NO.25; FR-H4: as shown in SEQ ID NO.26; The light chain nucleotide sequence of the second monoclonal antibody is shown in SEQ ID NO.27; The amino acid sequences of the light chain CDR and FR regions of the second monoclonal antibody are as follows: FR-L1: as shown in SEQ ID NO.28; CDRL1: as shown in SEQ ID NO.29; FR-L2: as shown in SEQ ID NO.30; CDR-L2: as shown in SEQ ID NO.31; FR-L3: as shown in SEQ ID NO.32; CDR-L3: as shown in SEQ ID NO.33; FR-L4: as shown in SEQ ID NO.34; The heavy chain nucleotide sequence of the second monoclonal antibody is shown in SEQ ID NO.35; The amino acid sequences of the heavy chain CDR and FR regions of the second monoclonal antibody are as follows: FR-H1: as shown in SEQ ID NO.36; CDR-H1: as shown in SEQ ID NO.37; FR-H2: as shown in SEQ ID NO.38; CDR-H2: as shown in SEQ ID NO.39; FR-H3: as shown in SEQ ID NO.40; CDR-H3: as shown in SEQ ID NO.41; FR-H4: as shown in SEQ ID NO.42.

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