Monoclonal antibody of mucus eubacterium and application thereof

Abstract

The invention provides a mucus eubacterium monoclonal antibody, the mucus eubacterium monoclonal antibody can identify mucus eubacterium, the monoclonal antibody comprises one of BT1 and BT2, a heavy chain variable region of the BT1 has three complementary determining regions of amino acid sequences as shown in SEQ ID NO: 5-SEQ ID NO: 7, and a heavy chain variable region of the BT2 has three complementary determining regions of amino acid sequences as shown in SEQ ID NO: 6-SEQ ID NO: 7. The light chain variable region has three complementary determining regions of amino acid sequences as shown in SEQ ID NO: 9-SEQ ID NO: 11; the heavy chain variable region of the BT2 has three complementary determining regions of amino acid sequences as shown in SEQ ID NO: 13 to SEQ ID NO: 15; the light chain variable region has three complementary determining regions of amino acid sequences as shown in SEQ ID NO: 17-SEQ ID NO: 19. The antibody can be applied to analysis, qualitative or quantitative analysis of metabolite of mucus eubacterium.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a kind of Eubacterium blautii monoclonal antibody and application thereof. BACKGROUND

[0002] Eubacterium blautii is a recently discovered genus of bacteria, which is classified from several abundant gastrointestinal bacteria, which were previously classified as Ruminococcus. Based on phenotypic and phylogenetic analysis, some species of Clostridium and Ruminococcus have also been reclassified as Blautia.

[0003] Eubacterium blautii is a gram-positive, non-spore-forming, coccobacillary bacterium, which occurs in pairs or chains, and most strains are non-sporulating. It is widely distributed in the feces and intestinal tract of mammals. The average size is 0.6-0.7 mm. It is strictly anaerobic, non-motile, and an important core genus in the mammalian intestinal tract. The optimum temperature and pH of most Eubacterium blautii strains are 37℃ and 7.0, respectively. Eubacterium blautii can use hydrogen and carbon dioxide to produce acetate. Acetic acid is a secondary energy source for intestinal epithelial cells, and also an energy source for muscle and brain tissue, can inhibit pathogenic bacteria, and has anti-inflammatory effects.

[0004] Culture experiments show that all Eubacterium blautii strains can utilize glucose, but different strains have different abilities to utilize sucrose, fructose, lactose, maltose, rhamnose and raffinose. The final products of Eubacterium blautii fermentation of glucose are acetic acid, succinic acid, lactic acid and ethanol, and the main biochemical tests show negative results for lecithin, lipase, catalase and indole.

[0005] The genus Blautia currently comprises a total of 20 validly published species, including: B. Hydrogenotrophica, B. coccoides, B. wexlerae, B. hansenii, B. producta. They were originally misclassified as Clostridium or Ruminococcus. The composition of the genus is constantly updated by the addition of new species and strains, but in general, the species in Blautia still form a relatively stable and coherent monophyletic branch. Different species are first discovered from different sources, but the main species are isolated from human feces. For example: Blautia hydrotrophica (B. hydrotrophica) and Blautia stercoris (B. stercoris) were first isolated from human feces.

[0006] The intestinal microbiota is a complex ecosystem that is associated with the development of host diseases, drug metabolism, immune system regulation and other processes. Blautia, as a dominant genus in the intestinal microbiota, has a significant correlation with host physiological dysfunction, such as obesity, diabetes, cancer, various inflammatory diseases, anorexia and malnutrition, and neurological diseases.

[0007] Therefore, in order to further study the related application of Eubacterium eligens, it is necessary to develop a product of Eubacterium eligens, which can be used for enriching Eubacterium eligens in feces, identifying metabolic products of Eubacterium eligens, and qualitative and quantitative research of Eubacterium eligens. SUMMARY

[0008] The purpose of the present application is to provide a monoclonal antibody of Eubacterium eligens and its application. The specific antibody of the target protein of Eubacterium eligens can be used to enrich Eubacterium eligens in feces, can be used for strain identification sequencing, and can be used for identification of metabolic products of Eubacterium eligens and qualitative and quantitative research.

[0009] To solve the above technical problems, the present application adopts the following technical solutions:

[0010] In the first aspect of the present application, a target protein of Eubacterium eligens is provided, the target protein of Eubacterium eligens has antigen immunogenicity, and the amino acid sequence of the target protein of Eubacterium eligens is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In the second aspect of the present application, a nucleic acid molecule is provided, the nucleic acid molecule encodes the target protein of Eubacterium eligens of claim 1, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO: 3 or SEQ ID NO: 4.

[0012] In the third aspect of the present application, a monoclonal antibody of Eubacterium eligens is provided, the monoclonal antibody can recognize Eubacterium eligens, and the monoclonal antibody comprises one of BT1 and BT2:

[0013] The heavy chain variable region of the BT1 has three complementarity determining regions of the amino acid sequence shown as SEQ ID NO: 5-SEQ ID NO: 7; the light chain variable region has three complementarity determining regions of the amino acid sequence shown as SEQ ID NO: 9-SEQ ID NO: 11;

[0014] The heavy chain variable region of the BT2 has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO: 13-SEQ ID NO: 15; the light chain variable region has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO: 17-SEQ ID NO: 19.

[0015] Further, the amino acid sequence of the heavy chain variable region of the BT1 is shown in SEQ ID NO: 8; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the BT2 is shown in SEQ ID NO: 16; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20.

[0016] Further, the monoclonal antibody further comprises:

[0017] The monoclonal antibody has the same function after one or more amino acids are substituted, deleted and / or added to the amino acid sequence of the monoclonal antibody;

[0018] or comprises a heavy chain variable region with an amino acid sequence derived from a Myxobacterium bacterium and having at least 90% homology with the heavy chain variable region; and a light chain variable region with an amino acid sequence having at least 90% homology with the light chain variable region;

[0019] or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0020] In a fourth aspect of the present application, a method for enriching Myxobacterium bacteria from feces is provided, the method comprising: using the antibody to capture Myxobacterium bacteria from feces.

[0021] Further, the method specifically comprises:

[0022] The carboxyl groups on the surface of the magnetic beads are activated to obtain activated magnetic beads;

[0023] The antibody is coupled to the activated carboxyl magnetic beads, and then magnetic separation is performed, the supernatant is aspirated, and the magnetic beads are resuspended with physiological saline after washing to obtain BT1 magnetic beads or BT2 magnetic beads;

[0024] The preliminarily treated fecal bacteria solution is taken, BT1 magnetic beads or BT2 magnetic beads or a mixture of the two kinds of magnetic beads are added, and mixed and incubated, the magnetic beads are separated by using a magnetic stand to remove the unbound microorganisms and the supernatant, and then the magnetic beads combined with Myxobacterium bacteria are resuspended with physiological saline, the antibody-labeled removal reagent is used to separate the magnetic beads from the Myxobacterium bacteria, and then the magnetic beads are collected by using a magnetic stand, and the supernatant is the Myxobacterium bacteria suspension.

[0025] In a fifth aspect of the application, the antibody is provided for use in the analysis, qualitative or quantitative analysis of metabolites of Veillonella.

[0026] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages:

[0027] 1. The application provides a monoclonal antibody of Veillonella bacteria and its application, the monoclonal antibody BT1 and BT2 are prepared by using a representative segment of a specific protein of Veillonella as an antigen, the advantage of the antibody mainly lies in the selection of the target protein in the early stage, gram-positive bacteria, the selection of the Veillonella bacteria S-layer protein (Surface-layer) and the molecular weight of the anchor protein binding region (LPxTG structure) in the transmembrane protein, and the homology cross of the species is high. The application selects two proteins with high homology and large molecular weight in the Veillonella surface protein, and uses the two proteins to prepare two specific antibodies BT1 and BT2 that recognize Veillonella bacteria by using hybridoma technology, and then the two antibodies are coupled to magnetic beads, and the two magnetic bead-antibody conjugates are used for enriching Veillonella bacteria, which can bind to the S-layer protein and the anchor protein binding region of the Veillonella bacteria surface protein, respectively, to enrich bacteria from different specific protein targets to improve the enrichment efficiency.

[0028] 2. The magnetic bead sorting method using a magnetic stand selected by the application has low cost and can preserve the activity of bacteria to a large extent, and the finally enriched Veillonella bacteria can be used for metabolite analysis of Veillonella bacteria, and can also be used for qualitative and quantitative analysis. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0030] Figure 1 It is an electrophoresis map of two purified antibodies;

[0031] Figure 2 It is the WB detection result of two monoclonal antibodies. DETAILED DESCRIPTION

[0032] The advantages and various effects of the present application will be more clearly presented hereinafter with specific embodiments and examples. Those skilled in the art should understand that these embodiments and examples are used to illustrate the present application, not to limit the present application.

[0033] Throughout this specification, unless otherwise specifically indicated otherwise, the terms used herein are understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. If there is a contradiction, the present specification takes priority.

[0034] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or obtained by existing methods.

[0035] The general idea of the present application is as follows:

[0036] 1. Expression and purification of two proteins from Myxobacterium bacteria: E. coli was used to express the N-terminal 1-412 aa of E5259_18370 (UniProt accession number A0A7G5MXS1) and the N-terminal 1-300 aa of SAMEA3545375_03028 (UniProt accession number A0A3S5A350), and the purity and concentration of the antigens were characterized.

[0037] E5259_18370 amino acid sequence s-layer protein (1-412 aa) SEQ ID NO. 1

[0038] MPMRKRVYSLVLCALLAASLLLSGCGRGIETEAETDAKDSVSDSLQKKIYQVFEPKKEEIPEADALQYVNGEYIPYGYESLTENQQKLYRQLLNGILEYKDMVTVDYCTEEDINLVNNMVFVDHPEFFWLDQQSYTFQGDADADTAGSVDLQLVYNIDKSEIESAKAGIEAAADQWISQVPPDTDTYGKIKYIYEFLSQNIAYDQSCPNNQNIQSVFLNQVTVCAGFSKATQYLLGKMGIFCTLVTGTAAPNNEEHAWNLVKIGDHYYYVDTTWANPGFSETQEGVVVQEISYTYLCCDASTLLATHTPDDLLPLPETVDDSYNYYKMNGTWYSYYDENEIYHILTNSIWEGKEREDFKFADAESYQQAVAAMVNGDLIERAVREAYQFGEGETYQWNIGYSDEDKLLTVYW

[0039] E5259_18370 nucleotide sequence SEQ ID NO. 2

[0040]

[0041] Amino acid sequence SEQ ID NO. 3 (1-300aa)

[0042] MIHNHSSGKYRLWRKVLSVCIAFAMICSVSISAFAVEASSETANENIETVQSGSAEDAEELTATEAENDQSVDEAQPEEPAVTTGDENTEEPTDVIEAPSEDPEQPEEPSVEPGEEPIQPADPTVTEQPEEPTVTEQPEEPGATPAGDADASVDEVQDVELEAQADAQSVQDIEQEQDPVAVQERWGSYQNYTGIAAIYYLATPDGIPESNDTQYWAPESDKSKLFGKINTNGAVWEKVDNKDKNIRDYVNNHVSTWPDGTAGSSWIVKRSNSSGNVDGKTYFNYILDSIWNAYKDNLGN

[0043] Nucleotide sequence SEQ ID NO. 4

[0044] ATGATCCATAATCACTCTTCCGGCAAATATCGTCTGTGGCGTAAGGTACTGAGCGTCTGCATTGCATTCGCTATGATTTGCTCTGTGTCTATCTCCGCGTTTGCAGTCGAAGCATCCTCTGAGACCGCTAACGAAAACATCGAGACCGTACAGTCTGGTTCTGCTGAGGACGCGGAAGAACTGACTGCAACCGAAGCCGAAAACGACCAGAGCGTGGACGAAGCTCAGCCAGAAGAACCAGCAGTAACGACGGGTGACGAAAACACCGAAGAGCCGACTGATGTCATCGAGGCGCCGTCCGAGGATCCGGAACAACCGGAAGAACCGTCTGTCGAGCCGGGCGAAGAACCAATCCAGCCGGCAGATCCTACTGTCACCGAACAGCCGGAAGAGCCGACGGTTACTGAACAGCCGGAAGAACCAGGTGCTACGCCGGCCGGTGATGCTGATGCTAGCGTTGATGAAGTTCAGGATGTGGAACTGGAAGCGCAAGCAGACGCACAAAGCGTTCAGGACATTGAGCAGGAACAGGATCCTGTGGCGGTGCAAGAACGCTGGGGTTCCTACCAAAACTATACCGGCATTGCCGCTATCTACTACCTGGCCACTCCGGACGGTATCCCGGAATCCAACGATACCCAGTACTGGGCCCCGGAATCTGATAAAAGCAAACTGTTCGGTAAAATCAACACCAACGGTGCGGTATGGGAGAAAGTGGATAACAAAGACAAGAACATCCGCGACTACGTTAACAATCACGTGTCCACCTGGCCGGATGGCACCGCGGGCAGCAGCTGGATTGTTAAACGCTCCAACTCTTCTGGCAACGTAGACGGTAAAACCTATTTCAACTATATCCTGGACTCCATCTGGAATGCCTACAAAGACAACCTGGGCAAC

[0045] 2. Immunization and cell fusion: Two recombinant proteins were used to immunize mice, respectively. After the completion of immunization, the tail vein blood of mice was taken to detect the titer and potency of the serum. The spleen and myeloma cells SP2 / 0 of the mouse with high potency were fused to screen the cell strains recognizing E5259_18370 and SAMEA3545375_03028, respectively.

[0046] 3. Ascites preparation and antibody purification: The hybridoma cell strains were domesticated by using serum-free medium. The supernatant was purified by affinity chromatography to obtain the antibody, and the purity was identified by SDS-PAGE.

[0047] 4. Cell strain sequencing: The RNA of the cell strain was extracted, and the cDNA was obtained by reverse transcription PCR. The variable region sequence of the antibody was determined by DNA sequencing using mouse IgG antibody light chain and heavy chain variable region universal primers.

[0048] 5. Magnetic bead sorting and Eubacterium limosum enrichment: The antibodies of E5259_18370 and SAMEA3545375_03028 were coupled to carboxyl magnetic beads, respectively, to separate Eubacterium limosum from the fecal suspension. The purity and yield of Eubacterium limosum were compared to find the best sorting condition. The magnetic beads containing Eubacterium limosum were obtained by washing after being attracted by a magnetic stand. The antibody and magnetic beads were dissociated by papain enzyme digestion. The magnetic beads were collected by a magnetic stand, and the supernatant was the Eubacterium limosum suspension.

[0049] 6. Eubacterium limosum counting and culture: The blood cell counter was used for counting, and the same amount of Eubacterium limosum was used for spread culture and colony counting.

[0050] To improve the screening efficiency, the sorting strategy of the present patent mainly includes two kinds of monoclonal antibody coupled magnetic beads. One is the antibody binding to the specific surface protein E5259_18370 of Eubacterium limosum, and the other is the antibody binding to the specific surface protein SAMEA3545375_03028 of Eubacterium limosum to capture the bacteria.

[0051] The present application will be described in detail below in combination with examples and experimental data. If the specific technology or condition is not specified in the examples, it is carried out according to the technical method or condition described in the literature, standard or other technical materials in the field, or according to the product instruction. The methods described in this part are conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used in this paper have the same meaning as known by those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be used in the implementation of the related technology involved in the present application.

[0052] Example 1: Detection of protein recombinant expression purity and concentration

[0053] The codon-optimized nucleotide sequences of E5259_18370 and SAMEA3545375_03028, SEQ ID NO. 2, SEQ ID NO. 4, were expressed in E. coli BL21, and the proteins were purified by nickel column after ultrasonic disruption and centrifugation. The protein A280 concentration was measured by ultramicro spectrophotometer, and the protein purity was analyzed by SDS-PAGE. The concentration and purity results of the two proteins after purification are shown in Table 1.

[0054] The amino acid sequence of E5259_18370 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2.

[0055] The amino acid sequence of SAMEA3545375_03028 is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4.

[0056] Table 1

[0057] Protein name Purification tag A280 concentration SDS-PAGE protein purity E5259_18370 His 1.5 mg / ml 92％ SAMEA3545375_03028 His 1.4 mg / ml 93％

[0058] Example 2: Mouse immunization and detection of antiserum titer and hybridoma cell fusion

[0059] E5259_18370 and SAMEA3545375_03028 proteins were mixed with Freund's adjuvant, respectively, and then emulsified in a homogenizer. The first immunization dose was 50 ug protein per mouse, and the adjuvant was Freund's complete adjuvant. The interval between the second and third immunizations was 3 weeks. The second to fourth immunization dose was 50 ug protein per mouse, and the adjuvant was Freund's incomplete adjuvant. The interval between the second and third immunizations was 2 weeks. Mouse tail vein blood was taken to detect serum antibody titer. Generally, mice with a titer of 1:10,000 or more were selected for fusion. The immunization method is shown in Table 2:

[0060] Table 2: Mouse immunization procedure

[0061]

[0062]

[0063] Three days before fusion, the mice were immunized with 50 ug of protein per mouse without adjuvant. After the mice were bled from the eye, the spleen cells from the mice with good immune response were washed with PBS and mixed with SP2 / 0 cells. The spleen cells and SP2 / 0 cells were mixed at a ratio of 10:1, centrifuged at 1000 rpm for 5 minutes, and then the cells were mixed and tapped to loosen the cell mass. Then, 1 ml of PEG 1450 was added to the cell mass in a 37°C water bath. After the PEG 1450 was added, the mixture was reacted in the 37°C water bath for 2 minutes, and then 20 ml of RPMI-1640 was slowly added along the wall of the tube to terminate the reaction. After the reaction was terminated, the cells were centrifuged at 800 rpm for 5 minutes, and then the residual liquid was removed. The cells were resuspended in DMEM complete medium containing HAT, and then the cells were pipetted into a 96-well cell culture plate. Cell colonies were observed 3 days after the fusion, and the medium was changed 7 days after the fusion.

[0064] According to the growth of the cells, the size of the colonies was observed, and when the size of the colonies was about 1 / 4 of the size of the bottom of the well, the cells were considered to be ready for detection. 100 μL of the supernatant was taken and detected by indirect ELISA. Positive wells with high OD values and good colony states were selected for subcloning.

[0065] The positive cell colonies obtained by screening were diluted to 1 cell per well by limiting dilution using HT medium. The cells were plated in a 96-well cell culture plate, and when the single clone cells grew to a medium size with a density of about 10 4 cells or more, the titer was detected. Then, the positive cell wells were selected again for repeated subcloning. When the supernatant of all the microwells was positive, the same subcloning was performed again until the positive hybridoma cell strains were obtained again. The cell strains were numbered BT1 and BT2. The titer of the antibodies was detected by indirect ELISA, as shown in Table 3. The subtypes of the two monoclonal antibodies were identified using an antibody subtype identification kit, and the results are shown in Table 4:

[0066] Table 3: Titer determination of two monoclonal antibodies

[0067]

[0068]

[0069] Table 4: Subtype determination of two monoclonal antibodies

[0070] Cell line number Subtype BT1 IgG3 BT2 IgG2b

[0071]

Example 3

[0072] One week before inoculation of the hybridoma cells, BALB / C mice were injected intraperitoneally with Freund's incomplete adjuvant at 0.5 ml per mouse, and then each mouse was inoculated with 5 x 10 6The positive hybridoma cells were cultured, and the ascites were collected after 7-12 days to determine the antibody titer. The collected ascites were centrifuged at 10,000 rpm for 10 min to remove the cell components and other precipitates, and the supernatant was collected and detected for the antibody titer, aliquoted, and stored at -80°C for standby use. The antibody sample to be purified was loaded onto a Protein A-sepharose affinity chromatography column at a flow rate of 0.5 mL / min, the antibody was combined with Protein A, and finally eluted with an eluent to obtain the antibody, which was identified for purity by SDS-PAGE, as shown in Figure 1

[0073]

Example 4

[0074] The BT1 and BT2 hybridoma cells were cultured, lysed, and total RNA and mRNA were extracted from the lysate. The mRNA was reverse transcribed to synthesize cDNA by using random hexamer primers (5'-P-d(NNNNNN)-3' N=G, A, T or C), and then two rounds of nested PCR were performed: the first-strand cDNA was used as a template for amplification, the forward primer was complementary to the sequence of the corresponding heavy chain and light chain leader sequence, and the reverse primer was a sequence in the heavy chain and light chain constant region.

[0075] Heavy chain forward primer: CGGCCCAGCCGGCC

[0076] Heavy chain reverse primer: TGAACCGCCTCCACC

[0077] Light chain forward primer: GGTTCCACTGGT

[0078] Light chain reverse primer: GTGCAGCATCAGC

[0079] 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, and extension at 72°C for 60 s, for 40 PCR cycles of extension; and final extension at 72°C for 5 min.

[0080] The second round of amplification produced gene products with restriction enzyme cutting sites (EcoR I and Hind III), which were ligated to the pMD19-T cloning vector, and then sequencing and analysis were performed to obtain the antibody light chain and heavy chain variable region sequences of BT1 and BT2.

[0081] Heavy chain forward primer: TGAATTCCGGCCCAGCCGGCC, heavy chain reverse primer: TAAGCTTTGAACCGCCTCCACC

[0082] Light chain forward primer: TGAATTCGGTTCCACTGGT, light chain reverse primer: TAAGCTTGTGCAGCATCAGC

[0083] ​Amino acid sequence of the heavy chain variable region of monoclonal antibody BT1

[0084] LASTATGVHSQVQLQQPGAELVRPGASVKLSCKASGYTFT TNSFC WVK QRPGQGLEWIG FVYFWETD LCNTSFRPE KATLTVDKSSSTAYIHLTSLTSEDSA VYYCAR AIAHN WGQGTTLTVSPASTTPPSV (SEQ ID NO: 8)

[0085] Note: Complementarity determining regions (CDRs) are bolded and underlined

[0086] FR-H1 : QVQLQQPGAELVRPGASVKLSCKASGYTFT

[0087] CDR-H1 : TNSFC (SEQ ID NO: 5)

[0088] FR-H2: WVKQRPGQGLEWIG

[0089] CDR-H2: FVYFWETDLCNTSFRPE (SEQ ID NO: 6)

[0090] FR-H3: KATLTVDKSSSTAYIHLTSLTSEDSAVYYCAR

[0091] CDR-H3: AIAHN (SEQ ID NO: 7)

[0092] FR-H4: WGQGTTLTVSP

[0093] Amino acid sequence of the light chain variable region of monoclonal antibody BT1

[0094] GVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISY RGWTWPENNAEWCF T WNQQKPGQPPKLLIY PP QYVDQ GIPARFSGSGSGTDFTLNIHPVEEEDAATY YC SSVYDEFNQ YGGRTWMEIKRKSTAPKCI (SEQ ID NO: 12)

[0095] Note: Complementarity determining regions (CDRs) are bolded and underlined

[0096] FR-L1 : DIVLTQSPASLAVSLGQRATISY

[0097] CDR-L1 : RGWTWPENNAEWCFT (SEQ ID NO: 9)

[0098] FR-L2: WNQQKPGQPPKLLIY

[0099] CDR-L2: PPQYVDQ (SEQ ID NO: 10)

[0100] FR-L3: GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC

[0101] CDR-L3: SSVYDEFNQ (SEQ ID NO: 11)

[0102] FR-L4: YGGRTWMEIK

[0103] Amino acid sequence of the heavy chain variable region of monoclonal antibody BT2

[0104] EVQLVESGGGLVKPGGSLKLSCAASGFTFS ENNPN WVRQTPEKRLEWV A CVWEAAQNCNNFEWPRA RFTISRDNAKNNLYLQMSSLKSEDTAMYYCAR NNNAKVEC WGQGTTLTVSA (SEQ ID NO: 16)

[0105] Note: Complementarity determining regions (CDRs) are bolded and underlined

[0106] FR-H1 : EVQLVESGGGLVKPGGSLKLSCAASGFTFS

[0107] CDR-H1 : ENNPN (SEQ ID NO: 13)

[0108] FR-H2: WVRQTPEKRLEWVA

[0109] CDR-H2: CVWEAAQNCNNFEWPRA (SEQ ID NO: 14)

[0110] FR-H3: RFTISRDNAKNNLYLQMSSLKSEDTAMYYCAR

[0111] CDR-H3: NNNAKVEC (SEQ ID NO: 15)

[0112] FR-H4: WGQGTTLTVSA

[0113] Amino acid sequence of the light chain variable region of monoclonal antibody BT2

[0114] MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTMSCRWWT WAANTVCSRCCAGWYQQKPGQSPKLLIYTGQCKDWGVPDRFTGSGSGTDF TLTISSVKAEDLAVYYCCCNNWTFTYFGGGTKLEIK (SEQ ID NO: 20) Note: Complementarity determining regions (CDRs) are bolded and underlined

[0115] FR-L1 : DIVMSQSPSSLAVSVGEKVTMSC

[0116] CDR-L1 : RWWTWAANTVCSRCCAG (SEQ ID NO: 17)

[0117] FR-L2: WYQQKPGQSPKLLIY

[0118] CDR-L2: TGQCKDW (SEQ ID NO: 18)

[0119] FR-L3: GVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC

[0120] CDR-L3: CCNNWTFTY (SEQ ID NO: 19)

[0121] FR-L4: FGGGTKLEIK

[0122] Example 5: Myxococcus bacteria enrichment

[0123] 1. Carboxyl activation of magnetic bead surface

[0124] After mixing the magnetic beads, take 100 μL of the Mag COOH magnetic beads (70113-5, Suzhou Beaver Bio) into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, and wash twice with 200 μL of MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20) by magnetic separation, then remove the supernatant; quickly add freshly prepared 100 μL of EDC solution (10 mg / mL, dispersed in the above MEST solution) and 100 μL of NHS solution (10 mg / mL, dispersed in the above MEST solution) to the centrifuge tube containing the magnetic beads, vortex to fully suspend the magnetic beads, and activate at 25°C for 30 min, keeping the magnetic beads in suspension during this period (can be inverted and mixed using a vertical mixer); after the above steps, the carboxyl groups on the surface of the magnetic beads have been activated and can be covalently coupled with biological ligands containing primary amino groups. (The activated state should not be stored for a long time, and it is recommended to perform coupling immediately)

[0125] 2. Covalent coupling of magnetic beads and antibodies

[0126] Replace the monoclonal antibody BT1 and the monoclonal antibody BT2 buffer with 15 mM MES buffer pH 6.0, dilute the antibodies with MES buffer to 2 mg / mL, take 200 ug of the antibodies and 100 μL of the above activated carboxyl magnetic beads (10 μm in diameter), and react at 25°C for 2 h, or couple at 25°C for 1 h and then place at 4°C overnight, keeping the magnetic beads in suspension during the coupling period (can be inverted and mixed using a vertical mixer); magnetic separation, aspirate the supernatant and simultaneously detect the remaining antibody content in the supernatant, calculate the amount and concentration of the antibody coupled to the magnetic beads, and continue to wash the magnetic beads 2 to 3 times with physiological saline to obtain BT1 magnetic beads and BT2 magnetic beads.

[0127] Take the above two kinds of magnetic beads (BT1 magnetic beads and BT2 magnetic beads), mix according to the mass ratio of 1:1. Add 5 grams of feces to physiological saline (such as 5 grams of feces to 25 mL of physiological saline) at a ratio of 1:5, filter through gauze, and collect the preliminarily treated fecal bacteria liquid. Take the preliminarily treated fecal bacteria liquid, add 0.1 mg of BT1 magnetic beads, BT2 magnetic beads or a mixture of the two kinds of magnetic beads respectively, mix and incubate at 37℃ for 0.5 h, separate the magnetic beads using a magnetic stand, and remove the unbound microbial and supernatant. Then resuspend the magnetic beads combined with the Myxococcus bacteria in physiological saline, mix with the antibody-labeled removal reagent, i.e. 0.05% papain (S10011, source leaf biological), at 37℃ for 0.5 h, for cutting the Fc and Fab of the mouse monoclonal antibody, so that the magnetic beads and Myxococcus bacteria are separated, and then collect the magnetic beads using a magnetic stand, and the supernatant is the Myxococcus bacteria suspension. Dilute the Myxococcus bacteria, add dropwise to a blood cell counting plate, and count under a microscope. Calculate the yield, and then use the two kinds of magnetic beads antibodies at a mass ratio of 1:1 for Myxococcus bacteria enrichment. The results are shown in Table 4, which prove that the effect of the two kinds of magnetic bead antibody conjugates is greater than that of one kind.

[0128] Table 4 Antibody conjugated magnetic bead combination type and yield

[0129] Figure 2 Combination type Yield 1 BT1 1.2 x 10 5 ]]> 2 BT2 1.3 x 10 5 ]]> 3 BT1 + BT2 1.5 x 10 5 ]]>

[0130] Example 6, Myxococcus bacteria culture and sequencing

[0131] Dilute the isolated Myxococcus bacteria to 10 3 -10 4 individuals / mL, then coat on American Type Culture Collection 2722 medium: trypsin soybean broth / agar supplemented medium, 37℃, 80% nitrogen-20% carbon dioxide condition p H7.0 anaerobic culture for 24 h. Observe the colony characteristics: spherical or ellipsoidal, in pairs or strands, most strains are non-spore, average size 0.6-0.7 mm. Pick 20 single colonies, use the colonies as templates for PCR amplification, the PCR selects the upstream primer sequence as 5'

[0132] -AGAGTTTGATCCTGGCTCAGPCR-3', the downstream primer sequence is 5'

[0133] -3'. PCR reaction system: DNA template (10 ng / μL) 1 μL, upstream and downstream primers (10 μmol / L) 2 μL each, 10 x PCR Buffer 5 μL, dNTPs (2.5 mmol / L) 4 μL, Taq enzyme (5 U / μL) 0.5 μL, ddH2O 35.5 μL. PCR reaction conditions: 94°C 10 min; 94°C 1 min, 56°C 1 min, 72°C 25 s, 72°C 10 min, 30 cycles. After the reaction, the results were identified by agarose gel electrophoresis, the target band was gel recovered and purified, and nucleotide sequencing was performed. The sequencing results were subjected to BLAST comparison in the NCBI database, and the results showed that the 16S rRNA gene sequences of the 20 colonies had 97% homology with the Eubacterium limosum (GenBank: EF036467.1), and therefore the isolated strain was determined to be Eubacterium limosum.

[0134] Example 7, WB identification of Eubacterium limosum bacteria antibody

[0135] Sample preparation: Take the identified Eubacterium limosum 104 / mL 2ml centrifuge tube, add 200ul RIPA lysis buffer to extract total protein, centrifuge at 10000rpm for 5min, add equal volume of 2 x loading buffer, boil in boiling water for 5min, aliquot and store at -20°C;

[0136] Electrophoresis: configure SDS-PAGE gel according to the standard protein electrophoresis method, load 15ul per well, run at 200V constant voltage for 30min;

[0137] Membrane transfer: use wet membrane transfer instrument for electric transfer membrane method, transfer the protein in the gel to PDVF membrane, transfer at 150mA constant current for 20-30min; blocking: take out the membrane, wash with PBST three times, each for 5min (horizontal shaking table shaking); take out the membrane, immerse in blocking solution at 37°C for 2h or overnight at 4°C;

[0138] Primary antibody incubation: take out the membrane, wash with PBST three times, each for 5min (horizontal shaking table shaking); take out the membrane, immerse in the primary antibody dilution diluted with 1% casein, at 37°C for 1h (general primary antibody dilution 1:1000);

[0139] Secondary antibody incubation: take out the membrane, wash with PBST three times, each for 5min (horizontal shaking table shaking); take out the membrane, immerse in the secondary antibody dilution diluted with 1% casein, at 37°C for 1h; (goat anti-mouse-HRP 1:5000).

[0140] Color development: DAB color development reaction

[0141] Data analysis: The molecular weight and net optical density of the target bands on the membrane were analyzed by using the gel image processing system. The results are as follows Figure 2 As shown above, it can be seen that the application uses two antibodies to capture Veillonella bacteria, has strong specificity and can improve the enrichment efficiency, can be applied to the analysis of metabolic products of Veillonella, and can also be used for qualitative and quantitative analysis.

[0142] Finally, it should be noted that the terms "comprising", "including", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements, but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0143] Although the preferred embodiments of the application have been described, those skilled in the art will, upon acquiring the basic creative concept, make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the application.

[0144] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A Veillonella spp. bacterial target protein, characterized in that, The target protein of the Myxobacterium bacterium has antigen immunogenicity, and the amino acid sequence of the target protein of the Myxobacterium bacterium is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the target protein of the Myxobacterium bacterium of claim 1, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

3. A bacterium monoclonal antibody against Veillonella bacteria, characterized in that, The monoclonal antibody can recognize the Myxobacterium bacterium, and the monoclonal antibody comprises one of BT1 and BT2: The heavy chain variable region of the BT1 has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO: 5-SEQ ID NO: 7; and the light chain variable region has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO: 9-SEQ ID NO: 11; The heavy chain variable region of the BT2 has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO: 13-SEQ ID NO: 15; and the light chain variable region has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO: 17-SEQ ID NO:

19.

4. The Veillonella bacterium monoclonal antibody according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region of the BT1 is shown in SEQ ID NO: 8; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region of the BT1 is shown in SEQ ID NO: 16; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

20.

5. The Veillonella bacterium monoclonal antibody according to claim 3, characterized by, The monoclonal antibody further comprises: The monoclonal antibody has the same function after one or more amino acids in the amino acid sequence of the monoclonal antibody are substituted, deleted and / or added; or comprises a heavy chain variable region derived from the Myxobacterium bacterium and having an amino acid sequence with at least 90% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence with at least 90% homology with the light chain variable region; or the antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

6. A method of enriching a Veillonella bacterium from feces, characterized by, The method comprises: capturing the Myxobacterium bacterium from feces by using the antibody of any one of claims 3-5.

7. The method of claim 6, wherein, The method specifically comprises: activating the carboxyl group on the surface of the magnetic beads to obtain activated magnetic beads; coupling the antibody of any one of claims 3-5 with the activated carboxyl magnetic beads, magnetically separating, aspirating the supernatant, resuspending the magnetic beads with physiological saline after washing, to obtain BT1 magnetic beads or BT2 magnetic beads; adding the BT1 magnetic beads or the BT2 magnetic beads or a mixture of the two kinds of magnetic beads to the preliminarily treated fecal bacteria liquid, uniformly mixing and incubating, separating the magnetic beads by using a magnetic stand to remove the unbound microbial and the supernatant, resuspending the magnetic beads combined with the Myxobacterium bacterium with physiological saline, separating the magnetic beads from the Myxobacterium bacterium by using an antibody labeling removal reagent, and collecting the magnetic beads by using a magnetic stand, and the supernatant is the Myxobacterium bacterium suspension.

8. The antibody of any one of claims 3-5 is used in the analysis, qualitative or quantitative analysis of the metabolites of Myxobacterium.

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