Monoclonal antibody against mcherry and use thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a kind of anti-mCherry monoclonal antibody and its application, the monoclonal antibody can recognize mCherry protein, the monoclonal antibody includes heavy chain variable region and light chain variable region;The light chain variable region has three complementarity determining regions with the amino acid sequence shown in SEQ ID NO:1-3;The heavy chain variable region has three complementarity determining regions with the amino acid sequence shown in SEQ ID NO:8-10.The anti-mCherry monoclonal antibody of the present application can specifically recognize mCherry protein, antibody specificity is good and titer is high.The anti-mCherry monoclonal antibody provided in the present application has good recognition specificity to in vitro purification human mCherry protein, intracellular exogenous expression mCherry protein;Compared with polyclonal antibody, the monoclonal antibody recognition specificity is stronger.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a monoclonal antibody against mCherry and its applications. Background Technology

[0002] Fluorescent proteins (GFPs) are essential optical imaging tools in the life sciences. They can be used for live-cell imaging and to label protein expression. Red fluorescent protein (RFP) was first reported in 1999, and its advantages over green fluorescent protein (GFP) are obvious. First, RFP can be used in conjunction with GFP to solve some scientific problems that GFP alone cannot address. Second, RFP has a longer excitation and emission wavelength, and most importantly, it has low background noise during intracellular imaging. The earliest RFP used for research, DsRed, was cloned from corals, but it had many drawbacks. For example, DsRed's oligomeric state is tetrameric, which can easily form multimers during protein fusion, affecting the target protein. Furthermore, it has a long maturation time and is prone to cytotoxicity within cells. These defects limited its applications, prompting scientists to modify its structure.

[0003] mCherry, a variant of red fluorescent protein, is a red fluorescent dye widely used in biotechnology as a tracer, including for molecular labeling and localization of cellular components. mCherry exhibits low cytotoxicity due to its color and photostability of its monomer molecules. It is superior to other fluorescent protein tags, with maximum excitation and emission wavelengths of 587 nm and 610 nm, respectively. Currently, commercially available antibodies against mCherry fluorescent protein are mainly polyclonal antibodies, such as Anti-mCherry Antibody (ab183628, abcam). Existing monoclonal antibodies are subject to foreign restrictions, are expensive, and cannot be obtained in large quantities.

[0004] Currently, there is a need to develop a monoclonal antibody against mCherry. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a monoclonal antibody against mCherry and its uses, which can specifically recognize mCherry protein. The antibody exhibits good specificity and high titer, and compared with polyclonal antibodies, this monoclonal antibody has stronger recognition specificity.

[0006] In a first aspect of the invention, a monoclonal antibody against mCherry is provided, the monoclonal antibody being capable of recognizing the mCherry protein, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

[0007] The light chain variable region has three complementarity-determining regions as shown in the amino acid sequences of SEQ ID NO:1-3;

[0008] The heavy chain variable region has three complementarity-determining regions as shown in the amino acid sequences of SEQ ID NO:8-10.

[0009] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16.

[0010] Furthermore, the light chain architecture sequence is shown in SEQ ID NO:4 to 7; the heavy chain architecture sequence is shown in SEQ ID NO:11 to 14.

[0011] Furthermore, the monoclonal antibody also includes:

[0012] The monoclonal antibody is modified by substituting, deleting, and / or adding one or more amino acids to obtain an antibody with the same function.

[0013] Alternatively, it may include a heavy chain variable region having an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology with the light chain variable region.

[0014] Alternatively, an antibody obtained by attaching a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0015] Furthermore, V H and / or V L The amino acid sequence can be 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the above sequence. It has a V-shaped structure similar to the above sequence. H and V L V regions with high (i.e., 80% or higher) homology H and V L Antibodies in the region can be obtained through mutagenesis, and then the retained function of the encoded altered antibody can be detected using the functional assays described herein.

[0016] The monoclonal antibodies include: human antibodies, humanized or chimeric antibodies.

[0017] Furthermore, the variable region gene can be converted into the scFv gene, once the encoding V is obtained... H and V L These DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes.

[0018] In these operations, the encoding V L or V H The DNA fragment is effectively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. As used herein, "effective linking" means that two DNA fragments are linked together such that the amino acid sequences encoded by both DNA fragments remain within the reading frame.

[0019] In a second aspect of the invention, a nucleic acid molecule encoding the anti-mCherry monoclonal antibody is provided, the nucleic acid molecule comprising a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the intact light chain.

[0020] In a third aspect of the invention, an expression vector comprising the aforementioned nucleic acid molecule is provided, the expression vector being capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0021] In a fourth aspect of the invention, an engineered bacterium or eukaryotic host cell comprising the expression vector described above is provided.

[0022] In a fifth aspect of the invention, the use of the aforementioned anti-mCherry monoclonal antibody in the preparation of mCherry protein detection reagents or kits is provided.

[0023] In a sixth aspect of the invention, the use of the aforementioned anti-mCherry monoclonal antibody in the preparation of a quality control antibody for an mCherry protein colloidal gold detection kit is provided.

[0024] In a seventh aspect of the invention, a colloidal gold rapid test strip for mCherry protein is provided, comprising:

[0025] Base plate,

[0026] The sample absorption pad, binding pad, chromatography matrix, and absorbent pad are bonded to the base plate and overlapped in sequence; wherein...

[0027] The surface of the conjugation pad is coated with a colloidal gold complex containing the anti-mCherry monoclonal antibody; a control line C is provided on the side of the chromatography matrix near the conjugation pad, and a detection line T is provided on the side of the chromatography matrix near the absorbent pad; the control line C is coated with anti-mouse IgG secondary antibody; and the detection line T is coated with the anti-mCherry monoclonal antibody.

[0028] The present invention has the following advantages and beneficial effects:

[0029] The monoclonal antibody against mCherry of the present invention can specifically recognize mCherry protein, and the antibody has good specificity and high titer.

[0030] The monoclonal antibody against mCherry provided by this invention has good recognition specificity for both in vitro purified human mCherry protein and in vitro and intracellularly expressed mCherry protein; compared with polyclonal antibodies, this monoclonal antibody has stronger recognition specificity. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This refers to the mCherry protein used to immunize mice in Example 1;

[0033] Figure 2 The titer of anti-mCherry protein monoclonal antibody was determined by ELISA; the x-axis represents the antibody dilution ratio, and the y-axis represents the A450 absorbance.

[0034] Figure 3 To identify the specific recognition interactions between polyclonal and monoclonal antibodies against mCherry protein and the mCherry protein using Western blotting experiments. The sample was a purified His-tagged mCherry fusion protein;

[0035] Figure 4 The left image shows the U2OS cell line that stably expresses mCherry, and the right image shows the detection results of the Anti-mCherry monoclonal antibody. Detailed Implementation

[0036] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0037] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning 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 this invention pertains. In the event of any conflict, this specification shall prevail.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0039] The following will provide a detailed description of the monoclonal antibody of this application, its preparation method, and its application effects, in conjunction with examples and experimental data. Specific experimental conditions and methods not specified in the following examples are generally performed according to conventional conditions, such as those described in books like J. Sambrook et al. (eds.), *Molecular Cloning: A Laboratory Manual* (3rd edition), Science Press, 1992; and DL Spector et al., *Cellular Laboratory Manual*, Science Press, 2001, or according to the manufacturer's recommendations.

[0040] Example 1: Monoclonal Antibody and its Preparation Method

[0041] 1. Construction of recombinant protein vectors and prokaryotic expression of proteins

[0042] (1) The mCherry CDS sequence was cloned into the Pet42b vector to obtain the recombinant vector Pet42b-mCherry.

[0043] The primer pair sequences used are:

[0044] Forward primer 5'-AAGAAGGAGATATACATATGGTGAGCAAGGGCGAGGAGGA-3';

[0045] Reverse primer 5'-TGGTGGTGGTGGTGCTCGAGCTTGTACAGCTCGTCCATGCCG-3';

[0046] The NdeI-XhoⅠ restriction site was cloned into the Pet42b vector.

[0047] (2) Protein expression and purification:

[0048] ① Plasmid transformation:

[0049] The constructed mCherry prokaryotic expression plasmid was transformed into BL21 competent cells and plated on LB plates containing kanamycin resistance. After single colonies grew, single colonies were picked and shaken.

[0050] ② Shaking and expression:

[0051] Shake the culture overnight, then transfer to a large volume the next morning, diluting 1:100 to 1:50. Shake at 37°C until OD reaches 0.50. 600 =0.4-0.8, induced with IPTG (0.1-0.3mM), expression induced at 16-25℃ for 24 hours.

[0052] ③ Harvesting & Splitting of Mycelium:

[0053] The induced bacterial culture was collected using a centrifuge at 4°C, washed once with Milli-Q water, and then an appropriate amount of lysis buffer, proteasome inhibitor, lysozyme, and PMSF (1:100) were added. The culture was then placed on ice at 30°C and sonicated for lysis. After lysis, the culture was centrifuged twice at high speed at 4°C for 30 minutes each time, and the supernatant was collected.

[0054] ④ Combining Ni NTA beads:

[0055] Wash the Ni NTA beads three times with lysis buffer, centrifuging at 2500 rpm for 3 min each time. After washing, add the beads to the supernatant and incubate at 4°C for 4-6 hours.

[0056] ⑤ Wash beads & wash off protein:

[0057] Centrifuge to remove supernatant, wash Ni NTAbeads with lysis buffer for 30 minutes, then wash twice with washing buffer for 30 minutes each time. After washing, aspirate the liquid and elute the protein with elution buffer for 4-6 hours.

[0058] ⑥ Concentrated & Dialyzed Protein

[0059] If the protein concentration is low, use a concentration tube to concentrate it, replacing the PBS buffer as you concentrate; if the protein concentration is high, you can dialyze directly with PBS.

[0060] ⑦ Protein storage

[0061] After dialysis, the protein is quantified by Coomassie brilliant blue staining, then mixed with 10-20% glycerol, flash-frozen in liquid nitrogen, and stored at -80°C.

[0062] (3) Protein purification reagent formulation

[0063] Lysis buffer: Tris-HCl: 100mM, NaCl: 300mM, NP-40: 0.05%, pH = 8.0;

[0064] Washing buffer: Tris-HCl: 100mM, NaCl: 300mM, Imidazole: 20-50mM, NP-40: 0.05%, pH=8.0;

[0065] Elution buffer: Tris-HCl: 100mM, NaCl: 300mM, Imidazole: 250mM, NP-40: 0.05%, pH = 8.0;

[0066] Protein purification diagram as shown Figure 1 As shown.

[0067] 2. Animal immunization

[0068] Purified mCherry protein was used as the antigen to immunize mice. Three 6-8 week old Balb / C mice were selected. Freund's complete adjuvant was used for the first master injection, and Freund's incomplete adjuvant was used for subsequent booster injections. Both adjuvants were thoroughly mixed with an equal volume of antigen before injection. Immunization was performed via multiple abdominal injections. The immunization dose was 100 μg antigen per mouse for the master injection and 50 μg antigen per mouse for the booster injection.

[0069] The immune cycle is shown in Table 1;

[0070] Table 1

[0071] deal with Immunization time Immunization dose primary immunization 2020-3-20 0.10mg / per Strengthen immunity 2020-4-5 0.05mg / per Strengthen immunity 2020-4-20 0.05mg / per Antibody test 2020-5-5 —— Strengthen immunity 2020-5-6 0.025mg / per Cell fusion 2022-12-24 ——

[0072] 3. Antiserum detection

[0073] (1) A small amount of blood was taken from the tail vein of a mouse to prepare antiserum.

[0074] (2) ELISA method was used to detect the antiserum titer.

[0075] 4. Cell fusion and subcloning

[0076] (1) Preparation of myeloma cells

[0077] One week before fusion, SP2 / 0 cells were revived and cultured normally to the logarithmic growth phase.

[0078] (2) Spleen cell preparation

[0079] Select mice for fusion, euthanize them by cervical dislocation on the day of fusion, harvest spleens, and collect and count spleen cells according to standard procedures.

[0080] (3) Cell fusion

[0081] Myeloma cells and spleen cells were mixed at a ratio of 1:3 to 1:10, and cell fusion was performed using standard procedures. The cells were then cultured in HAT DMEM complete medium. Hybridoma cells were visible 3 days after fusion. On day 7, the medium was replaced with half HAT complete medium, and on day 8, it was replaced with half HT medium. Screening and testing began approximately 10 days after fusion.

[0082] Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope. Multiple growing hybridoma cells were observed, proving that the fusion operation was successful.

[0083] (4) Fusion screening

[0084] 100 μL of cell supernatant was aspirated per well for indirect ELISA detection. Positive wells were identified based on the ELISA results. A single-channel pipette was used to pick up any positive wells detected on the entire plate for a second test to further confirm their positive status.

[0085] (5) Subcloning

[0086] Two rounds of subcloning were performed on the positive well cells from the second screening. Because the positive well cell lines obtained from the first subcloning are not yet stable and may contain multiple hybridoma cells, it is generally believed that the hybridoma cells after the second subcloning are single cell lines and are confirmed as positive.

[0087] The cells in the positive wells were first subcloned and diluted into multiple wells. They were then cultured in HT DMEM medium and observed under a microscope after about 7 days. Wells with clonal growth were detected by indirect ELISA, and wells with high OD values ​​were selected as positive wells. Cells from the positive wells were picked for a second subcloning to detect stable positive hybridoma cell lines, which were then used as cells for the final preparation of monoclonal antibodies. The cells were then expanded to obtain hybridoma cell lines.

[0088] 5. Ascites preparation and antibody purification

[0089] (1) Preparation of ascites

[0090] The positive cells were cultured and injected into the peritoneal cavity of Balb / C mice (sensitized with Freund's incomplete adjuvant). Abdominal distension was observed in the mice within 7-10 days, indicating the presence of ascites. The ascites was promptly aspirated when significant ascites was observed.

[0091] (2) Purification of ascites fluid

[0092] The ascites fluid from the above cells was purified, and the purity of the purified antibody was greater than 90%.

[0093] (3) Purification by ammonium octanoate sulfate + DEAE ion column method

[0094] (4) Centrifuge the ascites fluid, aspirate the pale yellow liquid and calculate the volume. Dilute it 1:3 with 4 times the volume of 60mM acetate buffer (pH 4.0), add caprylic acid dropwise (final concentration of 25μL / mL to dilute the ascites fluid), stir at room temperature for 30min, and then let it stand at 4℃ for more than 2h to allow it to fully precipitate.

[0095] (5) 10000 r / min, 4℃, 20 min, collect the supernatant, add 1 / 10 volume of 10×PBS (0.1M)

[0096] (pH 7.4). Add 0.277 g of solid ammonium sulfate (0.291 g / mL for 45% saturated ammonium sulfate at 0°C) to each milliliter of the above mixture and let it stand for at least 60 minutes.

[0097] (6) 10000 r / min, 4℃, 20 min, discard the supernatant, dissolve the precipitate in a small amount of PBS. Dialyze the PBS overnight at 4℃.

[0098] (7) Antibody concentration and purity were determined. The antibody concentration was found to be 1.5 mg / mL. The purity of the purified antibody was determined using the Coomassie brilliant blue staining assay. Figure 2 The results showed heavy and light chain bands, with no other impurities, indicating high antibody purity.

[0099] 6. Antibody light and heavy chain sequencing

[0100] (1) Culture hybridoma cells

[0101] After reviving the hybridoma cell line, culture it until the cell number expands to approximately 1 × 10⁻⁶. 7 Centrifuge at 1000 rpm for 5 minutes and collect the cells.

[0102] (2) Extracting RNA from cells

[0103] Under a clean bench environment, add 1 mL of Trizol reagent to the centrifuged cells, let stand for 5 min, add 200 L of chloroform, shake vigorously for 15 sec, let stand at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the supernatant to a new EP tube, add 0.5 mL of isopropanol, let stand at -20℃ for 10 min, centrifuge at 12000 rpm for 10 min. Discard the supernatant, add 1 mL of 75% ethanol, centrifuge at 7500 rpm for 5 min, dry the precipitate, and add 50 μL of RNase-free double-distilled water. Agarose gel electrophoresis is used to identify and quantify the purity, and the sample is stored at -80℃ for later use.

[0104] (3) Preparation of cDNA by reverse transcription

[0105] 1 μL total cellular RNA, 6 μL RNase-free ddH2O, 0.5 μL oligo dT Primer, 0.5 μL Prime ScriptRT Enzyme Mix I, and 2 μL 5× Prime Script Buffer were mixed and incubated at 37°C for 15 min and then at 85°C for 5 s.

[0106] (4) Amplify cDNA

[0107] Mouse IgG V H V L Primer library was used to amplify the above cDNA separately. 20 μL of 2×PCR mix, 2 μL of cDNA, 2 μL of upstream primer, 2 μL of downstream primer, and water were added to a final volume of 40 μL. PCR was performed under the following conditions: 98℃ for 5 min, 98℃ for 30 s of denaturation, 63℃ for 20 s of annealing, 72℃ for 25 s of extension, and 40 cycles followed by a final extension at 72℃ for 5 min.

[0108] (5) Agarose gel electrophoresis and gel recovery

[0109] The PCR products were subjected to agarose gel electrophoresis. The electrophoresis results were observed, and the amplification products with molecular weights of 700-800bp and 1400-1600bp were sent for sequencing.

[0110] Table 2 Amino acid sequence information

[0111]

[0112]

[0113] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15:

[0114] MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYL

[0115] QKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK.

[0116] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16:

[0117] MGWSWIFLFLLSVIAGVQSQVQLQQSGAELVRPGASVTLSCEASGYTFTDYEMHWVRQT

[0118] PVHGLEWIGAVDPETGGTAYNQKFKGKATLTADKSSSTAYMELRRSLTSGDSAVYFCTGFVFDFWGQGTTLTVSS.

[0119] Example 2: Antibody titer detection

[0120] The antibody titer was determined using an ELISA assay. 50 ng of antigen (prokaryotically purified mCherry protein) was coated onto each well of the plate. The purified monoclonal antibody was then added according to... Figure 2 The absorbance of A450 was measured for each well after proportional dilution, and the results are shown in Table 3. Figure 2 As shown;

[0121] Table 3

[0122]

[0123] Example 3: Antibody Specificity Detection

[0124] 1. Antibody specificity was detected using in vitro purified mCherry protein.

[0125] His-tagged mCherry protein was purified in vitro in prokaryotes, and the recognition specificity of the purified antibody was detected using Western blotting. Figure 3 As shown, the left and right figures respectively show the specificity of anti-mCherry polyclonal and monoclonal antibodies detected by Western blotting experiments. The results indicate that the anti-mCherry monoclonal antibody has good recognition specificity for mCherry.

[0126] 2. Detecting antibody specificity at the cellular level

[0127] A cell line stably expressing the mCherry gene was constructed in the human U2OS cell line, and the specificity of the monoclonal antibody was identified by Western blotting. Figure 4 As shown. The method for constructing a cell line overexpressing the mCherry gene in this embodiment of the invention is as follows: the mCherry gene sequence is cloned into the PB511B-1 vector (with EcoRI and BamHI restriction sites), and transfected into cells together with the PB210PA-1 plasmid encoding a transposase. Cell lines stably expressing the mCherry gene are obtained through puromycin selection. Figure 4 It is known that monoclonal antibodies can specifically detect mCherry protein expression.

[0128] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against mCherry, characterized in that: The monoclonal antibody can recognize mCherry protein, and the monoclonal antibody includes a heavy chain variable region and a light chain variable region; The light chain variable region has three complementary determining regions, wherein the amino acid sequence of L-CDR1 is shown in SEQ ID NO:1, the amino acid sequence of L-CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO:

3. The heavy chain variable region has three complementary determinant regions, wherein the amino acid sequence of H-CDR1 is shown in SEQ ID NO:8, the amino acid sequence of H-CDR2 is shown in SEQ ID NO:9, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO:

10.

2. The anti-mCherry monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

16.

3. The anti-mCherry monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the light chain architecture FR-L1 is shown in SEQ ID NO:4, the amino acid sequence of FR-L2 is shown in SEQ ID NO:5, the amino acid sequence of FR-L3 is shown in SEQ ID NO:6, and the amino acid sequence of FR-L4 is shown in SEQ ID NO:

7. The amino acid sequence of the heavy chain architecture FR-H1 is shown in SEQ ID NO:11, the amino acid sequence of FR-H2 is shown in SEQ ID NO:12, the amino acid sequence of FR-H3 is shown in SEQ ID NO:13, and the amino acid sequence of FR-H4 is shown in SEQ ID NO:

14.

4. A nucleic acid molecule encoding a monoclonal antibody against mCherry as described in any one of claims 1-3, characterized in that, The nucleic acid molecules include nucleic acid molecules encoding the heavy chain and nucleic acid molecules encoding the complete light chain.

5. An expression vector comprising the nucleic acid molecule of claim 4, characterized in that: The expression vector can express the nucleic acid in prokaryotic or eukaryotic host cells.

6. An engineered bacterium comprising the expression vector of claim 5.

7. A eukaryotic host cell comprising the expression vector of claim 5.

8. Use of the anti-mCherry monoclonal antibody according to any one of claims 1-3 in the preparation of mCherry protein detection reagents or kits.

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