Monoclonal antibody against copgfp and use thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a monoclonal antibody against CopGFP and application thereof, the monoclonal antibody can recognize CopGFP protein, and the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO:3; and the heavy chain variable region has three complementarity determining regions with the amino acid sequences shown in SEQ ID NO:8-SEQ ID NO:10. The monoclonal antibody against CopGFP of the present application can specifically recognize CopGFP protein, and the antibody has good specificity and high titer. The monoclonal antibody against CopGFP of the present application has good recognition specificity for in-vitro purified human CopGFP protein and intracellular exogenous expression CopGFP protein; compared with a polyclonal antibody, the monoclonal antibody has stronger recognition specificity.

Description

Technical Field

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

[0002] Green fluorescent protein (GFP) is a β-barrel-shaped protein composed of 238 amino acids with a molecular weight of approximately 27 kDa. GFP was isolated from the crystal jellyfish *Aequorea victoria*. GFP can convert the blue fluorescence emitted by the jellyfish's bioluminescent protein through energy transfer to green fluorescence via chemical reactions. GFP has an excitation wavelength of 488 nm and an emission peak at approximately 507 nm. Furthermore, high-resolution crystal structure studies of GFP have allowed scientists to manipulate its protein structure and create color variants that emit at different wavelengths. Enhanced green fluorescent protein (EGFP) is a mutant obtained by replacing the phenylalanine (Phe) at position 64 of GFP with leucine (Leu), exhibiting a fluorescence intensity more than six times greater than GFP. CopGFP is derived from... copepoda Pontellina plumata (Arthropoda; Crustacea; Maxillopoda; Copepoda) The obtained green fluorescent protein was 1.3 times brighter than EGFP.

[0003] Various tools have been derived from fluorescent proteins, among which fluorescent biosensors are widely used. These biosensors respond to different states and signaling molecules within an organism by detecting the presence or absence of fluorescence or changes in fluorescence color, and have seen extensive development. Currently, various biosensors have been developed, but yellow-green fluorescence remains the dominant type. CopGFP has been widely used in the construction of lentiviral packaging plasmids. Currently, commercially available antibodies against fluorescent proteins are mainly anti-GFP, such as EPR14104 (ab183734). There is a need to develop a monoclonal antibody against CopGFP. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a monoclonal antibody against CopGFP and its uses, which can specifically recognize CopGFP protein. The antibody exhibits good specificity and high titer, and compared with polyclonal antibodies, this monoclonal antibody has stronger recognition specificity.

[0005] In a first aspect of the invention, there is a monoclonal antibody against CopGFP, the monoclonal antibody recognizing the CopGFP protein, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region:

[0006] The light chain variable region has three complementarity-determining regions as shown in the amino acid sequences of SEQ ID NO:1-SEQ ID NO:3;

[0007] The heavy chain variable region has three complementarity-determining regions of the amino acid sequence as shown in SEQ ID NO:8-SEQ ID NO:10.

[0008] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:16.

[0009] Furthermore, the light chain architecture sequence is shown in SEQ ID NO:4~7; the heavy chain architecture sequence is shown in SEQ ID NO:11~14.

[0010] Furthermore, the monoclonal antibody also includes:

[0011] The monoclonal antibody is modified by substituting, deleting, and / or adding one or more amino acids to obtain an antibody with the same function.

[0012] 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.

[0013] Alternatively, an antibody obtained by attaching a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0014] 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.

[0015] The monoclonal antibodies include: human antibodies, humanized or chimeric antibodies.

[0016] 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.

[0017] 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.

[0018] In a second aspect of the invention, a nucleic acid molecule encoding the aforementioned anti-CopGFP monoclonal antibody is provided, the nucleic acid molecule comprising a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0019] In a third aspect of the invention, an expression vector comprising the nucleic acid is provided, the expression vector being capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0020] In a fourth aspect of the invention, an engineered bacterium or eukaryotic host cell for the expression vector described above is provided.

[0021] In a fifth aspect of the invention, the use of the aforementioned anti-CopGFP monoclonal antibody in the preparation of a CopGFP protein detection reagent or kit is provided.

[0022] In a sixth aspect of the invention, the use of the aforementioned anti-CopGFP monoclonal antibody in the preparation of a quality control antibody for a CopGFP protein colloidal gold detection kit is provided.

[0023] In a seventh aspect of the invention, a colloidal gold rapid detection test strip for CopGFP protein is provided, comprising:

[0024] Base plate,

[0025] The sample absorption pad, binding pad, chromatography matrix, and absorbent pad are bonded to the surface of the base plate and overlapped in sequence; wherein...

[0026] The surface of the conjugation pad is coated with a colloidal gold complex containing the anti-CopGFP monoclonal antibody described in section 4; 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-CopGFP monoclonal antibody described in section 4.

[0027] The present invention has the following advantages and beneficial effects:

[0028] The monoclonal antibody against CopGFP provided by this invention can specifically recognize the CopGFP protein, and the antibody has good specificity and high titer.

[0029] The monoclonal antibody against CopGFP provided by this invention has good recognition specificity for both in vitro purified human CopGFP protein and in vitro and intracellularly expressed CopGFP protein; compared with polyclonal antibodies, this monoclonal antibody has stronger recognition specificity. Attached Figure Description

[0030] 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.

[0031] Figure 1 The CopGFP protein used to immunize mice in Example 1;

[0032] Figure 2 The titer of anti-CopGFP monoclonal antibody was determined by ELISA; the x-axis represents the antibody dilution ratio, and the y-axis represents the A450 absorbance.

[0033] Figure 3 To identify the specific recognition interactions between polyclonal and monoclonal antibodies against CopGFP protein and CopGFP protein in Western blotting experiments, the sample was purified CopGFP fusion protein with a His tag.

[0034] Figure 4 The left image shows the U2OS cell line that stably expresses CopGFP, and the right image shows the detection results of the Anti-CopGFP monoclonal antibody. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1: Monoclonal Antibody and its Preparation Method

[0040] 1. Construction of recombinant protein vectors and prokaryotic expression of proteins

[0041] (1) CopGFP The CDS sequence was cloned into the Pet42b vector to obtain the recombinant vector Pet42b- CopGFP .

[0042] The primer pair sequences used are:

[0043] Forward primer 5'-aagaaggagatatacatATGGAGAGCGACGAGAGCGGCCTGCCC-3';

[0044] Reverse primer 5'-tggtggtggtggtgctcgagGCGAGATCCGGTGGAGCCGGGTCC-3';

[0045] Cloned into the Pet42b vector NdeI - XhoⅠ Enzyme cleavage site.

[0046] (2) Protein expression and purification:

[0047] ① Plasmid transformation:

[0048] The constructed CopGFP 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.

[0049] ② Shaking and expression:

[0050] 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 1 / 2. 600 =0.4-0.8, induced with IPTG (0.1-0.3 mM), expression induced at 16-25 ℃ for 24 hours.

[0051] ③ Harvesting & Splitting of Mycelium:

[0052] The induced bacterial culture was collected by centrifugation 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 min each time, and the supernatant was collected.

[0053] ④ Combining Ni NTA beads:

[0054] 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.

[0055] ⑤ Wash beads & wash off protein:

[0056] Centrifuge to remove supernatant, wash Ni NTA beads 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.

[0057] ⑥ Concentrated & Dialyzed Protein

[0058] 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.

[0059] ⑦ Protein storage

[0060] After dialysis, the protein is quantified by Coomassie brilliant blue staining, then 10-20% glycerol is added, followed by flash freezing in liquid nitrogen and storage at -80°C.

[0061] (3) Protein purification reagent formulation

[0062] Lysis buffer: Tris-HCl: 100 mM, NaCl: 300 mM, NP-40: 0.05%, pH=8.0;

[0063] Washing buffer: Tris-HCl: 100 mM, NaCl: 300 mM, Imidazole: 20-50 mM, NP-40: 0.05%, pH=8.0;

[0064] Elution buffer: Tris-HCl: 100 mM, NaCl: 300 mM, Imidazole: 250 mM, NP-40: 0.05%, pH=8.0;

[0065] Protein purification diagram as shown Figure 1 As shown.

[0066] 2. Animal immunization

[0067] Purified CopGFP 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. All injections were thoroughly mixed with an equal volume of antigen. 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 injections.

[0068] The immune cycle is shown in Table 1;

[0069] Table 1

[0070]

[0071] 3. Antiserum detection

[0072] (1) A small amount of blood was taken from the tail vein of a mouse to prepare antiserum.

[0073] (2) ELISA method to detect antiserum titer.

[0074] 4. Cell fusion and subcloning

[0075] (1) Preparation of myeloma cells

[0076] One week before fusion, SP2 / 0 cells were revived and cultured normally to the logarithmic growth phase.

[0077] (2) Spleen cell preparation

[0078] 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.

[0079] (3) Cell fusion

[0080] 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.

[0081] 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.

[0082] (4) Fusion screening

[0083] 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.

[0084] (5) Subcloning

[0085] 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.

[0086] 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.

[0087] 5. Ascites preparation and antibody purification

[0088] (1) Preparation of ascites

[0089] 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.

[0090] (2) Purification of ascites

[0091] The ascites fluid from the above cells was purified, and the purity of the antibody after purification was greater than 90%.

[0092] (3) Purification by ammonium octanoate sulfate + DEAE ion column method

[0093] (4) Centrifuge the ascites fluid, aspirate the pale yellow liquid and calculate the volume. Dilute it 1:3 with 4 times the volume of 60 mM acetate buffer (pH 4.0), add caprylic acid dropwise (final concentration of 25 μL / mL to dilute the ascites fluid), stir at room temperature for 30 min, and then let it stand at 4 ℃ for more than 2 h to allow it to precipitate fully.

[0094] (5) At 10,000 r / min, 4 °C, for 20 min, collect the supernatant and add 1 / 10 volume of 10×PBS (0.1 M, 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 mL of the above mixture and let it stand for at least 60 min.

[0095] (6) 10000 r / min, 4 ℃, 20 min, discard the supernatant, dissolve the precipitate in a small amount of PBS. Dialyze to PBS overnight at 4 ℃.

[0096] (7) Detection of antibody concentration and purity. The antibody concentration was measured to be 1.5 mg / mL. The purity of the purified antibody was detected 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.

[0097] 6. Antibody light and heavy chain sequencing

[0098] (1) Culture of hybridoma cells

[0099] 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 min and collect the cells.

[0100] (2) Extracting cellular RNA

[0101] Under a clean bench environment, add 1 mL of Trizol reagent to the centrifuged cells, let stand for 5 min, add 200 μL of chloroform, shake vigorously for 15 sec, let stand at room temperature for 5 min, centrifuge at 12000 rpm for 15 min, aspirate the upper aqueous layer 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. Store at -80℃ for later use.

[0102] (3) Preparation of cDNA by reverse transcription

[0103] 1 μL total cellular RNA, 6 μL RNase-free ddH2O, 0.5 μL oligo dT Primer, 0.5 μL PrimeScript RT Enzyme Mix I, and 2 μL 5×Prime Script Buffer were mixed and incubated at 37 °C for 15 min and 85 °C for 5 s.

[0104] (4) Amplify cDNA

[0105] Mouse IgG V H V LPrimer 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 for denaturation, 63 ℃ for 20 s for annealing, 72 ℃ for 25 s for extension, 40 cycles, followed by a final extension at 72 ℃ for 5 min.

[0106] (5) Agarose gel electrophoresis and gel recovery

[0107] The PCR products were subjected to agarose gel electrophoresis. The electrophoresis results were observed. The amplification products with molecular weights of 700-800bp and 1400-1600bp were sent for sequencing.

[0108] Table 2 Amino acid sequence information

[0109]

[0110] The amino acid sequence of the light chain variable region is as follows:

[0111] MDFQVQIFSFFLLMSASVIMSRGQIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIK (SEQ ID NO: 15).

[0112] The amino acid sequence of the heavy chain variable region is as follows:

[0113] MAVLALLLCLVAFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLNDYGVHWVRQPPGKGLEWLGLIWAGGSTNYNLALMSRLSISKDNKSQVFLKMNSLQTDDTAMYYCARGGYGSIYYFDYWGQGTTLTVSS (SEQ ID NO: 16).

[0114] Example 2: Antibody titer detection

[0115] The antibody titer was determined using an ELISA assay. 50 ng of antigen (prokaryotically purified CopGFP 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;

[0116] Table 3

[0117]

[0118] Example 3: Antibody Specificity Detection

[0119] 1. Antibody specificity was detected using in vitro purified CopGFP protein.

[0120] His-tagged CopGFP protein was purified in vitro in prokaryotes, and the recognition specificity of the purified antibody was detected by Western blotting. Figure 3 As shown, the left and right figures respectively show the specificity of anti-CopGFP polyclonal and monoclonal antibodies detected by Western blotting experiments. The results indicate that the anti-CopGFP monoclonal antibody has good recognition specificity for CopGFP.

[0121] 2. Detecting antibody specificity at the cellular level

[0122] Constructing in human U2OS cell lines CopGFP Cell lines with stable gene expression, and the specificity of monoclonal antibodies was identified by Western blotting, such as... Figure 4 As shown in the embodiments of the present invention. CopGFP The method for constructing cell lines with gene overexpression is as follows: CopGFP The gene sequence was cloned into the PB511B-1 vector (the restriction enzyme sites are...). EcoR I and BamH I The cells were co-transfected with the PB210PA-1 plasmid encoding the transposase, and stable expression was obtained after puromycin selection. CopGFP gene The cell line. From Figure 4 It is known that monoclonal hCOPGFP antibody can specifically detect CopGFP protein expression.

[0123] 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 CopGFP, characterized in that: The monoclonal antibody recognizes the CopGFP protein and 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 CDR-L1 is shown in SEQ ID NO:1, the amino acid sequence of CDR-L2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

3. The heavy chain variable region has three complementary determinant regions, wherein the amino acid sequence of CDR-H1 is shown in SEQ ID NO:8, the amino acid sequence of CDR-H2 is shown in SEQ ID NO:9, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO:

10.

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

16.

3. The monoclonal antibody against CopGFP according to claim 1, characterized in that: The amino acid sequences of the light chain's structural sequence FR-L1 are shown in SEQ ID NO:4, FR-L2 in SEQ ID NO:5, FR-L3 in SEQ ID NO:6, and FR-L4 in SEQ ID NO:7; the amino acid sequences of the heavy chain's structural sequence FR-H1 are shown in SEQ ID NO:11, FR-H2 in SEQ ID NO:12, FR-H3 in SEQ ID NO:13, and FR-H4 in SEQ ID NO:

14.

4. A nucleic acid molecule encoding the monoclonal antibody against CopGFP according to any one of claims 1 to 3, characterized in that: The nucleic acid molecules include nucleic acid molecules encoding the heavy chain variable region and nucleic acid molecules encoding the light chain variable region.

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

6. An engineered bacterium or eukaryotic host cell comprising the expression vector of claim 5.

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

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