CN117510629A

CN117510629A - Recombinant antibody for resisting beta-actin protein and application thereof

Info

Publication number: CN117510629A
Application number: CN202311568292.4A
Authority: CN
Inventors: 潘炜松; 贾箫琪; 吴川; 陈刚; 徐超
Original assignee: Hunan Nuohe New Biotechnology Co ltd
Current assignee: Hunan Nuohe New Biotechnology Co ltd
Priority date: 2023-11-23
Filing date: 2023-11-23
Publication date: 2024-02-06

Abstract

The invention relates to a recombinant antibody of an anti-beta-actin protein and application thereof, wherein the antibody, a heavy chain CDR1, comprises an amino acid sequence shown in SEQ ID NO. 1; a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2; a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO. 3; a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO. 4; a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO. 5; the light chain CDR3 comprises a sequence shown as SEQ ID NO. 6, the affinity of the antibody provided by the invention with beta-actin protein is high and reaches 9.219E-8, the activity of the purified anti-beta-actin monoclonal antibody is high and the detection sensitivity is high, in particular to the antibody with heavy chain sequence shown as SEQ ID NO. 9 and light chain sequence shown as SEQ ID NO. 10.

Description

Recombinant antibody for resisting beta-actin protein and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant antibody for resisting beta-actin protein and application thereof.

Background

Immunoblotting (WB) is one of the most common methods in molecular biology to detect the presence of specific proteins in complex biological extracts. WB must control the Loading to ensure comparability of the target protein changes, and usually a relatively constant amount of protein is selected as an internal reference (Loading control) that is commonly distributed in all tissues, because the change in the target protein expression is only credible if the expression level of the internal reference is the same among wells, and the internal reference antibody is an antibody capable of specifically recognizing the internal reference protein, which is used to detect the internal reference protein, and is often used to correct experimental errors in the protein quantification process and verify whether the steps of protein transfer, color development, etc. are normal. When a scientific research person selects an reference antibody, the reference antibody is mainly selected according to own needs, particularly the needs of experimental application, and the titer of the reference antibody is often an important reference factor, even a decisive factor.

The internal reference beta-actin, namely beta actin, consists of 375 amino acids, has high conservation and relatively stable expression in cells, and is widely used as an internal reference in experiments such as real-time quantitative PCR, western blotting and the like, so that the recombinant antibody of the anti-beta-actin protein is also an internal reference antibody widely applied. The existing commercially available recombinant antibodies against the beta-actin protein have few varieties and low detection sensitivity, so that the recombinant antibodies against the beta-actin protein with high antibody titer and high detection sensitivity are searched, the change of the specific protein expression quantity in complex organisms can be detected more accurately, and the accuracy of experiments such as real-time quantitative PCR and Western blotting can be improved.

Disclosure of Invention

Aiming at the defects or improvement demands of the prior art, the invention provides a recombinant antibody of an anti-beta-actin protein and application thereof, and aims to stimulate mice by taking the recombinant beta-actin protein as an antigen, find a recombinant antibody of the anti-beta-actin protein with high titer and sensitive detection in the bodies, clone and sequence the variable region genes of the antibody to obtain the amino acid sequences of the heavy chain and the light chain of the antibody, and prepare the antibody of the recombinant monoclonal beta-actin protein based on the amino acid sequences, thereby solving the technical problems of few recombinant antibody types of the existing anti-beta-actin protein and insufficient detection sensitivity of the commercially available beta-actin antibody.

To achieve the above object, according to one aspect of the present invention, there is provided a recombinant antibody against β -actin protein, comprising complementarity determining regions of 3 heavy chains: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and complementarity determining regions of 3 light chains: light chain CDR1, light chain CDR2, and light chain CDR3;

the heavy chain CDR1 comprises an amino acid sequence A-Y-F-M-N shown in SEQ ID NO. 1;

the heavy chain CDR2 comprises an amino acid sequence R-I-N-P-Y-N-A-Y-N-F-Y-N-Q-K-F-K-G shown in SEQ ID NO. 2;

the heavy chain CDR3 comprises an amino acid sequence E-G-E-Y-S-G-T-F-P-Y-G-M-D-Y shown in SEQ ID NO 3;

the light chain CDR1 comprises an amino acid sequence K-A-S-Q-N-A-G-N-I-I-A shown in SEQ ID NO. 4;

the light chain CDR2 comprises an amino acid sequence L-A-S-Y-R-Y-S shown in SEQ ID NO. 5

The light chain CDR3 comprises a sequence Q-Q-Y-K-N-S-P-Y-T shown in SEQ ID NO. 6.

Preferably, the recombinant antibody of the anti-beta-actin protein, the heavy chain variable region of the antibody comprises any amino acid sequence with more than 90 percent of identity with the amino acid sequence shown in SEQ ID NO. 7;

or one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 7.

Preferably, the recombinant antibody against β -actin protein, the heavy chain variable region of which antibody comprises any one of the amino acid sequences having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the amino acid sequence shown in SEQ ID No. 7, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID No. 7.

Preferably, the recombinant antibody of the anti-beta-actin protein, the light chain variable region of the antibody comprises any amino acid sequence with more than 90 percent of identity with the amino acid sequence shown in SEQ ID NO. 8; or one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 8.

Preferably, the recombinant antibody against β -actin protein, the antibody, the light chain variable region thereof, comprises any one of an amino acid sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the amino acid sequence shown in SEQ ID No. 8, or an amino acid mutation having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids compared to the amino acid sequence shown in SEQ ID No. 8.

Preferably, the recombinant antibody against the β -actin protein, the amino acid mutation of which is a conservative mutation, comprises a substitution, insertion or deletion of the amino acid sequence.

Preferably, the recombinant antibody of the beta-actin protein has the heavy chain amino acid sequence shown as NO. 9 and the light chain amino acid sequence shown as NO. 10.

According to another aspect of the present invention there is also provided a biological material comprising recombinant antibodies encoding an anti-beta-actin protein according to the present invention, the biological material comprising an expression vector, an expression cassette, a host cell, an engineering bacterium or a hybridoma cell line.

According to another aspect of the invention there is also provided the use of a recombinant antibody against a β -actin protein according to the invention in the preparation of an anti- β -actin antibody fusion protein detection reagent.

According to another aspect of the present invention there is also provided a test kit comprising a recombinant antibody against a β -actin protein according to the present invention.

In general, the above technical solutions conceived by the present invention, compared with the prior art, enable the following beneficial effects to be obtained:

the affinity of the recombinant antibody against the beta-actin protein reaches 9.219E-8, and the purified anti-beta-actin monoclonal antibody has high activity and high detection sensitivity, thereby being beneficial to improving the accuracy of experiments such as real-time quantitative PCR, western blotting and the like.

Drawings

FIG. 1 is an amino acid sequence of heavy and light chains of the recombinant antibodies prepared in example 3;

FIG. 2 is a graph of Western blotting results; wherein A is the anti-beta-actin recombinant antibody provided by the invention, and B is the commercial beta-actin (murine monoclonal antibody); 1. 2 and 3 are the antigen diluted to 4, 2 and 1ng respectively.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

Terminology and definition:

the following terms used in this application have the following meanings, unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art.

The term "amino acid" refers to a naturally occurring or non-naturally occurring carboxy alpha-amino acid. The term "amino acid" as used herein may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V), threonine (Thr, T). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocysteine, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

In the present invention, peptides, polypeptides, proteins are not strictly distinguished and may in some cases be used interchangeably, generally referring to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic. The polypeptide may also comprise non-amino acid components such as carbohydrate groups, metal ions or carboxylic acid esters. The non-amino acid component may be added by the cell expressing the polypeptide and may vary with cell type. A polypeptide is defined in the present invention with respect to its amino acid backbone structure or the nucleic acid encoding it. Such as the addition of carbohydrate groups, is not generally specified, but may be present. All polypeptide sequences are written according to commonly accepted practices, with the α -N-terminal amino acid residue on the left and the α -C-terminal amino acid residue on the right. The term "N-terminus" as used herein refers to the free alpha-amino group of an amino acid in a polypeptide, and the term "C-terminus" refers to the free alpha-carboxylic acid end of an amino acid in a polypeptide. A polypeptide ending with a group at the N-terminus refers to a polypeptide that carries a group on the alpha-amino nitrogen of the N-terminal amino acid residue. An amino acid ending with a group at the N-terminus refers to an amino acid bearing a group on the alpha-amino nitrogen.

Antibodies, which are a functional protein, are also called immunoglobulins (Ig), and can be simply distinguished as albumin (albumin) and globulin (globulin). The whole antibody is composed of four polypeptide chains, namely two light chains (L chain) with the same structure and two heavy chains (H chain) with the same structure, and the four chains form a typical Y-shaped antibody structure. Amino acid sequence studies have shown that the structure of about 3/4 of the C-terminal end of the light chain and the C-terminal end of the heavy chain of antibodies (exemplified by IgG) are highly conserved, known as the conserved region (Constant region) and the N-terminal end of the corresponding light chain and heavy chain have extremely high amino acid sequence diversity, known as the variable region (variable region); relatively short sequences are distributed within these variable regions, exhibiting extremely strong diversity, known as high frequency mutation regions, three hypervariable regions within the variable regions of the light and heavy chains, respectively, and subsequent studies have demonstrated that the hypervariable regions within the heavy and light chain antibody regions constitute the antigen binding sites of antibody molecules, which are also known as complementarity-determining region (CDRs) of antibody molecules because they are complementary to epitope structures. However, antigens have many epitopes, one antibody can specifically bind to one epitope, however the affinities between different antibodies and epitopes differ.

The invention stimulates mice through beta-actin antigen, and discovers a beta-actin antibody with high titer in the mice, and the titer of ascites antibody can reach 9.13 multiplied by 10 ⁶ . Specifically, recombinant beta-actin protein is used as beta-actin antigen, BALB/c mice are immunized for multiple times, a large number of hybridoma cell strains are prepared, positive cells of secreted antibodies are screened and cloned, five stable beta-actin resistant hybridoma cell strains are obtained finally, ascites are prepared, monoclonal antibodies are purified and obtained, one hybridoma cell 5G9 with higher secreted antibody titer is screened out through antibody titer determination, and experimental results show that the titer of ascites antibodies secreted by the hybridoma cell 5G9 can reach 9.13 multiplied by 10 ⁶ Is significantly higher than other 4 hybridoma cell strains; the amino acid sequences of the heavy chain and the light chain of the antibody are obtained through gene cloning and sequencing of the variable region of the antibody, and the complementarity determining regions of the heavy chain are analyzed: CDR1: A-Y-F-M-N; CDR2: R-I-N-P-Y-N-A-Y-N-F-Y-N-Q-K-F-K-G; CDR3: E-G-E-Y-S-G-T-F-P-Y-G-M-D-Y; complementarity determining regions of the light chain: CDR1: K-A-S-Q-N-A-G-N-I-I-A; CDR2: L-A-S-Y-R-Y-S CDR3: Q-Q-Y-K-N-S-P-Y-T; recombinant antibodies were prepared according to the amino acid sequences of the heavy and light chains of the antibodies obtained.

Based on this finding, the present invention provides a recombinant antibody against β -actin protein, comprising a complementarity determining region comprising 3 heavy chains: heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and complementarity determining regions comprising 3 light chains: light chain CDR1, light chain CDR2, light chain CDR3;

The light chain CDR3 comprises the sequence Q-Q-Y-K-N-S-P-Y-T shown in SEQ ID NO. 6.

Further, the antibody, the heavy chain variable region of which comprises any one of the amino acid sequences having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 7, or having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 7;

preferably a sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% sequence identity, or having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations; the amino acid mutations, preferably conservative mutations, include substitutions, insertions or deletions of the amino acid sequence.

The antibody, the light chain variable region of which comprises any one amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 8, or has one or more amino acid mutations compared with the amino acid sequence shown in SEQ ID NO. 8;

In some embodiments, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO. 9 and the light chain amino acid sequence is shown in SEQ ID NO. 10.

Proteins are composed of amino acids, of which 20 are used, which, although changing the type of the individual amino acids in the protein, do not affect the function of the protein, e.g.leucine at position 17 of the cytochrome C peptide chain of yeast, while wheat is isoleucine, which is changed but the cytochrome C can be unaffected. The amino acids of the active site of the protein are mainly affected by the functions of the protein, the amino acids of other sites are not greatly affected by the change, or the changed amino acids are similar to the original amino acids (such as hydrophobic) in nature and also have little effect on the functions of the protein, and the amino acid sequences obtained by carrying out individual amino acid mutation on the premise of maintaining the functions of the antibody according to the amino acid mutation principle still belong to the protection scope of the invention.

The invention also provides a biological material containing a nucleic acid sequence encoding a recombinant antibody or fragment thereof against a beta-actin protein, the biological material comprising an expression vector, an expression cassette, a host cell, an engineering bacterium or a hybridoma cell strain. Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence, "operably linked" refers to a coding sequence that is linked to the regulatory sequence in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct expression of the protein of interest in a suitable host cell, and include promoters, enhancers and other expression control elements.

In addition, the invention also provides application of the recombinant antibody of the anti-beta-actin protein in preparing an anti-beta-actin antibody fusion protein detection reagent.

In another aspect, the invention also provides a detection kit comprising a recombinant antibody against a β -actin protein according to the invention.

The following are examples

EXAMPLE 1 preparation of recombinant beta-actin protein fragments

1. Antigen fragment selection

According to the coding gene sequence (NCBI ReferenceSequence:NM_ 001101.4) of the beta-actin protein in NCBI, the genes for expressing the beta-actin recombinant protein, which are respectively provided with NdeI enzyme cutting sites and XhoI enzyme cutting sites at the 5 'end and the 3' end, are artificially synthesized, the DNA sequence of the genes is shown as SEQ ID NO. 11, and the coded amino acid sequence is shown as SEQ ID NO. 12. The NdeI and XhoI double enzyme digestion is utilized to obtain an digested beta-actin DNA fragment and a pET-21a (+) DNA fragment, the beta-actin DNA fragment is connected to a pET-21a (+) expression vector by using T4 ligase, and the correct pET-21a (+) -beta-actin expression vector is finally obtained through gene sequence determination, and the pET-21a (+) vector is provided with a 6 XHis tag, so that the purification can be performed by using a Ni column.

2. Expression and purification of recombinant beta-actin protein fragments

100ul of competent cells stored at-80℃were removed, and after thawing, 5ul of plasmid was added, and after ice bath for 30min, heat shock at 42℃for 90 seconds, ice bath for 5min, 800. Mu.L of non-resistant LB medium was added. Resuscitates at 37℃and 200rpm for 60min, plating and overnight incubation. From the transformed plate, the single clone was picked into 5ml of LB liquid medium, cultured at 37℃and 200rpm overnight, and then maintained. 5ul of bacterial liquid is taken into 5ml of LB liquid culture medium, and the culture is carried out overnight for resuscitation. The cultured bacterial liquid is transferred to 500ml LB liquid culture medium for mixing, the temperature is 37 ℃, the rpm is 200, and the bacterial liquid is cultured until the bacterial liquid reaches OD ₆₀₀ =0.5-0.8, induction was performed at 37 ℃ for 4h with addition of IPTG (0.5 mM), 120rpm. The cells were collected by centrifugation at 5000g for 10min with ice bath for 10min, and the supernatant was discarded. The precipitate was blown off with 20-30ml of 50mM Tris-HCl (pH 8.0) (containing 0.5M sodium chloride), sonicated to remove cells, centrifuged at 12000rpm for 20 minutes, the supernatant was collected and passed through a 0.22mM filter, and the sample was applied to a Ni-Ni column for purification, followed by elution with 50mM Tris-HCl (pH 8.0) (containing 0.5M sodium chloride) containing 50mM imidazole, 250mM imidazole, 500mM imidazole, respectively, to obtain beta-actin protein.

Example 2 screening of antibodies to high titers of beta-actin proteins

1. Antigen immunization

The beta-actin protein obtained in example 1 was diluted to 1mg/mL and emulsified by mixing with an equal volume of Freund's complete adjuvant (appearance: amber cell suspension; composition: paraffin Oil 85%, mannide Monooleate%, mycobacterium smegmatis mg/mL). The emulsion is subcutaneously injected into BALB/c mice at a dosage of 0.15mL, the immunity is enhanced in the abdominal cavity (equal amount of antigen is mixed with Freund's incomplete adjuvant) after 14 days of the first immunization, and after the immunity is enhanced to four needles, tail blood is collected for potency detection, and the potency reaches the fusion requirement. 3 days before fusion, the same dose of antigen is used for intraperitoneal injection to boost immunity, and the immunization method is the same.

2. Preparation of hybridoma cell lines

(1) Preparation of feeder cells

Feeding fine with BALB/c mouse abdominal macrophagesAnd (5) cells. 1 day before fusion, BALB/c mice were sacrificed by pulling the neck, immersed in 75% alcohol, aseptically manipulated in a super clean bench to shear off the abdominal skin, expose the peritoneum, and injected with 5mL of RPMI 1640 basal medium by syringe and repeatedly rinsed. The washing solution was recovered, centrifuged at 1000rpm for 5 minutes, and the pellet was resuspended in RPMI 1640-selective medium (HAT-containing RPMI 1640 complete medium). Cell concentration was adjusted to 1X 10 ⁵ mu.L/well of 96-well plate, 150. Mu.L/well, 37℃and 5% CO were added per mL ₂ Culturing overnight.

(2) Preparation of immune spleen cells

Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a plate, rinsed once with RPMI 1640 basal culture solution, placed on a nylon mesh of a small beaker, and ground and filtered to prepare a cell suspension. Centrifuging, discarding supernatant, re-suspending RPMI 1640 basic culture solution, repeating for three times to obtain immune spleen cells, and counting.

(3) Preparation of myeloma cells

After 8-azaguanine screening, the mouse myeloma cells Sp2/0 are cultured to the logarithmic phase, two large bottles are taken to prepare cell suspension, the cell suspension is centrifuged, the supernatant is discarded, the cell suspension is resuspended by using RPMI 1640 basic culture solution, and myeloma cells are obtained by repeating the steps for three times if needed, and the number of the myeloma cells is counted.

(4) Cell fusion and HAT selection hybridomas

Myeloma cells and immune splenocytes were mixed at a ratio of 1:10, washed 1 time with RPMI 1640 basal medium in a 50mL plastic centrifuge tube, and centrifuged at 1200rpm for 10 minutes. The supernatant was discarded, the cells were mixed well, 1mL of 50% PEG 1500 was slowly added for fusion, and after 1 minute of fusion, 15mL of RPMI 1640 basal medium was added to terminate the cell fusion. Centrifuge at 1000rpm for 5-10 minutes. The supernatant was discarded, and the culture broth was gently resuspended in 50mL of RPMI 1640, halved in 10 96-well plates, 50. Mu.L/well, 37℃and 5% CO ₂ Culturing. Culturing was carried out until the sixth day, and the HAT medium (the complete medium of RPMI 1640 containing HAT) was changed twice.

(5) Hybridoma cell screening

a. Antigen coating beta-actin antigen was diluted to a final concentration of 1. Mu.g/mL with 50mM carbonate buffer, pH 9.6. 100 μl/well was added to 96-well plates at 4deg.C overnight.

b. Closing: 0.02M pH 7.2PBS,0.15mL/well with 1% nonfat milk powder, blocked for 2 hours at 37℃for detection.

c. Sample adding: on the seventh day after fusion, 0.1mL of the cell supernatant was taken in the 96-well assay plate at 37℃for 30 minutes.

d. Washing: washing with water for six times.

e. Adding enzyme-labeled anti-antibody, namely adding 2000 times of horseradish peroxidase-labeled goat anti-mouse IgG, and 30 minutes at 37 ℃.

f. Washing: washing with water for six times

g. Color development: 100. Mu.L of a phosphate buffer containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH 5.0 and citric acid was added to each well at 37℃for 15 minutes.

h. Terminating the reaction: adding dilute sulfuric acid solution, 50 mu L per well

i. The absorbance at 450nm was read by the microplate reader. RPMI 1640 complete culture solution was used as a negative control, and positive cells were obtained at a ratio of the measured value to the control value of ∈ 2.0.

The positive cell holes of the secreted antibody are cloned by a limiting dilution method on a 96-well culture plate with 1 cell/well, the positive holes are screened and cloned three times continuously according to the upper method, after the expansion culture, the positive cells are frozen in a culture solution containing 10% DMSO, and the cell density is 10 ⁶ And each mL. Five stable anti-beta-actin hybridoma cell strains are obtained, and are respectively named as A, B, C, D, E.

3. Preparation of monoclonal antibodies

Selecting BALB/c mice with 6-8 weeks of robustness, and injecting 0.5mL of pristane into the abdominal cavity of each mouse; intraperitoneal injection 1X 10 after 10 days ⁶ And a hybridoma cell. Ascites can be generated 7-10 days after inoculating cells, the health condition and the symptoms of the ascites of animals are closely observed, the mice are killed before death as much as possible, the ascites is sucked into a test tube by a dropper, and generally 5-10 mL of ascites can be obtained by one mouse. Collecting ascites, centrifuging to obtain supernatant, and storing in a refrigerator at-20deg.C. The ascites supernatant was diluted with 3 volumes of PBS and filtered through filter paper. The resulting filtrate was applied to a protein G affinity chromatography column equilibrated with PBS at a flow rate of 1 mL/min. Then washed with PBS at a flow rate of 1mL/min without being absorbed by protein GAttached substances up to OD ₂₈₀ The absorbance at nm reaches baseline. The antibody was eluted with a 0.1M glycine eluent (pH 2.5) and recovered. The recovered solution was neutralized with 0.1M Tris (pH 8.8), and the antibody concentration was adjusted to an appropriate concentration by ultrafiltration, and sub-packaged and frozen at-20 ℃.

4. Antibody titer determination

The titers of the 5 hybridoma cells and the secreted ascites antibodies were detected by an indirect ELISA method. The specific experimental steps are as follows:

(1) Antigen coating: diluting the beta-actin antigen to 1 mug/mL, 100 mug/Kong Jiazhi in a 96-well reaction plate, and overnight at 4 ℃;

(2) Washing: washing the plate with washing liquid for 5 times, and finally drying;

(3) Closing: adding 150 mu L/hole sealing liquid, and covering a 37 ℃ incubator for incubation for 1.5-2 hours;

(4) Washing: washing the plate with washing liquid for 5 times, and finally drying;

(5) Sample adding: adding cell culture supernatant and ascites diluted into different gradients into a coated 96-well reaction plate, respectively, 100 mu L/well, and covering a 37 ℃ incubator for 1 hour (simultaneously making a negative control well and a positive control well);

(6) Washing: washing the plate with washing liquid for 5 times, and finally drying;

(7) Adding enzyme-labeled anti-antibody: adding horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (diluted 6000 times with blocking solution), 100 μl/hole, and incubating in a 37 ℃ incubator for 1 hr;

(8) Washing: washing the plate with washing liquid for 5 times, and finally drying;

(9) Color development: preparing the color development liquid TMB at 100 mu L/hole, and performing light-shielding reaction at 37 ℃ for 30 minutes;

(10) Terminating the reaction: 2M sulfuric acid, 50. Mu.L/well, was added to each reaction well;

(11) Microplate reader reading: measured with 450nm and 630nm wavelengths. Antibody titer results are shown in table 1 below.

TABLE 1 determination of different antibody titers

Beta-actin cell strain	Hybridoma cell culture supernatant titers	Ascites antibody titre
			6E3	5.27×10 ³	6.57×10 ⁵
3C10	4.78×10 ⁴	4.24×10 ⁶
			2D3	4.29×10 ³	5.52×10 ⁵
5G9	8.54×10 ⁴	9.13×10 ⁶
			4C7	3.23×10 ⁴	3.79×10 ⁶

As shown in Table 1, the antibody to beta-actin secreted by hybridoma cell line 5G9 has a potency of 9.13X10 ⁶ Significantly higher than the antibodies secreted by the other 4 hybridoma cell lines.

EXAMPLE 3 preparation of recombinant antibodies

1. Antibody variable region gene cloning and sequencing

Total RNA was extracted from hybridoma cell line 5G9 secreting anti-beta-actin monoclonal antibody of example 2, and first strand cDNA synthesis was performed using SMARTERTM RACE cDNA Amplification Kit expression cassette and SMARTER II A oligo nucleotide and 5' -CDS primer in the expression cassette, and the obtained first strand cDNA product was used as a PCR amplification template. The light chain genes were amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP) and mIgG CKR primers and the heavy chain genes were amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP) and mIgG CHR primers. Purifying and recovering by agarose gel electrophoresis, adding A reaction of the product by rTaq DNA polymerase, inserting into pMD-18T vector, transforming into DH5 alpha competent cells, taking 4 clones of heavy chain and light chain gene clone respectively after bacterial colony growth, and sequencing by the company of the family Praeparatae.

The heavy and light chain sequences of the antibody obtained by the hybridoma cell line 5G9 are shown in FIG. 1, and the complementarity determining regions of the heavy chain are analyzed:

CDR1：A-Y-F-M-N；

CDR2：R-I-N-P-Y-N-A-Y-N-F-Y-N-Q-K-F-K-G；

CDR3：E-G-E-Y-S-G-T-F-P-Y-G-M-D-Y。

complementarity determining regions of the light chain:

CDR1：K-A-S-Q-N-A-G-N-I-I-A；

CDR2：L-A-S-Y-R-Y-S；

CDR3：Q-Q-Y-K-N-S-P-Y-T。

2. preparation of recombinant antibodies for affinity analysis and Activity characterization

(1) Affinity analysis

Enzyme-free indirect method was used as data in the same manner as for activity identification, and the coating was used as four gradients of 1. Mu.g/ml, 0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.125. Mu.g/ml; antibodies were loaded from a 2-fold gradient dilution of 1000ng/ml to 0.97656 ng/ml. The OD values corresponding to the different antibody concentrations at the coating-free concentration were obtained. Under the same coating concentration, the antibody concentration is plotted with the antibody concentration as an abscissa and the OD value as an ordinate, and the antibody concentration at the maximum OD value of 50% is calculated according to a fitting equation; the formula is introduced: k= (n-1)/(2 x (n x Ab '-Ab)), where Ab and Ab' represent the antibody concentration at 50% of the maximum OD value at the corresponding coating concentration (Ag, ag '), respectively, n = Ag/Ag'; every two coating concentrations can be combined to calculate a K value, and finally six K values can be obtained, the average value is obtained, and the reciprocal is obtained to be the affinity constant KD. The affinity analysis data of the purified anti-beta-actin monoclonal antibody are shown in the table 2 below.

TABLE 2 affinity assay results for purified beta-actin monoclonal antibodies

Sample name	KD
		5G9	9.219E-8
3C10	4.145E-7
		2D3	9.289E-6
6E3	5.118E-7
		4C7	7.834E-7

As shown in Table 2, the affinity of the purified beta-actin monoclonal antibody 5G9 is obviously better than that of the other 4 hybridoma cells.

(2) Activity assay

a. Coating: diluting the beta-actin antigen to 1 mug/mL with 50mM carbonate buffer coating solution for coating the microwell plate, wherein each well is 100 mug, and the temperature is 4 ℃ overnight;

b. washing: washing the washing solution with PBST for 2 times, and drying;

c. closing: blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking;

d. sample adding: adding diluted anti-beta-actin monoclonal antibody 5G9, carrying out 5-fold specific dilution from 1000ng/ml, loading sample, 100 mu L/hole, and carrying out 37 ℃ for 30min (part of supernatant 1 h);

e. washing: washing with PBST washing solution for 5 times, and drying;

f. adding enzyme-labeled anti-antibody: adding horseradish peroxidase-labeled goat anti-mouse IgG (immunoglobulin G) into each hole at 100 mu L and 37 ℃ for 30min;

g. washing: washing with PBST washing solution for 5 times, and drying;

h. color development: urea peroxide (50 μl/well) was added, and tetramethylbenzidine (50 μl/well) was added for 10min;

i. and (3) terminating: adding dilute hydrochloric acid to terminate the reaction, wherein the concentration is 50 mu L/hole;

microplate reader reading: OD was read on the microplate reader at 450nm (reference 630 nm). The purified anti-beta-actin monoclonal antibody activity is identified, and the result is shown in the table 3 below.

TABLE 3 identification of beta-actin monoclonal antibody Activity after purification

Sample concentration ng/ml	1000	200	40	8	1.6	0.32	0
								Beta-actin antibody 5G9	2.924	2.086	0.816	0.168	0.122	0.084	0.024

EXAMPLE 4 Western blotting validation of recombinant antibodies

Western blotting verification is synchronously carried out by using the anti-beta-actin protein recombinant antibody prepared by the invention and a commercial anti-beta-actin protein monoclonal antibody (A5316). The antigen was diluted to 4, 2, 1ng and incubated with β -actin antibody at a concentration of 0.1 μg/mL. Firstly, protein electrophoresis is carried out, electrotransfer is carried out after the electrophoresis is finished, and the beta-actin recombinant antibody prepared by the invention and a commercial anti-beta-actin protein monoclonal antibody (A5316) are respectively used as primary antibodies, and sheep anti-mouse IgG is used as secondary antibodies, so that the immunoblotting analysis result is shown in figure 2.

In FIG. 2, A is the recombinant antibody against beta-actin provided by the present invention, B is commercially available beta-actin (murine monoclonal antibodies; 1, 2, 3 are antigen diluted to 4, 2, 1ng, respectively).

As shown in FIG. 2, compared with the commercial beta-actin antibody, the beta-actin recombinant antibody provided by the invention has higher detection sensitivity, and has obvious reaction bands when the antigen is diluted to 1ng, which indicates that the anti-beta-actin protein recombinant antibody prepared by the invention has higher binding efficiency and higher affinity with the antigen.

The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.

Claims

1. A recombinant antibody against β -actin protein, comprising 3 heavy chain complementarity determining regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and complementarity determining regions of 3 light chains: light chain CDR1, light chain CDR2, and light chain CDR3;

the heavy chain CDR1 comprises an amino acid sequence A-Y-F-M-N shown in SEQ ID NO. 1; the heavy chain CDR2 comprises an amino acid sequence R-I-N-P-Y-N-A-Y-N-F-Y-N-Q-K-F-K-G shown in SEQ ID NO 2; the heavy chain CDR3 comprises an amino acid sequence E-G-E-Y-S-G-T-F-P-Y-G-M-D-Y shown in SEQ ID NO 3;

the light chain CDR1 comprises an amino acid sequence K-A-S-Q-N-A-G-N-I-I-A shown in SEQ ID NO. 4; the light chain CDR2 comprises an amino acid sequence L-A-S-Y-R-Y-S shown in SEQ ID NO. 5 The light chain CDR3 comprises the sequence Q-Q-Y-K-N-S-P-Y-T shown in SEQ ID NO. 6.

2. The recombinant antibody against β -actin protein according to claim 1, wherein the heavy chain variable region of the antibody comprises the amino acid sequence:

including any amino acid sequence having more than 90% identity to the amino acid sequence shown in SEQ ID NO. 7, or having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 7.

3. The recombinant antibody against β -actin protein according to claim 2, wherein the heavy chain variable region of the antibody comprises any one of an amino acid sequence having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the amino acid sequence shown in SEQ ID No. 7, or an amino acid mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compared to the amino acid sequence shown in SEQ ID No. 7.

4. The recombinant antibody against β -actin protein according to claim 1, wherein the antibody, the light chain variable region, comprises the amino acid sequence:

including any amino acid sequence having more than 90% identity to the amino acid sequence shown in SEQ ID NO. 8, or having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 8.

5. The recombinant antibody against β -actin according to claim 4, wherein the antibody light chain variable region comprises any one of the amino acid sequences having at least 91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identity to the amino acid sequence shown in SEQ ID No. 8, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the amino acid sequence shown in SEQ ID No. 8.

6. The recombinant antibody against β -actin protein according to any one of claims 2 to 5, wherein the amino acid mutation is a conservative mutation, including a substitution, insertion or deletion of the amino acid sequence.

7. The recombinant antibody against β -actin according to claim 1, wherein the heavy chain amino acid sequence is shown in No. 9 and the light chain amino acid sequence is shown in No. 10.

8. A biological material comprising a recombinant antibody encoding an anti- β -actin protein according to any one of claims 1 to 7, characterized in that the biological material comprises an expression vector, an expression cassette, a host cell, an engineering bacterium or a hybridoma cell line.

9. Use of the recombinant antibody against β -actin according to any one of claims 1 to 7 for the preparation of an anti- β -actin antibody fusion protein detection reagent.

10. A test kit comprising the recombinant anti- β -actin antibody of any one of claims 1 to 7.