CN117567607B

CN117567607B - Anti-CREG monoclonal antibody 20F6 and preparation method and application thereof

Info

Publication number: CN117567607B
Application number: CN202311129054.3A
Authority: CN
Inventors: 闫承慧; 何皓; 韩雅玲; 杜忠波; 关鸣
Original assignee: Shenzhen Guanghe Biotechnology Co ltd; General Hospital of Shenyang Military Region
Current assignee: Shenzhen Guanghe Biotechnology Co ltd; General Hospital of Shenyang Military Region
Priority date: 2023-09-04
Filing date: 2023-09-04
Publication date: 2024-07-05
Anticipated expiration: 2043-09-04
Also published as: CN117567607A

Abstract

The invention provides an anti-CREG monoclonal antibody 20F6, a preparation method and application thereof. The anti-CREG monoclonal antibody 20F6 includes a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region, the amino acid sequence of the CDR1 of the heavy chain variable region is shown as SEQ ID 1, the amino acid sequence of the CDR2 is shown as SEQ ID 2, and the amino acid sequence of the CDR3 is shown as SEQ ID 3; the light chain comprises a light chain variable region, the amino acid sequence of the light chain variable region CDR1 is shown as SEQ ID:21, the amino acid sequence of the CDR2 is shown as SEQ ID:22, and the amino acid sequence of the CDR3 is shown as SEQ ID: 23. The anti-CREG monoclonal antibody 20F6 provided by the invention can be specifically combined with CREG protein, can specifically detect CREG protein, and provides a key raw material for an in vitro diagnostic reagent for cardiovascular diseases.

Description

Anti-CREG monoclonal antibody 20F6 and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to an anti-CREG monoclonal antibody 20F6, and a preparation method and application thereof.

Background

In 1998, university of Harvard, gill taught that a transcriptional regulatory Gene, designated the E1A activator Gene (Cellular Repressor ofE A-stimulated Gene, CREG), was found in Hela cells, and the encoded CREG protein was a secreted glycoprotein containing 220 amino acids with 3M 6P glycosylation sites, primarily localized to lysosomes and the perinuclear endoplasmic reticulum. The N-terminus of CREG protein contains a small signal peptide, which is responsible for guiding CREG to transport to cell membrane and then to secrete to the outside of cell. Thus, the expression of CREG protein was detected in cell supernatants cultured in vitro and in human blood.

CREG proteins are widely expressed in differentiated and mature tissues and cells, and play an important role in the proliferation, migration, apoptosis, differentiation and other processes of various cells. There is evidence that CREG protein is an important myocardial protective factor involved in cardiomyocyte differentiation maintenance and homeostasis regulation. In addition, there is a report that CREG protein is highly expressed in serum of patients with cardiovascular disease, which may be an early diagnostic index of cardiovascular disease. Based on the existing research evidence, it is speculated that if the expression level of CREG protein in human serum can be detected, the method has important significance for diagnosis, progress and prognosis judgment of cardiovascular disease patients. Therefore, in order to detect the content of CREG protein in serum of patients with cardiovascular diseases, development and research of a monoclonal antibody capable of specifically binding to CREG protein are of great importance.

Disclosure of Invention

The invention provides an anti-CREG monoclonal antibody 20F6, a preparation method and application thereof, wherein the anti-CREG monoclonal antibody 20F6 can be specifically combined with CREG protein, can specifically detect the CREG protein, and provides a key raw material for an in vitro diagnosis reagent of cardiovascular diseases.

According to a first aspect of the present invention, there is provided an anti-CREG monoclonal antibody 20F6, the anti-CREG monoclonal antibody 20F6 comprising a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region, the heavy chain variable region comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 of the heavy chain variable region is shown as SEQ ID 1, the amino acid sequence of the CDR2 of the heavy chain variable region is shown as SEQ ID 2, and the amino acid sequence of the CDR3 of the heavy chain variable region is shown as SEQ ID 3; the light chain comprises a light chain variable region, the light chain variable region comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 of the light chain variable region is shown as SEQ ID:21, the amino acid sequence of the CDR2 of the light chain variable region is shown as SEQ ID:22 (specific amino acid sequence: RAS), and the amino acid sequence of the CDR3 of the light chain variable region is shown as SEQ ID: 23.

Preferably, the nucleotide sequence encoding the CDR1 of the heavy chain variable region is shown as SEQ ID 11, the nucleotide sequence encoding the CDR2 of the heavy chain variable region is shown as SEQ ID 12, and the nucleotide sequence encoding the CDR3 of the heavy chain variable region is shown as SEQ ID 13; the nucleotide sequence for coding the CDR1 of the light chain variable region is shown as SEQ ID 31, the nucleotide sequence for coding the CDR2 of the light chain variable region is shown as SEQ ID 32 (the specific nucleotide sequence is AGGGCATCC), and the nucleotide sequence for coding the CDR3 of the light chain variable region is shown as SEQ ID 33.

Preferably, the heavy chain variable region further comprises framework regions FR1, FR2, FR3 and FR4, the amino acid sequence of the framework region FR1 of the heavy chain variable region is shown as SEQ ID:4, the amino acid sequence of the framework region FR2 of the heavy chain variable region is shown as SEQ ID:5, the amino acid sequence of the framework region FR3 of the heavy chain variable region is shown as SEQ ID:6, and the amino acid sequence of the framework region FR4 of the heavy chain variable region is shown as SEQ ID:7; the light chain variable region also comprises framework regions FR1, FR2, FR3 and FR4, the amino acid sequence of the framework region FR1 of the light chain variable region is shown as SEQ ID:24, the amino acid sequence of the framework region FR2 of the light chain variable region is shown as SEQ ID:25, the amino acid sequence of the framework region FR3 of the light chain variable region is shown as SEQ ID:26, and the amino acid sequence of the framework region FR4 of the light chain variable region is shown as SEQ ID: 27.

Preferably, the nucleotide sequence encoding the heavy chain variable region framework region FR1 is shown as SEQ ID:14, the nucleotide sequence encoding the heavy chain variable region framework region FR2 is shown as SEQ ID:15, the nucleotide sequence encoding the heavy chain variable region framework region FR3 is shown as SEQ ID:16, and the nucleotide sequence encoding the heavy chain variable region framework region FR4 is shown as SEQ ID:17; the nucleotide sequence for coding the framework region FR1 of the light chain variable region is shown as SEQ ID:34, the nucleotide sequence for coding the framework region FR2 of the light chain variable region is shown as SEQ ID:35, the nucleotide sequence for coding the framework region FR3 of the light chain variable region is shown as SEQ ID:36, and the nucleotide sequence for coding the framework region FR4 of the light chain variable region is shown as SEQ ID:37.

Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID 9; the amino acid sequence of the light chain variable region is shown in SEQ ID 29.

Preferably, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 19; the nucleotide sequence for coding the light chain variable region is shown as SEQ ID 39.

Preferably, the amino acid sequence of the heavy chain is shown in SEQ ID 10; the amino acid sequence of the light chain is shown as SEQ ID NO. 30.

Preferably, the nucleotide sequence encoding the heavy chain is shown as SEQ ID 20; the nucleotide sequence for coding the light chain is shown as SEQ ID 40.

Preferably, the anti-CREG monoclonal antibody 20F6 is prepared by the following steps:

s1, coupling CREG protein molecules with carrier proteins to prepare an immunogen protein;

s2, immunizing a rabbit by utilizing an immunogenic protein to obtain B cells of the rabbit;

s3, culturing and screening rabbit B cells to obtain positive cloned B cells, extracting RNA of the anti-CREG monoclonal antibody from the positive cloned B cells, and carrying out reverse transcription and amplification to obtain nucleic acid for encoding heavy chain of the anti-CREG monoclonal antibody and nucleic acid for encoding light chain of the anti-CREG monoclonal antibody;

s4, connecting nucleic acid for encoding the heavy chain of the anti-CREG monoclonal antibody and nucleic acid for encoding the light chain of the anti-CREG monoclonal antibody into a vector to obtain an expression vector;

s5, introducing the expression vector into a host cell by means of cotransfection or transformation, culturing for 72-96 hours, collecting cell supernatant, and purifying the cell supernatant to obtain the CREG-resistant monoclonal antibody 20F6.

According to a second aspect of the present invention, there is provided a nucleic acid molecule encoding the above-described anti-CREG monoclonal antibody 20F 6.

According to a third aspect of the present invention there is provided a recombinant expression vector comprising a nucleic acid molecule as described above.

Preferably, the recombinant expression vector further comprises at least one of a first signal peptide and a second signal peptide; a first signal peptide is operably linked to the heavy chain; a second signal peptide is operably linked to the light chain.

Preferably, the amino acid sequence of the first signal peptide is shown in SEQ ID 8 and the amino acid sequence of the second signal peptide is shown in SEQ ID 28.

Preferably, the nucleotide sequence encoding the first signal peptide is shown in SEQ ID 18 and the nucleotide sequence encoding the second signal peptide is shown in SEQ ID 38.

According to a fourth aspect of the present invention there is provided a host cell comprising a nucleic acid molecule as described above or a recombinant expression vector as described above.

According to a fifth aspect of the present invention, there is provided the use of the above-described anti-CREG monoclonal antibody 20F6 in the preparation of a kit or chip for detecting CREG proteins.

According to a sixth aspect of the present invention, there is provided a kit for detecting CREG protein, the kit comprising the above-mentioned anti-CREG monoclonal antibody 20F6.

The invention has the beneficial effects that:

1. The anti-CREG monoclonal antibody 20F6 provided by the invention can be specifically combined with CREG protein, has high antibody affinity activity and high reaction titer with CREG protein, can specifically detect CREG protein, and provides a key raw material for an in vitro diagnostic reagent for cardiovascular diseases.

2. Compared with the traditional method of firstly obtaining hybridoma cells and then injecting the hybridoma cells into an animal body through an abdominal cavity so as to generate a monoclonal antibody in the animal body, the anti-CREG monoclonal antibody 20F6 provided by the invention is obtained by firstly preparing a recombinant expression vector, then transfecting or transforming the recombinant expression vector into a host cell, and finally carrying out recombinant expression, a large number of animals are not required to be adopted for experiments, and the preservation stability of the recombinant expression vector of the anti-CREG monoclonal antibody 20F6 is higher, so that the amplified production is facilitated to prepare a large number of anti-CREG monoclonal antibodies 20F6.

3. In the process of preparing the CREG-resistant monoclonal antibody 20F6, rabbits are adopted as immunized animals, and the rabbits have the unique advantages in antigen presentation of small molecules due to large spleen and high gene diversity, so that more excellent positive clones can be screened more easily in the subsequent positive clone screening, the screening efficiency is improved, the cost is low, and the operation is simpler and more convenient.

Drawings

FIG. 1 is a graph showing the results of detection of an anti-CREG antibody 20F6 in rabbit antisera by ELISA.

FIG. 2 is a diagram showing the results of detection of the anti-CREG antibody 20F6 in rabbit antisera by SDS-PAGE.

FIG. 3 is a graph showing the results of detecting anti-CREG antibody 20F6 in rabbit antisera by HPLC.

Detailed Description

The technical features of the technical solution provided in the present invention will be further clearly and completely described in connection with the detailed description below, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The embodiment provides a method for immunizing animals by using CREG antigen and screening positive clones, which comprises the following specific steps:

1. Coupling CREG protein molecule with carrier protein (bovine serum albumin, BSA) to obtain the immunogenic protein.

2. Subcutaneous multipoint injection is carried out on the back and the abdomen of New Zealand white rabbits (4.1-5.4 kg) by using an immunogenic protein, repeated immunization is carried out every 2 weeks to generate an anti-CREG antibody (20F 6) in the rabbits for 3 times, wherein, when the immunogenic protein is injected, the immunogenic protein and an adjuvant are firstly mixed according to the mass ratio of 1:1 were mixed and then injected, the amount of the first immunization was 750 μg/dose, the amount of the subsequent immunization was 350 μg/dose, the first immunization was performed with complete adjuvant (ex Sigma, cat# F5881), and the subsequent immunization was performed with incomplete adjuvant (ex pegbaolone, cat# KX 0210047Q-10).

3. 1 Week after the end of immunization, rabbit antisera were taken and detected by enzyme-linked immunosorbent assay (ELISA), the specific procedure is as follows: adding streptavidin solution into a 96-well plate, adding three primers shown in table 1 (wherein primer 20F6-1 is a positive control, primer 20F6-2 and primer 20F6-3 are negative controls, and primers 20F6-1, 20F6-2 and 20F6-3 are all biotinylated, i.e., biotin-bound Biotin, and primer 20F6-1 contains antibody 20F 6-modified RNA) into the 96-well plate, and incubating at 37 ℃ for 2 hours; CREG protein with the concentration of 5 mu g/mL is taken as an antigen, added into a 96-well plate in the amount of 50 mu L/well, placed overnight at 4 ℃, poured off and washed; 100 mu L of 5% BSA is added into each hole for blocking, and the mixture is placed for 0.5h at room temperature and washed; the rabbit antiserum was diluted to give 8 concentration gradients of anti-CREG antibody 20F6 (1. Mu.g/mL, 0.3333. Mu.g/mL, 0.1111. Mu.g/mL, 0.0370. Mu.g/mL, 0.0123. Mu.g/mL, 0.0041. Mu.g/mL, 0.0014. Mu.g/mL, 0.0005. Mu.g/mL), then added to a 96-well plate in an amount of 50. Mu.L/well, incubated at 37℃for 2 hours, washed, developed, terminated, and the reaction was assayed at a wavelength of 450nm as an OD value plotted on the abscissa at a concentration of anti-CREG antibody 20F6 and an OD450 on the ordinate, and the results are shown in FIG. 1, wherein wells to which no rabbit antiserum was added but an equivalent amount of deionized water (50. Mu.L/well) was added were used as a blank (NC).

As can be seen from FIG. 1, rabbit antisera were able to specifically recognize the primer modified with the anti-CREG antibody 20F 6.

TABLE 1 three primers for performing ELISA experiments

4. The back and abdomen of New Zealand white rabbits were subjected to subcutaneous multipoint injection with 200. Mu.g of the immunogen protein to boost the immunity once, and then antibodies (anti-CREG antibodies) in the rabbit antiserum were extracted, and the anti-CREG antibodies in the rabbit antiserum were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and High Performance Liquid Chromatography (HPLC), the detection results of SDS-PAGE are shown in FIG. 2, and the detection results of HPLC are shown in FIG. 3.

FIG. 2, panel A, shows the detection result (reduced form) of the addition of mercaptoethanol as a reducing agent to an electrophoresis sample when the anti-CREG antibody in the rabbit antiserum is detected by SDS-PAGE, and FIG. 2, panel B, shows the detection result (non-reduced form) of the electrophoresis sample without the reduction of the reducing agent due to the complete reduction of the anti-CREG antibody in the electrophoresis sample to a monomer by the addition of the reducing agent. As can be seen from the inner panel A of FIG. 2, the anti-CREG antibody in the electrophoresis sample was reduced to a monomer with a size of about 50KD, and as can be seen from the inner panel B of FIG. 2, the anti-CREG antibody in the electrophoresis sample without reducing agent was about 100-120 KD, and had two bands, which indicated that the anti-CREG antibody in the electrophoresis sample was a binary protein. As can be seen from FIG. 3, the peak time of the anti-CREG antibody in the rabbit antiserum was between 7.5 and 9 minutes. The results of fig. 2 and 3, taken together, demonstrate that antibodies extracted from rabbit antisera are anti-CREG antibodies as claimed in the present invention.

5. Three days later, taking rabbit spleen, separating lymphocytes from the spleen, carrying out fluorescent marking on B lymphocytes by using CREG protein marked by Fluorescein Isothiocyanate (FITC), enriching the cells by using a flow cytometer, then separating the cells into 96-well plates, culturing the cells for 10-14 days one by one, taking cell supernatant, carrying out ELISA verification experiments by referring to the operation of the step 3, screening out the holes with high titers and good specificity of the anti-CREG antibody (20F 6) as positive holes, wherein the B lymphocytes in the positive holes are the B cells of positive clones.

Example 2

The positive clone B lymphocytes obtained by screening in example 1 were subjected to antibody expression and purification, and the specific procedures were as follows:

1. The B lymphocytes of the positive clones were collected and lysed, and the RNA was extracted and reverse transcribed into cDNA, and the cDNA obtained by reverse transcription was amplified by PCR using heavy chain amplification primers and light chain amplification primer pairs shown in table 2, wherein the PCR reaction conditions are shown in table 3.

TABLE 2 heavy chain amplification primers and light chain amplification primers

TABLE 3 PCR reaction conditions

2. Amplifying the cDNA of the corresponding positive clone through the PCR reaction to obtain a natural paired rabbit monoclonal antibody heavy chain gene (VH) and a natural paired light chain gene (VL), determining the sequences of the VH and the VL through sequencing, wherein the gene sequence of a heavy chain variable region is shown as SEQ ID 19, and the corresponding amino acid sequence is shown as SEQ ID 9; the gene sequence of the light chain variable region is shown as SEQ ID 39, and the corresponding amino acid sequence is shown as SEQ ID 29; the gene sequence of the coding heavy chain is shown as SEQ ID 20, the corresponding amino acid sequence is shown as SEQ ID 10, the gene sequence of the coding light chain is shown as SEQ ID 40, and the corresponding amino acid sequence is shown as SEQ ID 30.

3. The heavy chain gene and the light chain gene were separately connected to pcDNA3.1 vector (purchased from Invitrogen corporation) by homologous recombination, and CHO cells (purchased from the university of Wuhan's classical culture collection) were co-transfected after endotoxin-free plasmid extraction for 72 to 96 hours, and after transfection, the cells were removed by centrifugation, and the cell supernatants were collected.

4. Purifying the cell-expressed supernatant by using Protein A affinity gel resin to obtain an antibody (anti-CREG monoclonal antibody 20F 6), wherein the amino acid sequence of a heavy chain variable region CDR1 of the anti-CREG monoclonal antibody 20F6 is shown as SEQ ID 1, the amino acid sequence of a heavy chain variable region CDR2 is shown as SEQ ID 2, the amino acid sequence of a heavy chain variable region CDR3 is shown as SEQ ID 3, the amino acid sequence of a light chain variable region CDR1 is shown as SEQ ID 21, the amino acid sequence of a light chain variable region CDR2 is shown as SEQ ID 22 (specific amino acid sequence is RAS), and the amino acid sequence of a light chain variable region CDR3 is shown as SEQ ID 23.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention, but these modifications or substitutions are all within the scope of the present invention.

Claims

1. An anti-CREG monoclonal antibody 20F6, characterized in that: including heavy and light chains;

The heavy chain comprises a heavy chain variable region, the heavy chain variable region comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 of the heavy chain variable region is shown as SEQ ID 1, the amino acid sequence of the CDR2 of the heavy chain variable region is shown as SEQ ID 2, and the amino acid sequence of the CDR3 of the heavy chain variable region is shown as SEQ ID 3;

The light chain comprises a light chain variable region, the light chain variable region comprises a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 of the light chain variable region is shown as SEQ ID:21, the amino acid sequence of the CDR2 of the light chain variable region is shown as SEQ ID:22, and the amino acid sequence of the CDR3 of the light chain variable region is shown as SEQ ID: 23.

2. The anti-CREG monoclonal antibody 20F6 according to claim 1, wherein: the heavy chain variable region further comprises framework regions FR1, FR2, FR3 and FR4, the amino acid sequence of the framework region FR1 of the heavy chain variable region is shown as SEQ ID 4, the amino acid sequence of the framework region FR2 of the heavy chain variable region is shown as SEQ ID 5, the amino acid sequence of the framework region FR3 of the heavy chain variable region is shown as SEQ ID 6, and the amino acid sequence of the framework region FR4 of the heavy chain variable region is shown as SEQ ID 7;

The light chain variable region further comprises framework regions FR1, FR2, FR3 and FR4, the amino acid sequence of the framework region FR1 of the light chain variable region is shown as SEQ ID:24, the amino acid sequence of the framework region FR2 of the light chain variable region is shown as SEQ ID:25, the amino acid sequence of the framework region FR3 of the light chain variable region is shown as SEQ ID:26, and the amino acid sequence of the framework region FR4 of the light chain variable region is shown as SEQ ID: 27.

3. The anti-CREG monoclonal antibody 20F6 according to claim 1, wherein: the amino acid sequence of the heavy chain is shown as SEQ ID 10;

the amino acid sequence of the light chain is shown as SEQ ID 30.

4. A nucleic acid molecule encoding the anti-CREG monoclonal antibody 20F6 of any one of claims 1-3.

5. A recombinant expression vector, characterized in that: the recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. The recombinant expression vector of claim 5, wherein: the recombinant expression vector also encodes at least one of a first signal peptide and a second signal peptide;

the first signal peptide is operably linked to the heavy chain;

the second signal peptide is operably linked to the light chain.

7. The recombinant expression vector of claim 6, wherein: the amino acid sequence of the first signal peptide is shown as SEQ ID 8, and the amino acid sequence of the second signal peptide is shown as SEQ ID 28.

8. A host cell, characterized in that: the host cell comprising the nucleic acid molecule of claim 4 or the recombinant expression vector of any one of claims 5-7.

9. The use of an anti-CREG monoclonal antibody 20F6 according to any one of claims 1-3 in the preparation of a kit or chip for detecting CREG proteins.

10. A kit for detecting CREG protein, characterized in that: an anti-CREG monoclonal antibody 20F6 according to any one of claims 1-3.