CN112662632B

CN112662632B - ADORA2A-AS1 monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN112662632B
Application number: CN202011608671.8A
Authority: CN
Inventors: 王树珑
Original assignee: Jiangsu Qinke Biological Research Center Co ltd
Current assignee: Jiangsu Qinke Biological Research Center Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-06-07
Anticipated expiration: 2040-12-30
Also published as: CN112662632A

Abstract

The invention provides an ADORA2A-AS1 monoclonal antibody, a preparation method and application thereof. The invention adopts the general microbiological center preserved in China Committee for culture Collection of microorganisms with the preservation number: the hybridoma cell of CGMCC No.21099 is used for preparing an ADORA2A-AS1 monoclonal antibody, and can be used for detecting ADORA2A-AS1 in a sample.

Description

ADORA2A-AS1 monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an ADORA2A-AS1 monoclonal antibody and a preparation method and application thereof.

Background

The ADORA2A-AS1(AAS1_ HUMAN) protein is a latest protein which is not researched much and is discovered by scientists at present, and may have a certain relation with the pathogenesis of common Acne (Genome-side association study ingredients novel below grade importance loci for segment Acme vulgaris, U.S. Commun.2014 Jun 13). During the acne vulgaris development phase, ADORA2A-AS1 exhibited high levels of expression, which reflects a role for ADORA2A-AS1 in the early development of colon cancer (Nat commun.2014 Jun 13). However, monoclonal antibodies against ADORA2A-AS1 are not currently available on the market.

Disclosure of Invention

In view of the above disadvantages of the prior art, the present invention aims to provide an ADORA2A-AS1 monoclonal antibody, and a preparation method and a use thereof, which fill the blank of the prior art.

The invention provides a hybridoma cell strain HOO57-ADORA2A-AS1 which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No. 21099.

In another aspect, the invention provides an ADORA2A-AS1 monoclonal antibody secreted and produced by the hybridoma cell strain HOO56-IRG 1.

Further, the ADORA2A-AS1 monoclonal antibody is IgG.

In another aspect, the invention provides the use of the ADORA2A-AS1 monoclonal antibody in the preparation of a formulation for detecting ADORA2A-AS1 in a sample.

Further, the detection is an immunoblotting or immunohistochemical assay.

In another aspect, the invention provides a kit for detecting ADORA2A-AS1 in a sample, wherein the kit contains the ADORA2A-AS1 monoclonal antibody.

In another aspect, the present invention provides a method for preparing the ADORA2A-AS1 monoclonal antibody, the method comprising the steps of:

1) providing an immunogen: adopting polypeptide with an amino acid sequence shown as SEQ ID NO.1 as immunogen and crosslinking with carrier protein to obtain crosslinked immunogen;

2) preparing hybridoma cells: immunizing animals by cross-linked immunogen, taking spleen of the immunized animals to fuse with mouse myeloma cells to prepare hybridoma cells and culturing;

3) preparing a monoclonal antibody: the hybridoma cells are injected into the abdominal cavity of a mouse, and the monoclonal antibody is extracted from ascites.

Further, the polypeptide shown in SEQ ID NO.1 is further modified to obtain the polypeptide shown in SEQ ID NO.2, and the polypeptide shown in SEQ ID NO.2 is crosslinked with carrier protein.

Further, the carrier protein is Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA).

Further, the N end of the cross-linked immunogen is covalently cross-linked with the amino group of the carrier protein through a cross-linking agent.

Furthermore, the cross-linked immunogen and the immune adjuvant are mixed and emulsified, and then are injected subcutaneously at multiple points on the back of a mouse, and the serum titer is more than 1: 10000 after more than two times of boosting immunity.

Further, the method for extracting monoclonal antibodies is the prior art, and a person skilled in the art can know how to extract monoclonal antibodies by the prior knowledge.

Further, the method comprises a step 4) of purifying the monoclonal antibody.

Further, the purification includes protein a affinity purification and peptide affinity purification.

As described above, the ADORA2A-AS1 monoclonal antibody, the preparation method and the application thereof have the following beneficial effects:

the ADORA2A-AS1 monoclonal antibody provided by the invention can effectively recognize ADORA2A-AS 1.

Cell name: hybridoma cell

Preservation time: 12 month and 04 days 2020

The preservation unit: china general microbiological culture Collection center

The preservation number is: CGMCC No.21099

And (4) storage address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

Drawings

FIG. 1 is a Western blot chart, and the band of about 17kDa detected in lane B is the signal of ADORA2A-AS1 protein.

Detailed Description

A polypeptide having the sequence: MEQDWQPGEEVTPGPEP (SEQ ID NO. 1). The monoclonal antibody of anti-ADORA 2A-AS1 prepared by the polypeptide can specifically recognize natural ADORA2A-AS1 protein in tissues or cells in immunoblotting (Western blot) and Immunohistochemical (IHC) analysis. The anti-ADORA 2A-AS1 antibody is obtained by the following steps:

the method comprises the following steps: analysis and design of polypeptide sequence: the amino acid sequence of the ADORA2A-AS1 protein is subjected to epitope analysis by using DNAstar software, indexes such AS hydrophilicity, antigenicity, surface possibility, flexibility and the like are mainly evaluated, and the amino acid sequence of the ADORA2A-AS1 protein, which is 18 amino acids from 1-18 positions, is finally determined AS the amino acid sequence of the synthetic polypeptide, wherein the sequence is MEQDWQPGEEVTPGPEP (SEQ ID NO.1), in combination with the practical experience of preparing antibodies in the past.

Step two: polypeptide synthesis and cross-linking: synthesizing target polypeptide by using an ACT396 full-automatic multi-channel polypeptide synthesizer, and identifying by using a mass spectrum;

to enhance the antigenicity of the polypeptide, the ADORA2A-AS1 polypeptide was crosslinked to the carrier protein KLH using a Sulfo-SMCC crosslinking method.

Step three: polypeptide immunization and hybridoma preparation: mixing and emulsifying the crosslinked ADORA2A-AS1-KLH and Freund's adjuvant, carrying out intradermal injection immunization on the back of a BalB/C mouse, and repeatedly boosting the immunization until the titer of antibodies detected by blood sampling reaches the standard.

Step four: : preparing a hybridoma: three days after booster immunization, mice were sacrificed, spleens were ground and mixed with mouse myeloma cells in a 10: 1 ratio and PEG fusion. Culturing in HAT and HT culture medium in sequence, obtaining monoclonal by limiting dilution method, detecting OD value of supernatant by indirect ELISA, selecting cell strain of monoclonal cell strain with high OD value, and performing amplification culture.

Step five: preparing ascites: BalB/C mice were injected intraperitoneally with sterile paraffin oil, and 7 days later, the prepared hybridomas were injected intraperitoneally. After the abdomen of the mouse rises for 7-10 days, the ascites of the mouse is extracted.

Step six: antibody purification: separating ascites from mouse, purifying the antibody with Protein A, and further purifying with peptide affinity to obtain the target antibody.

The anti-ADORA 2A-AS1 monoclonal antibody was identified by the following steps: the antibody obtained from ADORA2A-AS1 was used AS a primary antibody for antigen detection using standard immunoblotting procedures, confirming that the antibody is capable of interacting with the denatured native ADORA2A-AS1 protein.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art. Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Examples

1) Analysis and design of polypeptide sequences

The amino acid sequence of the ADORA2A-AS1 protein is subjected to epitope analysis by using DNAstar software, indexes such AS hydrophilicity, antigenicity, surface possibility, flexibility and the like are mainly evaluated, and then the amino acid sequence of the ADORA2A-AS1 protein, which is MEQDWQPGEEVTPGPEP (SEQ ID NO.1), is finally determined by considering the amino acid structure complexity, the easy oxidation degree, the synthesis difficulty, the amino acid type and distribution and the like in combination with the actual experience of preparing antibodies in the past. Meanwhile, in order to ensure the affinity purification of the later polypeptide cross-linked carrier protein and the peptide, a cysteine C is added at the N terminal, and the sequence of the finally synthesized polypeptide is CMEQDWQPGEEVTPGPEP (SEQ ID NO.2)

2) Polypeptide synthesis and crosslinking

Synthesizing: the ACT396 full-automatic multi-channel polypeptide synthesizer automatically synthesizes target polypeptides according to a programmed program, dissolves the synthesized polypeptides in 50% acetonitrile, adopts a mass spectrometer for identification, and confirms the obtained polypeptides as the target polypeptides.

And (3) crosslinking: Sulfo-SMCC as a cross-linker cross-linked the carrier protein KLH with the synthetic polypeptide: 10mg of KLH was dissolved in 0.5ml of ultrapure water; 3mg of sulfo-SMCC was dissolved in 0.5ml of ultrapure water, and the pH was adjusted to about 7 with 3M NaOH. In the case of mixing, the sulfo-SMCC solution was slowly added dropwise to the KLH solution, and the reaction was stirred and mixed at room temperature for 30 min. The reacted sulfo-SMCC/KLH mixture was loaded onto a Sephadex G25 column equilibrated in advance with equilibration buffer (0.05M PB, pH6.0) for 30min, and the light gray eluate, i.e., the activated sulfo-SMCC/KLH solution, was collected. 2mg of the crosslinked polypeptide was dissolved in 200. mu.l of PBS (pH7.3), 0.2 volume of the sulfo-SMCC/KLH complex solution was added to the polypeptide solution, the pH was adjusted to 7.3, the mixture was shaken at room temperature for 4 hours, frozen at-70 ℃ and lyophilized in a lyophilizer for 24 hours. Polypeptide crosslinking efficiency is determined by detecting polypeptide sulfydryl before and after crosslinking through an Ellman method, and the European Union rate is 99.3 percent.

3) Polypeptide immunization and antisera preparation

100. mu.g of the crosslinked KLH-polypeptide was dissolved in 100. mu.l of phosphate buffer (0.01M PBS) and emulsified well (until no diffusion in water) by adding an equal volume of Freund's complete adjuvant. The mice are immunized by healthy BalB/C mice with the age of 6 weeks and the weight of 20-25g, and the immunization is carried out by intradermal injection on the back, and at least more than 3 points are required. After 2 weeks of the first immunization, 100. mu.g of the polypeptide was dissolved in 100. mu.l of phosphate buffer (0.01M PBS), and the resulting solution was thoroughly emulsified with an equal amount of incomplete Freund's adjuvant and then subjected to intradermal immunization, and the immunization was required to be performed by intradermal injection into the back as a first booster immunization at least at 3 points. 2 weeks after the second immunization, a second boost was performed, the method and requirements were identical to the first boost. After 1 week, tail blood is taken, the ELISA plate is coated with non-crosslinked synthetic polypeptide, and the titer of immune serum is detected by an indirect ELISA method. The booster immunization and the titer determination are repeated until the serum titer reaches more than 1: 10000. After the potency reached the requirement, 100ug of polypeptide was dissolved in 0.5ml phosphate buffer (0.01M PBS) and injected intraperitoneally as a booster.

4) Hybridoma cell preparation

(1) Cell fusion: three days after boosting, the eyeballs of the mice were bled and the mice were sacrificed. Soaking in 75% alcohol for sterilization, taking mouse spleen from an ultra-clean bench, grinding with a 200-mesh steel sieve, and adding DMEM to constant volume to 10 ml. The ground spleen was then dispersed into single cells by pipetting. Counting 10ul of the mixed solution, and calculating the total number of splenocytes. Following myeloma cell: the spleen cells were added with the corresponding amount of myeloma cells at 1:10, mixed well, centrifuged at 1000r/min for 5 minutes and the supernatant discarded. The mixed cells were flicked to disperse them loosely and placed in a water bath at 37 ℃. Add 1ml PEG1500 with a pipette while stirring for 1 min. Stirring for 1min, stirring PEG gently, and standing for 90 s. 1ml of serum-free DMEM was added while stirring, PEG was stirred up, and the reaction was stopped. 40ml serum-free DMEM was added with stirring, centrifuged at 1000rpm/min for 5min, the PEG washed off, and the supernatant discarded.

(2) And (3) cell culture screening: the bottom fused cells were gently blown up with 2ml of fused cell culture solution, and made up to 50ml with HAT culture solution (HAT in DMEM containing 15% calf serum). The cells were evenly distributed into 96-well plates containing feeder cells, 100. mu.l/well. After 5 days, the supernatant in the 96-well plate was discarded and replaced with a newly prepared HAT medium (HAT in DMEM containing 15% calf serum). After 3 days of fluid change, 100ul of supernatant per well was placed on an ELISA plate coated with the antigen, and the positive value of the supernatant was measured by indirect ELISA. And taking the hole with the high positive value, replacing HAT culture solution, and rechecking after one day. The positive wells were subjected to limiting dilution with HT medium (HT in DMEM containing 15% calf serum) and distributed evenly in 96-well plates containing feeder cells at 100. mu.l/well. One week later, the plate positive values were checked, and the monoclonal with high positive values was subcloned in DMEM containing 15% calf serum. After the positive monoclonal subcloning, the positive rate of the pore plate supernatant reaches 100%, the cell strain with high positive value and large cell group area is taken for amplification culture, and the obtained hybridoma cell is sent to the China general microbiological culture Collection center for preservation, with the preservation number: CGMCC No. 21099.

5) Preparation of monoclonal antibodies

One week before cell killing, 3 healthy 8-week-old BalB/C female mice were injected with 1ml of paraffin oil per abdominal cavity. When the cells were in log phase, the cells in the cell vial were counted by blowing down 3 x 10⁶The cells are put into a centrifuge tube and centrifuged for 5min at 1000 r/min. The supernatant was discarded, 5ml of sterile phosphate buffer (0.01M PBS) was added, cells were blown off with a pipette gun, 30ml of phosphate buffer (0.01M PBS) was supplemented, and the mixture was centrifuged at 1000rpm for 5 min. 3ml of phosphate buffer (0.01M PBS) was added to the centrifuge tube and mixed well, and 1ml of the mixture was injected into the abdominal cavity of each mouse. One week after injection, the neck was cut off and the mice were sacrificed, soaked in 75% alcohol for sterilization, the abdominal epithelium was cut off with scissors, and the syringe was inserted into the abdominal cavity to pick up the abdominal membraneAnd sucking the ascites into a centrifuge tube, and centrifuging for 5min at 3500 r/min. The supernatant was collected, mixed with the binding buffer at 1:1 and filtered through a 0.45um filter.

6) Antibody affinity purification

(1) And (3) TIgG purification: 10ml of 50% Protein-agarose suspension was applied to a 30ml column using a liquid dispenser, the top and bottom caps were removed, the volume of the column after the liquid had flowed out was 5ml, and then the column was washed 3 times with 25ml deionized water. 10ml of the corresponding serum was removed, mixed with 2ml of PBS and applied to a 30ml column, and the serum sample was allowed to flow out by vortexing on a rotary mixer at room temperature (20-25 ℃) for 1 hour. The column was washed 3 times with 20ml of purified wash solution and eluted with 10ml of eluent.

(2) Peptide affinity purification: 1ml of Sulfo-link gel suspension (0.5ml of gel) was added to the column, and after the column liquid drained, the column was washed with 4ml of coupling buffer. The synthesized ADORA2A-AS1 polypeptide was dissolved in 1ml of coupling buffer and added to the column, 1ml of coupling buffer was added to the column, and the mixture was mixed by inversion at room temperature for 1 hour. The column was washed with 6ml of coupling buffer, then 3ml of blocking solution was added and mixed well for 1 hour at room temperature. The column was washed 3 times, then 6ml IgG and 3ml PBS were added to the column, and mixed by inversion at room temperature for 1 hour. The column was washed 3 times with PBS and then eluted with 2ml of eluent. The obtained purified antibody was packed in a dialysis bag and dialyzed at 4 ℃. Dialyzed overnight, and then centrifuged at 4000rpm X35 min to remove the precipitate, and the supernatant was collected. The titer of the antibody measured by an indirect ELISA method is 1: 32000 the protein concentration was measured by the Bradford method and was 0.98 mg/ml.

7) Immunoblot analysis

Preparing SDS-PAGE gel according to a standard method, sequentially adding 5 mul of lysate with the protein concentration of 5mg/ml, keeping the pressure constant for about 30 minutes at 80V, changing the voltage to 160V when a sample runs through the concentrated gel and is basically in a straight line, stopping electrophoresis until a bromophenol blue indicator completely runs out of the separation gel (about 60 minutes), and performing constant-pressure 100V electric conversion to 80 minutes by adopting an electric membrane conversion method to convert the membrane to a PVDF membrane. The obtained ADORA2A-AS1 antibody was used AS a primary antibody, and the antibody obtained by the above-described membrane transfer was hybridized with an antigen at a concentration of 1.25. mu.g/ml at room temperature for 1 hour, then hybridized with an HRP-labeled goat anti-rabbit antibody at room temperature for 1 hour, developed by ECL color development, developed by X-ray in a dark room and exposed to light, and the immunoblotting results were AS shown in FIG. 1.

The results show that: the obtained ADORA2A-AS1 monoclonal antibody can effectively recognize ADORA2A-AS 1.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

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Claims

1. A hybridoma cell strain HOO57-ADORA2A-AS1, which is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation number of: CGMCC No. 21099.

2. The hybridoma cell line of claim 1, HOO56-IRG1 secretes ADORA2A-AS1 monoclonal antibody.

3. Use of the ADORA2A-AS1 monoclonal antibody of claim 2 in the preparation of a formulation for detecting ADORA2A-AS1 in a sample.

4. A kit for detecting ADORA2A-AS1 in a sample, said kit comprising the ADORA2A-AS1 monoclonal antibody of claim 2.