CN114591437B

CN114591437B - RAC1 protein monoclonal antibody and preparation method and application thereof

Info

Publication number: CN114591437B
Application number: CN202210151670.8A
Authority: CN
Inventors: 张浩洋; 石坚; 徐爱华; 朱国方
Original assignee: Shaoxing Shouren Medical Health Technology Co ltd
Current assignee: Shaoxing Shouren Medical Health Technology Co ltd
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2024-02-27
Anticipated expiration: 2042-02-18
Also published as: CN114591437A

Abstract

The application relates to the technical field of biological immunology, in particular to an RAC1 protein monoclonal antibody, a preparation method and application thereof, and provides the RAC1 protein monoclonal antibody, wherein the sequence of the RAC1 protein monoclonal antibody comprises the following components: the amino acid sequence of the heavy chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.2; the provided RAC1 protein monoclonal antibody has strong specificity, can accurately and rapidly detect the RAC1 protein, is combined with the RAC1 protein in specificity, ensures no cross reaction with other proteins of RHO protein family and the like, and is favorable for being widely applied to immunological detection.

Description

RAC1 protein monoclonal antibody and preparation method and application thereof

Technical Field

The application belongs to the technical field of biological immunology, and particularly relates to an RAC1 protein monoclonal antibody, a preparation method and application thereof.

Background

Rac1 is one of the Rho family members. The Rho family plays a role of a "molecular switch" in some basic functions of cells, and is involved in the processes of cell movement, actin recombination, malignant transformation of tumors, invasion and metastasis, regulation of transcription factors, apoptosis of cells, angiogenesis of tumors, and the like. The protein coded by the Rac1 gene is GTPase, belonging to RAS superfamily of small GTP binding protein; plays an important role in the aspects of cell movement and adhesion, cell proliferation and differentiation and apoptosis, tumor invasion and metastasis, immune regulation and the like.

Currently, immunological detection methods are used for detecting RAC1 protein, wherein the immunological detection methods comprise ELISA, fluorescence immunoadsorption, chemiluminescence and immunochromatography. The difficulty of immunological detection is to find antibodies with high accuracy and good stability. The antibodies adopted by the RAC1 protein at the present stage are all polyclonal antibodies, the polyclonal antibodies refer to the same antigenic determinant, and antibodies can be produced by several clones in the organism to form several monoclonal antibody impurities, so that the specificity of the detection of the RAC1 protein is poor, false positives are easy to occur, and accurate judgment is not facilitated.

Disclosure of Invention

The purpose of the application is to provide an RAC1 protein monoclonal antibody, a preparation method and application thereof, and aims to solve the problems that the RAC1 protein polyclonal antibody in the prior art is poor in specificity and is not beneficial to accurately and rapidly detecting the RAC1 protein.

In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:

in a first aspect, the present application provides an RAC1 protein monoclonal antibody, the sequence of which RAC1 protein monoclonal antibody comprises: the amino acid sequence of the heavy chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.2.

In a second aspect, the present application provides a method for preparing an RAC1 protein monoclonal antibody, where the method for preparing the RAC1 protein monoclonal antibody includes the following steps:

dissolving RAC1 protein in a buffer solution to obtain an RAC1 protein solution, and using the RAC1 protein solution to immunize a mouse to obtain an immunized mouse and immune serum;

respectively culturing spleen cells and myeloma cells of an immunized mouse, carrying out cell fusion treatment, and screening by adopting indirect ELISA and indirect competition ELISA to obtain hybridoma cell strains;

performing cell cloning on the hybridoma cell strain to obtain a strong positive monoclonal cell;

the strong positive monoclonal cells are inoculated into the body of a mouse, and the RAC1 protein monoclonal antibody is prepared by adopting an in-vivo ascites induction method.

In a third aspect, the present application provides an RAC1 protein monoclonal antibody or an application of an RAC1 protein monoclonal antibody prepared by a preparation method of an RAC1 protein monoclonal antibody in preparing a detection reagent of an RAC1 protein.

In a fourth aspect, the present application provides a kit for detecting an RAC1 protein, the kit comprising an RAC1 protein monoclonal antibody or an RAC1 protein monoclonal antibody prepared by the method for preparing an RAC1 protein monoclonal antibody.

The sequence of the RAC1 protein monoclonal antibody provided in the first aspect of the application comprises the following steps: amino acid sequence of heavy chain variable region of RAC1 protein monoclonal antibody sequence SEQ ID NO.1 and amino acid sequence of light chain variable region of RAC1 protein monoclonal antibody sequence SEQ ID NO.2; the provided RAC1 protein monoclonal antibody has strong specificity, can accurately and rapidly detect the RAC1 protein, is combined with the RAC1 protein in specificity, ensures no cross reaction with other proteins of RHO protein family and the like, and is favorable for being widely applied to immunological detection.

The preparation method of the RAC1 protein monoclonal antibody provided by the second aspect of the application takes RAC1 protein as a raw material to prepare the RAC1 protein monoclonal antibody, in the preparation process, immune spleen cells and mouse myeloma cells are fused by adopting a cell fusion technology, and screening is carried out by utilizing indirect ELISA and indirect competition ELISA pairs to obtain strong positive monoclonal cells; the method for preparing the RAC1 protein monoclonal antibody by in-vivo induced ascites is clear in flow, convenient to operate, simple in synthesis method and high in synthesis efficiency, the prepared RAC1 protein monoclonal antibody can be specifically combined with RAC1 protein, the antibody titer is ensured to have no cross reaction with other proteins of RHO protein family and the like, and the method is favorable for being widely applied to immunological detection.

The application of the RAC1 protein monoclonal antibody or the RAC1 protein monoclonal antibody prepared by the preparation method of the RAC1 protein monoclonal antibody in the detection reagent for preparing the RAC1 protein is beneficial to wide application because the RAC1 protein monoclonal antibody has high specificity, can be accurately combined with the RAC1 protein in the application process, ensures no cross reaction with other proteins of RHO protein family and the like.

The kit for detecting the RAC1 protein provided by the fourth aspect of the application comprises an RAC1 protein monoclonal antibody or the RAC1 protein monoclonal antibody prepared by the preparation method of the RAC1 protein monoclonal antibody, and can be accurately combined with the RAC1 protein in the application process due to high specificity of the RAC1 protein monoclonal antibody, ensures no cross reaction with other proteins of RHO protein family and the like, and is favorable for wide application.

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

In this application, the term "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood that, in various embodiments of the present application, the sequence number of each process does not mean that the sequence of execution is sequential, and some or all of the steps may be executed in parallel or sequentially, where the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application in the examples and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weights of the relevant components mentioned in the embodiments of the present application may refer not only to specific contents of the components, but also to the proportional relationship between the weights of the components, and thus, any ratio of the contents of the relevant components according to the embodiments of the present application may be enlarged or reduced within the scope disclosed in the embodiments of the present application. Specifically, the mass in the specification of the embodiment of the present application may be a mass unit well known in the chemical industry field such as μ g, mg, g, kg.

The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.

In a first aspect, embodiments of the present application provide an RAC1 protein monoclonal antibody, where the sequence of the RAC1 protein monoclonal antibody includes: the amino acid sequence of the heavy chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.2.

The sequence of the RAC1 protein monoclonal antibody provided in the first aspect of the embodiment of the application comprises: amino acid sequence of heavy chain variable region of RAC1 protein monoclonal antibody sequence SEQ ID NO.1 and amino acid sequence of light chain variable region of RAC1 protein monoclonal antibody sequence SEQ ID NO.2; the provided RAC1 protein monoclonal antibody has strong specificity, can accurately and rapidly detect the RAC1 protein, is combined with the RAC1 protein in specificity, ensures no cross reaction with other proteins of RHO protein family and the like, and is favorable for being widely applied to immunological detection.

Specifically, the sequence of the RAC1 protein monoclonal antibody comprises: the amino acid sequence of the heavy chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.2. Wherein SEQ ID NO.1 is EVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWRGGSTDYNTAFMSRLRITKDNSKSQVFFKMNSLQADD TAIYYCAKNSYGSRNFDVWGAGTTVTVSS; SEQ ID NO.2 is DIVMTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRL LIKYVSQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPVTFGAGTKLELK.

In some embodiments, the RAC1 protein monoclonal antibody belongs to the IgG subclass. IgG subclass refers to the immune complex formed by lgG1-lgG3 and antigen, and activates complement by classical pathway to exert functions such as lysis and cytolysis. The provided RAC1 protein monoclonal antibody activates complement through classical pathway, and plays roles of lysis, cytolysis and the like.

In some embodiments, the relative molecular mass of the RAC1 protein monoclonal antibody is 150-160 kDa. The provided RAC1 protein monoclonal antibody has small relative molecular mass and is beneficial to rapid preparation.

In some embodiments, the antibody titer of the RAC1 protein monoclonal antibody is 10E 4-10E 5, and the provided RAC1 protein monoclonal antibody has higher antibody titer, is favorable for specific binding with the RAC1 protein, and further improves the interaction effect with the RAC1 protein.

The second aspect of the embodiment of the present application provides a method for preparing an RAC1 protein monoclonal antibody, where the method for preparing the RAC1 protein monoclonal antibody includes the following steps:

s01, dissolving RAC1 protein in a buffer solution to obtain an RAC1 protein solution, and using the RAC1 protein solution to immunize mice to obtain immunized mice and immune serum;

s02, respectively culturing spleen cells and myeloma cells of the immunized mice, carrying out cell fusion treatment, and screening by adopting indirect ELISA and indirect competition ELISA to obtain hybridoma cell strains;

s03, performing cell cloning on the hybridoma cell strain to obtain a strong positive monoclonal cell;

s04, inoculating the strong positive monoclonal cells into a mouse body, and adopting an in-vivo ascites induction method to prepare the RAC1 protein monoclonal antibody.

The preparation method of the RAC1 protein monoclonal antibody provided by the second aspect of the embodiment of the application takes RAC1 protein as a raw material to prepare the RAC1 protein monoclonal antibody, in the preparation process, immune spleen cells and mouse myeloma cells are fused by adopting a cell fusion technology, and screening is carried out by utilizing indirect ELISA and indirect competition ELISA, so that strong positive monoclonal cells are obtained; the method for preparing the RAC1 protein monoclonal antibody by in-vivo induced ascites is clear in flow, convenient to operate, simple in synthesis method and high in synthesis efficiency, the prepared RAC1 protein monoclonal antibody can be specifically combined with RAC1 protein, the antibody titer is ensured to have no cross reaction with other proteins of RHO protein family and the like, and the method is favorable for being widely applied to immunological detection.

In step S01, RAC1 protein is dissolved in a buffer to obtain an RAC1 protein solution, and the RAC1 protein solution is used for immunizing mice to obtain immunized mice and immune serum.

In some embodiments, the buffer provided is a PBS buffer for dissolving the RAC1 protein to obtain a RAC1 protein solution. In some embodiments, the protein concentration of the RAC1 protein solution is 50-55 mg/mL and the pH of the RAC1 protein solution is 7.4-7.5.

In some embodiments, the resulting RAC1 protein solution has a protein concentration of 50mg/mL and a pH of 7.4.

In some embodiments, the step of using the RAC1 protein solution in the step of immunizing the mouse comprises primary immunizing and boosting the mouse with the RAC1 protein solution, wherein the number of boosting is 3-4.

In some embodiments, the step of primary immunization comprises: preparing an RAC1 protein solution into an antigen solution with the concentration of 0.1 mu g/mu L-10 mu g/mu L, uniformly mixing the antigen solution with an equal volume of Freund complete adjuvant to obtain a first antigen stock solution, and performing primary immunization on a mouse by adopting the first antigen stock solution;

the step of enhancing immunity comprises: the RAC1 protein solution is prepared into antigen solution with the concentration of 0.1 mu g/mu L-10 mu g/mu L, and the antigen solution is uniformly mixed with Freund incomplete adjuvant with the same volume to obtain second antigen stock solution, and the second antigen stock solution is adopted to strengthen the immunity of the mice.

Further, by primary immunization and booster immunization treatment, immunized mice are obtained, and immune serum of the immunized mice is obtained.

In some embodiments, the immune serum is screened using an indirect ELISA and an indirect competition ELISA, the screening step comprising: the RAC1 protein is taken as a coating antigen, and after dilution of serum multiple ratio, an indirect ELISA method is adopted to determine the antiserum titer; if the serum titer is higher than 10E4, the serum titer is measured by indirect competition ELISA using RAC1 protein standard, and the mouse with the highest affinity between the serum and the RAC1 protein is selected for preparing the monoclonal antibody.

In some embodiments, the serum multiple dilution comprises: and adopting PBS solution containing 5% of skim milk by mass fraction, and carrying out gradient dilution by 2-3 times.

In step S02, spleen cells and myeloma cells of the immunized mice are respectively cultured, and cell fusion treatment and screening are carried out to obtain hybridoma cell strains.

In some embodiments, providing an immunized mouse, collecting and grinding spleen of the immunized mouse to obtain a spleen cell suspension, and providing myeloma cells for culture, and maintaining the myeloma cells in a logarithmic growth phase to prepare a myeloma cell suspension; combining spleen cell suspension and myeloma cell suspension, centrifuging to collect precipitate, and performing cell fusion treatment by using polyethylene glycol to obtain fused cells.

In some embodiments, the cell fusion process comprises the steps of:

respectively sucking 6-6.5X10 ⁷ Spleen cells and 1.2-1.3X10-containing ⁷ Combining cell suspensions of the hybridoma cells, putting the cell suspensions into a centrifuge tube, centrifuging at 1000-1200 rpm for 5-6 min, discarding supernatant, and collecting cell sediment;

slowly adding 50-55 v/v% polyethylene glycol PEG 15001-1.2 mL into the cell sediment within 60-65 s, then slowly sucking the cell suspension into a pipette, and finishing sucking within 30-35 s; standing for 30-35 s, and slowly blowing the cell suspension back into the centrifuge tube within 30-35 s;

adding 25-26 mLDMEM culture medium into the cell suspension within 5-6 min to terminate the reaction; centrifuging at 1000-1200 rpm for 7-8 min, discarding supernatant, adding 20-22 mLHAT culture medium, and slightly blowing and sucking to suspend the precipitated cells; transferring into 96-well cell culture plate with feeder cells, placing 0.1 mL/well at 37-37.4deg.C and containing 5-6% CO ₂ Is cultured in an incubator; the fused cells were obtained.

In some embodiments, the cell number of spleen cells and hybridoma cells is (4.8-5): 1.

in some embodiments, the step of adding 25 to 26mLDMEM medium to the cell suspension over 5 to 6 minutes comprises: 1 mM LDMEM medium was added in 1min, 4 mM LDMEM medium was added in 2min, and the remaining 20 mM LDMEM medium was added in 3 min.

In some embodiments, the fused cells are screened to obtain hybridoma cell lines. Wherein the step of screening the fused cells comprises: detecting culture supernatants of all cloned growth holes by using an indirect competition ELISA method, selecting cell holes with high OD value, high inhibition rate and less cloning number for cloning, and performing expanded culture; and (3) screening the hybridoma cells by adopting indirect ELISA and indirect competition ELISA, and selecting hybridoma cell lines with high supernatant titer, good competition inhibition effect and vigorous growth for the next test.

In step S03, cell cloning is performed on the hybridoma cell strain, and a strong positive monoclonal cell is obtained.

In some embodiments, the resulting hybridoma cell line is subjected to cell cloning using limiting dilution to obtain a strong positive monoclonal cell.

In step S04, strong positive monoclonal cells are inoculated into the body of a mouse, and an RAC1 protein monoclonal antibody is prepared by adopting an in-vivo ascites induction method.

In some embodiments, monoclonal antibodies are prepared by in vivo induced abdominal water method, strong positive monoclonal cells are inoculated into mice, after 10-14 days, ascites are collected, supernatant is collected centrifugally, RAC1 protein monoclonal antibodies are obtained, and the monoclonal antibodies are packaged and frozen for later use.

The third aspect of the embodiment of the application provides an RAC1 protein monoclonal antibody or application of the RAC1 protein monoclonal antibody prepared by the preparation method of the RAC1 protein monoclonal antibody in preparing a detection reagent of the RAC1 protein.

The application of the RAC1 protein monoclonal antibody or the RAC1 protein monoclonal antibody prepared by the method for preparing the RAC1 protein monoclonal antibody in the detection reagent for preparing the RAC1 protein is beneficial to wide application because the RAC1 protein monoclonal antibody has high specificity, can be accurately combined with the RAC1 protein in the application process, ensures no cross reaction with other proteins of RHO protein family and the like.

The fourth aspect of the embodiment of the application provides a kit for detecting an RAC1 protein, the kit comprising an RAC1 protein monoclonal antibody or an RAC1 protein monoclonal antibody prepared by a preparation method of an RAC1 protein monoclonal antibody.

The kit for detecting the RAC1 protein provided by the fourth aspect of the embodiment of the application comprises an RAC1 protein monoclonal antibody or the RAC1 protein monoclonal antibody prepared by the preparation method of the RAC1 protein monoclonal antibody, and can be accurately combined with the RAC1 protein in the application process due to high specificity of the RAC1 protein monoclonal antibody, ensures no cross reaction with other proteins of RHO protein family and the like, and is favorable for wide application.

The following description is made with reference to specific embodiments.

Example 1

RAC1 protein monoclonal antibody and preparation method thereof

Wherein the sequence of the RAC1 protein monoclonal antibody comprises: amino acid sequence of heavy chain variable region of RAC1 protein monoclonal antibody sequence SEQ ID NO.1 and amino acid sequence of light chain variable region of RAC1 protein monoclonal antibody sequence SEQ ID NO.2.

The preparation method of the RAC1 protein monoclonal antibody comprises the following steps:

(1) Preparing an antigen: 100mg of RAC1 protein was weighed and dissolved in 1.8mL of PBS, the pH was adjusted to 7.4, and the volume was adjusted to 2mL to prepare a 50mg/mL solution of RAC1 protein.

(2) Animal immunization and potency determination

Taking a plurality of healthy BALB/c mice with the age of 6-8 weeks, respectively numbering, taking 50mg/ml antigen solution, uniformly mixing with equal volume of complete Freund's adjuvant, fully emulsifying, performing primary immunization according to 100 mug/mouse, performing booster immunization once every 2-4 weeks, preparing 0.1 mug/mug-10 mug/mug of antigen solution with normal saline during booster immunization, uniformly mixing with equal volume of incomplete Freund's adjuvant, fully emulsifying, and performing subcutaneous multipoint injection according to 100 mug/mouse back. After 7d of 3 rd-4 th booster immunization, obtaining serum after tail breaking and blood taking immunization; the serum was diluted 2-3 times in gradient with PBS solution pH7.4 containing 5% skim milk by mass fraction using RAC1 protein as coating source, and the antiserum titer was measured by indirect ELISA. If the serum titer is higher than 10E4, detecting indirect competition ELISA by using an RAC1 protein standard substance, and selecting a mouse with highest affinity between the serum and the RAC1 protein for preparing a monoclonal antibody; 3 days prior to fusion, the peritoneal cavity was boosted (without adjuvant, directly with antigen solution).

(3) Preparation and fusion of spleen cells and myeloma cells of immunized mice

Taking the mice with highest affinity of the serum (2) and the RAC1 protein, taking the eyeballs for blood collection after 3 days of booster immunization, collecting the serum for standby, killing the mice, taking out the spleen aseptically, and grinding the spleen to obtain spleen cell suspension. The cells were resuspended by washing 2-3 times with DMEM medium, adding a certain amount of DMEM medium, staining and counting. The recovered SP2/0 myeloma cells were cultured in complete medium containing 10% (v/v) fetal bovine serum. 1 day prior to fusion, cell exchange was performed and cells were maintained in the logarithmic growth phase. On the day of fusion, myeloma cells are collected, the supernatant is removed by centrifugation, the cells are resuspended by adding incomplete medium, and the cell viability is calculated after counting and counting after being stained with the phenol blue, and the cell viability is more than 90% and can be used for fusion.

Wherein the step of cell fusion comprises:

a) Cell suspensions containing 6×10E7 spleen cells and 1.2X10E7 SP2/0 cells were separately aspirated (ratio of two cell numbers is 5:1), combined and placed in a 50mL plastic centrifuge tube, centrifuged at 1000rpm for 5min, and the supernatant was discarded;

b) Flicking the bottom of the tube with finger to loosen the sediment into paste;

c) Taking 50% (v/v) polyethylene glycol PEG15001mL, rotating a centrifuge tube at a constant speed by a left hand, sucking 0.7mL of 50% PEG solution by a right hand by a 1mL pipette, slowly dripping along the rotating tube wall (which is close to cells as much as possible), controlling the time to be over 60s, then slowly sucking the cell suspension into the pipette, controlling the time to be over 30s, standing for 30s, slowly blowing the cell suspension back into the centrifuge tube, and controlling the time to be over 30 s;

d) Immediately, 25ml of medium was added to the mixture over 5min to terminate the reaction. The adding speed is as follows: 1mL was added in 1min, 4mL was added in 2min, and the remaining 20mL was added in 3 min. The centrifuge tube is gently rotated at a constant speed during liquid adding, and the operation is gentle at the moment;

e) Centrifugation at 1000rpm for 7min, removal of supernatant, addition of 20mLHAT medium, gentle pipetting to suspend the pelleted cells. At the moment, the operation should be gentle and no force is applied to blow;

f) Adding the cell suspension into 96-well cell culture plate with feeder cells, placing at 37deg.C and containing 5% CO at 0.1 mL/well ₂ Is cultured in an incubator;

g) Cell growth was observed and recorded daily. Half of HAT complete medium was used for each well at day 7 after fusion, half of HT complete medium was used for each well at day 14 after fusion, and then full medium was used.

(4) Selection of hybridoma cells

When the fused cell clone grows to 1/4-1/3 of the hole bottom area, the culture supernatant of all the cloned growth holes can be detected by an indirect competition ELISA method, and the cell holes with high OD value, large inhibition rate and small clone number are selected for cloning and are subjected to expansion culture.

Hybridoma cells were screened by indirect ELISA and indirect competition ELISA as follows:

indirect ELISA primary screening: 1. Mu.g/mL RAC1 protein solution diluted with coating buffer was added, 100. Mu.L/well coating ELISA plate overnight; after washing the plates, blocking with PBS solution containing 5% skimmed milk, 150. Mu.L/well, and incubating at 37deg.C for 2h; after washing the plates, 30. Mu.L/well of the cell culture supernatant was added, 70. Mu.L/well of PBST was added, and the plates were washed 5 times by incubation at 37 ℃; adding an enzyme-labeled goat anti-mouse secondary antibody, 100 mu L/hole, incubating for 1h at 37 ℃, and washing the plate for 5 times; adding tetramethyl benzidine TMB color development liquid into each hole at the temperature of 37 ℃ for 10-15 min; adding 50 mu L of stop solution (2M H2SO 4), and measuring absorbance at 450nm by an ELISA; the cell supernatants were assayed for positive (positive control OD450nm values greater than 2-fold negative control wells OD450nm values, positive serum highest dilution was used as ELISA titer of serum.

(5) And (3) performing cell cloning on the hybridoma cell strain to obtain a strong positive monoclonal cell.

And performing cell cloning on the obtained hybridoma cell strain by adopting a limiting dilution method to obtain a strong positive monoclonal cell.

(6) The strong positive monoclonal cells are inoculated into the body of a mouse, and the RAC1 protein monoclonal antibody is prepared by adopting an in-vivo ascites induction method.

Wherein, the monoclonal antibody is prepared by adopting an in-vivo induced abdominal water method, strong positive monoclonal cells are inoculated into a mouse body, after 10 to 14 days, ascites is collected, the supernatant is collected centrifugally, and the RAC1 protein monoclonal antibody is obtained, split charging and freezing preservation are carried out for standby.

Comparative example 1

RAC1 protein polyclonal antibody and preparation method thereof

The preparation method of the RAC1 protein polyclonal antibody comprises the following steps:

(1) Providing RAC1 protein and mixing with PBS, and preparing RAC1 protein antigen;

(2) Providing rabbits for experiments, and carrying out animal immunization on the rabbits by adopting RAC1 protein antigen, wherein the immunization adopts a lymph node injection method, and the specific steps comprise: (1) 50mg (about 0.30ml on each side) of live BCG vaccine is injected subcutaneously (or intradermally) at the plantar portion of the two hind legs of the rabbit, and after 7-10 days, the plantar and popliteal lymph nodes of the rabbit are enlarged; (2) 0.50ml of RAC1 protein antigen (containing 5mg/ml antigen, 1 000U/ml penicillin, 1000 μg/ml streptomycin) with complete adjuvant was injected into each of the two enlarged lymph nodes; (3) repeating the step (2) once after 14 days if necessary; (4) after 7 days more, 0.50ml (containing IgG5mg/ml, penicillin 1 000U/ml, streptomycin 1 000. Mu.g/ml) of the complete adjuvant-added RAC1 protein antigen was injected into each of the bilateral lymph nodes; (5) after 5-7 days, the auricular vein is sampled, the serum titer is measured, and whether the immunization is successful or not is observed.

(3) If the immunization is successful, collecting all serum of animals, and purifying to obtain the RAC1 protein polyclonal antibody.

Property measurement and result analysis

According to the measurement, the titer of the RAC1 protein monoclonal antibody prepared in the example 1 reaches 10E 4-10E 5, and the RAC1 protein monoclonal antibody prepared in the example 1 has strong binding capacity with an RAC1 antigen; the method can accurately and rapidly detect the RAC1 protein, specifically combines with the RAC1 protein, ensures no cross reaction with other proteins of RHO protein family and the like, and is favorable for being widely applied to immunological detection.

The foregoing description of the preferred embodiments of the present application is not intended to be limiting, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the present application.

SEQUENCE LISTING

<110> Shaoxing Ke medical health science and technology Co., ltd

<120> RAC1 protein monoclonal antibody, preparation method and application thereof

<130> 2022/02/18

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 118

<212> PRT

<213> Synthesis

<400> 1

Glu Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Thr Ala Phe Met

50 55 60

Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys Asn Ser Tyr Gly Ser Arg Asn Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 2

<211> 107

<212> PRT

<213> Synthesis

<400> 2

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Val Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Val

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

Claims

1. A RAC1 protein monoclonal antibody, characterized in that the sequence of said RAC1 protein monoclonal antibody comprises: the amino acid sequence of the heavy chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the RAC1 protein monoclonal antibody sequence is SEQ ID NO.2;

wherein SEQ ID NO.1 is EVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWRGGSTDYNTAFMSRLRITKDNSKSQVFFKMNSLQADDTAIYYCAKNSYGSRNFDVWGAGTTVTVSS;

SEQ ID NO.2 is DIVMTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPVTFGAGTKLELK.

2. The RAC1 protein monoclonal antibody according to claim 1, wherein the relative molecular mass of the RAC1 protein monoclonal antibody is 150-160 kDa.

3. The RAC1 protein monoclonal antibody according to claim 1, wherein the RAC1 protein monoclonal antibody has an antibody titer of 10e 4-10 e5.

4. Use of the RAC1 protein monoclonal antibody according to any one of claims 1-3 in the preparation of a detection reagent for RAC1 protein.

5. A kit for detecting RAC1 protein, characterized in that it comprises the RAC1 protein monoclonal antibody according to any one of claims 1 to 3.