CN114426581B

CN114426581B - Enolase monoclonal antibody and preparation method and application thereof

Info

Publication number: CN114426581B
Application number: CN202210011886.4A
Authority: CN
Inventors: 闫亚平; 郝文斌; 穆瑜; 张亚剑; 王璐; 刘龙月; 李科
Original assignee: Shaanxi Mybiotech Co ltd
Current assignee: Shaanxi Mybiotech Co ltd
Priority date: 2022-01-07
Filing date: 2022-01-07
Publication date: 2023-02-03
Anticipated expiration: 2042-01-07
Also published as: CN114426581A

Abstract

The invention relates to an enolase monoclonal antibody and a preparation method and application thereof, belonging to the technical field of biology. The monoclonal antibody 12D11 of the present invention comprises a light chain and a heavy chain, wherein the light chain belongs to kappa, the heavy chain belongs to IgG1, the nucleotide sequences of the 3 complementarity determining regions of the variable region of the light chain are respectively shown as SEQ ID nos. 1-3, and the nucleotide sequences of the 3 complementarity determining regions of the variable region of the heavy chain are respectively shown as SEQ ID nos. 4-6. The enolase monoclonal antibody provided by the invention has the advantages of good specificity, high titer and high purity, and can be used for specifically detecting ENO1, ENO2 and ENO3.

Description

Enolase monoclonal antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to an enolase monoclonal antibody and a preparation method and application thereof.

Background

Enolase (enolase) is a key enzyme in the glycolysis process in organisms, and in vertebrates, 3 isoenzymes, α, β, γ, exist for enolase. α -enolase (ENO 1), also known as non-neuronal enolase (nce), is present in many tissues; beta-enolase (ENO 3), also known as muscle-specific enolase (MSE), skeletal muscle enolase (skeletelmucolase), is found almost exclusively in muscle tissue; γ -enolase (ENO 2), also known as neuronal enolase (neuro-lass), is a neuron-specific enolase (NSE) that is found primarily in neurons and neuroendocrine tissues. The active form of Enolase is a dimer, i.e., composed of two subunits, and 5 combinations of forms, α α α, β β, γ γ, α β, α γ, are currently known.

Alpha-enolase (ENO 1) is a multifunctional protein that, in addition to acting as a rate-limiting enzyme in glycolysis, is also involved in other biological activities in the cell. The vital activities of the cells require energy supply, and the alpha-enolase maintains the ATP level of the cells by regulating the energy production process of the cells, so as to ensure the survival of the cells and the execution of physiological functions of the cells. Alpha-enolase is an important enzyme in the energy metabolism process, and the expression level and the activity of the alpha-enolase are also adaptively changed so as to meet the requirements of cells under different states. In addition, alpha-enolase, as an autoantigen, is a cytoplasmic enzyme, and can be expressed in tissues such as kidney, blood vessels, etc., and some cells such as endothelial cell membrane or bound to the cell surface. Alpha-enolase undergoes carbamoylornithine acidification and stimulates the body to produce specific autoantibodies, thus triggering the chronic inflammatory response of rheumatoid arthritis. In the body of a patient with autoimmune retinopathy, anti-alpha-enolase antibody exists, in addition, alpha-enolase also exists in human respiratory epithelial cells, and is used as an autoantigen to induce autoimmune reaction, so that the respiratory epithelial cells are damaged, and airway inflammatory reaction is induced. In addition, alpha-enolase, as an immunogenic tumor-associated antigen, has a poor prognosis and high malignancy of tumors such as lung cancer, and these suggest that alpha-enolase may be a biomarker for predicting the degree of tumor progression.

Enolase 2 (ENO 2), also known as Neuron Specific Enolase (NSE), is found primarily in the cytoplasm of neurons and neuroendocrine cells. Neuron-specific enolase is a nervous system-specific glycolytic enzyme that is ubiquitous in the cytoplasm of organisms and is sensitive to neuronal damage.

NSE was the earliest to be used as a tumor marker of small cell lung cancer, but in recent years, more and more research results show that NSE can be used as a specific marker of various types of neuron damage. In cerebrovascular diseases, especially in cerebral hemorrhage, people pay more and more attention to the cerebrovascular diseases. Determination of NSE is of great significance in diagnosis of cerebral hemorrhage, judgment of disease severity, estimation of prognosis, guidance of treatment and the like.

In brain injury diseases, NSE in nerve cells is released into cerebrospinal fluid and blood through a blood brain barrier, so that the NSE level in serum and the cerebrospinal fluid is increased, the release amount and the release speed of NSE in brain injuries with different degrees and different properties are different, and NSE is continuously increased in patients with wide brain injuries and increasingly serious secondary brain injuries, so that the level of the NSE can reflect the degree of primary brain injuries and the progress of secondary brain injuries. The detection of the NSE level of the serum can provide a new means for the diagnosis and prognosis judgment of cerebrovascular diseases such as craniocerebral injury, cerebral hemorrhage and the like. NSE has excellent potential as a long-term prognostic biomarker and therapeutic index for neurological intensive care.

Muscle specific enolase β -enolase (ENO 3), a marker for identifying muscle damage.

Serum enolase and muscle-specific enolase are specific enzymes for diagnosing Acute Myocardial Infarction (AMI). The total amount and the total activity of enolase are required to be detected in serological research, and the research shows that the total activity of the enolase in serum and the muscle-specific enolase thereof have the characteristics of early rise time and long duration, so that the method is not only favorable for early diagnosis of AMI, but also favorable for diagnosis of AMI with a later visit time, and the dynamic observation of the enzyme activity in a time sequence is favorable for understanding the course evolution of AMI.

Therefore, the detection of the enolase has very important guiding significance for disease diagnosis and prognosis judgment.

However, the traditional hybridoma cells are not easy to store, the cell state is deteriorated after a long time, even the cells are difficult to recover, the amount of the generated antibody is reduced, the sequence of the antibody is unknown, and the antibody cannot be deeply researched.

Disclosure of Invention

The invention aims to provide an enolase monoclonal antibody and a preparation method and application thereof. The enolase monoclonal antibody provided by the invention has the advantages of good specificity, high titer and high purity, and can be used for specifically detecting ENO1, ENO2 and ENO3.

The invention provides a monoclonal antibody 12D11 resisting human enolase, wherein the monoclonal antibody 12D11 comprises a light chain and a heavy chain, the light chain belongs to kappa, the heavy chain belongs to IgG1, the nucleotide sequences of 3 complementarity determining regions of a variable region of the light chain are respectively shown as SEQ ID NO. 1-3, and the nucleotide sequences of 3 complementarity determining regions of the variable region of the heavy chain are respectively shown as SEQ ID NO. 4-6.

Preferably, the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 12D11 is shown as SEQ ID NO.7, and the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody 12D11 is shown as SEQ ID NO. 8.

Preferably, the amino acid sequence of the variable region of the light chain of the monoclonal antibody 12D11 is shown as SEQ ID NO.9, and the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 12D11 is shown as SEQ ID NO. 10.

The invention also provides an expression vector of the monoclonal antibody 12D11 in the technical scheme.

Preferably, the backbone vector of the expression vector comprises pFUSE-CHIg-mG1 and pFUSE2ss-CLIg-mk.

The invention also provides a cell of the monoclonal antibody 12D11 in the technical scheme.

Preferably, the cell type comprises eukaryotic cells, including 293T cells or CHO cells.

The invention also provides a preparation method of the monoclonal antibody 12D11 in the technical scheme, which comprises the following steps: the heavy chain and light chain sequences of monoclonal antibody 12D11 were cloned into an expression vector, transfected into cells, and expressed to give monoclonal antibody 12D11.

The invention also provides application of the monoclonal antibody 12D11 in the technical scheme in preparation of a kit for specifically detecting enolase.

Preferably, the enolase includes non-neuronal enolase, muscle-specific enolase and neuron-specific enolase.

The invention provides an enolase monoclonal antibody. The enolase monoclonal antibody provided by the invention has the advantages of good specificity, high titer and high purity, and can specifically identify three antigens, namely ENO1, ENO2 and ENO3. Recombinant antibody expression vectors and cells can also be obtained through the sequences of heavy chain and light chain variable regions of the monoclonal antibody. The monoclonal antibody carrier obtained by the invention is easy to store and is easy to control the quality of the antibody production process; and a series of modification, more intensive research and wider application of the antibody are facilitated. The monoclonal antibody obtained by the preparation method can be used for other scientific researches such as enolase antigen detection and the like, and has very good application value and very important scientific research guidance significance.

Drawings

FIG. 1 shows the results of the measurement of the antibody titer of the immunized mouse provided by the present invention;

FIG. 2 shows the result of electrophoresis of the monoclonal antibody provided by the present invention;

FIG. 3 shows the result of the subtype identification of the monoclonal antibody provided by the present invention;

FIG. 4 shows the result of verifying the specificity of the monoclonal antibody by Western Blot provided by the present invention;

FIG. 5 shows the effect of monoclonal antibodies in immunofluorescence assay according to the present invention;

FIG. 6 shows the effect of monoclonal antibody tested by ELISA provided by the present invention;

FIG. 7 shows the expression of the recombinant monoclonal antibody in cells verified by immunofluorescence provided by the present invention.

Detailed Description

The invention provides a monoclonal antibody 12D11 resisting human enolase, wherein the monoclonal antibody 12D11 comprises a light chain and a heavy chain, the light chain belongs to kappa, the heavy chain belongs to IgG1, and the nucleotide sequences of 3 complementarity determining regions of a variable region of the light chain are respectively shown in SEQ ID NO. 1-3 (CDR 1: arg-Ser-Ser-Lys-Ser-Leu-Leu-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Tyr (SEQ ID NO. 1); CDR2: arg-Met-Ser-Asn-Leu-Ala-Ser (SEQ ID NO. 2), CDR3: met-Gln-His-Leu-Glu-Tyr-Pro-Phe-Thr (SEQ ID NO. 3)), and the nucleotide sequences of 3 complementarity determining regions of the variable region of the heavy chain are shown in SEQ ID NO.4 to 6, respectively (CDR 1: ser-Tyr-Asn-Met-His (SEQ ID NO. 4); CDR2: ala-Ile-Ser-Pro-Gly-Asn-Gly-Asp-Thr-Ser-Phe-Asn-Gln-Lys-Phe-Arg-Gly (SEQ ID NO. 5); CDR3: trp-Gly-Leu-Phe-Gly-Glu-Ala-Trp-Phe-Ala-Tyr (SEQ ID NO. 6)). The antibody is obtained by operations such as ENO2 gene synthesis, protein expression and purification, immunization of mice, monoclonal antibody titer determination, myeloma cell preparation, spleen cell preparation, cell fusion, ELISA detection of positive hybridoma cells, monoclonal antibody preparation, antibody subtype classification identification, monoclonal antibody sequence sequencing and the like. The antibody provided by the invention has good specificity and high titer on human enolase, has high purity, and can realize high-efficiency detection of ENO1, ENO2 and ENO3.

In the invention, the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 12D11 is shown as SEQ ID NO.7, and the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody 12D11 is shown as SEQ ID NO. 8.

In the invention, the amino acid sequence of the variable region of the light chain of the monoclonal antibody 12D11 is shown as SEQ ID NO.9, and the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 12D11 is shown as SEQ ID NO. 10.

The invention also provides an expression vector of the monoclonal antibody 12D11 in the technical scheme. In the present invention, the backbone vector of the expression vector preferably includes pFUSE-CHIg-mG1 and pFUSE2ss-CLIg-mk (both available from invitogen). In the invention, pFUSE-CHIg-mG1 is used for constructing a heavy chain expression vector, and pFUSE2ss-CLIg-mk is used for constructing a light chain expression vector. The method for constructing the expression vector of the present invention is not particularly limited, and any method known to those skilled in the art may be used.

The invention also provides a cell of the monoclonal antibody 12D11 in the technical scheme. In the present invention, the cell is preferably obtained by transfection using the above-described expression vector. In the present invention, the cell type preferably comprises eukaryotic cells, preferably comprising 293T cells or CHO cells.

The invention also provides a preparation method of the monoclonal antibody 12D11 in the technical scheme, which comprises the following steps: the heavy chain and light chain sequences of monoclonal antibody 12D11 were cloned into expression vectors, transfected into cells, and expressed to obtain monoclonal antibody 12D11.

In the present invention, the enolase includes non-neuronal enolase, muscle-specific enolase and neuron-specific enolase.

The enolase monoclonal antibody, the preparation method and the application thereof according to the present invention will be described in further detail with reference to the following specific examples, and the technical solution of the present invention includes, but is not limited to, the following examples.

Example 1

ENO2 protein preparation

Step one, construction of ENO2 prokaryotic expression vector

1. Searching an ENO2 gene sequence (SEQ ID NO. 11) with the sequence number of NM-001975.3 from a GenBank sequence database, synthesizing the gene sequence on a pET-32a vector, and connecting the target gene ENO2 to a pGEX4t-1 vector. ENO2 is synthesized on a pET-32a vector, marked as ENO2-32a, and the selected enzyme cutting sites are NdeI and NotI; ENO2 is connected to a pGEX4t-1 vector and is marked as ENO2-4t-1, and the selected enzyme cutting sites are EcoRI and NotI;

2. designing a primer, and amplifying a target band by PCR;

3. carrying out agarose gel electrophoresis on the PCR product, cutting and recovering the gel, and respectively carrying out enzyme digestion on the target fragment and the vector by using corresponding restriction enzymes and recovering;

4. connecting the target fragment and the vector by using homologous recombinase, and connecting for 15min at 50 ℃;

5. the ligation products were transformed into TOP10 competent cells and cultured overnight in an incubator at 37 ℃;

6. selecting a monoclonal antibody to a corresponding resistant LB culture medium, and carrying out shaking table overnight culture at 37 ℃;

7. and extracting plasmids, sequencing and comparing results.

The experimental result shows that the ENO2 prokaryotic expression vector is successfully constructed and is used for the subsequent preparation of ENO2 antigen and the preparation of protein required by detecting ENO2 antibody by a coating ELISA plate.

Step two, ENO2 protein expression and purification

1. Transferring the constructed plasmids ENO2-32a and ENO2-4t-1 into an escherichia coli BL21 (DE 3) expression competent cell;

2. selecting a single clone, inoculating the single clone in an LB culture medium, shaking the strain at 37 ℃ until the OD value is 0.5-1, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction, and expressing overnight at 16 ℃;

3. collecting thalli, carrying out ultrasonic disruption, centrifuging, collecting a supernatant, purifying ENO2 protein expressed by pET-30a by using Ni column affinity chromatography, and marking the purified protein as ENO2-his; the ENO2 purified protein expressed by pGEX4t-1 is marked as ENO2-GST, purified by a GST column, dialyzed and concentrated to obtain ENO2-his protein with high concentration as antigen for later use, and the ENO2-GST protein is used for coating an ELISA plate to detect ENO2 antibody.

Example 2

Obtaining hybridoma cells

Step one, immunizing mice by ENO2 antigen

1. Mixing 40ug of purified ENO2-his antigen with complete freund's adjuvant 1;

2. taking 5 female Balb/c mice of 6-8 weeks old, injecting 100ul (40 ug antigen) of the mixed antigen into the left rear calf muscle; three weeks later, a second immunization was performed with 40ug of antigen mixed with incomplete freund adjuvant 1, and the right posterior calf was intramuscularly injected with 100ul (40 ug of antigen) of the mixed antigen;

step two, ELISA is used for detecting the condition that the mouse generates the antibody

Collecting tail blood after three weeks after the second immunization, centrifuging to collect serum, and detecting the condition of the antibody generated by the mouse by ELISA, wherein the specific steps are as follows:

1. ELISA plates were coated with purified ENO2-GST protein, 100 ng/well, overnight at 4 ℃ and coating solution: 25mL carbonate buffer pH 9.6 (Na2CO30.03975 g, naHCO30.07325g, KH2PO40.00625 g);

PBST wash 3 times, each time for 3min, each time beat dry;

3.2% BSA full-well blocking, incubation at 37 ℃ for 1h;

PBST wash 3 times, each time for 3min, each time beat dry;

5. serum was diluted in multiple dilutions, at 1:4 ten thousand, 1:8 ten thousand, 1:16 ten thousand, 1:32 ten thousand, 1:64 ten thousand, 1, 128 ten thousand, 1, 256 ten thousand, 1, 512 ten thousand dilutions, 100ul per well added to the plate, 37 ℃ incubation for 1h, PBST washing 3 times, each time for 3min, each time beat dry;

6. goat anti-mouse IgG-HRP 1.

PBST was washed 3 times, patted dry, TMB was developed for 10min ₂ SO ₄ The reaction was terminated and absorbance at 450nm was measured. The results are shown in FIG. 1 and Table 1, and the antibody titer of mouse No. 33 is 1024 ten thousand or more and the antibody titer of mouse No. 44 is 500 ten thousand or more (No. 33 and No. 44 are numbers of two of 5 mice) relative to normal mice which were not immunized. From the above results, the antibody titers of mouse 33 and mouse 44 were high, and both of them were used in the subsequent experiments.

TABLE 1 determination of the antibody titer in immunized mice

Step three, cell fusion

1. Preparation of myeloma cells

The SP2/0 cells were recovered and subcultured with 15% fetal bovine serum DMEM for one week. For fusion, myeloma cells in the logarithmic growth phase were selected.

2. Preparation of spleen cells

Taking No. 33 and No. 44 mice with good immunization effect, removing eyeballs, collecting blood, separating serum, killing the mice by cervical dislocation, and soaking in 75% alcohol for 5min for disinfection; fixing the mouse in a super clean bench, taking out spleen, and removing adipose tissue and connective tissue adhered with cells by scissors; washing spleen with serum-free culture solution, placing on a cell filter screen, lightly grinding with an inner core of an injector, gently washing the filter screen with the serum-free culture solution, and collecting spleen cell suspension; centrifuging at 500g for 5min, washing cells for 3 times, discarding supernatant, suspending cell precipitate with serum-free DMEM culture solution, and counting for use.

3. Cell fusion

A37 ℃ water bath was prepared in a clean bench, and SP2/0 cells and splenocytes were added to a 50ml centrifuge tube at a ratio of 1. Centrifuging at 500g for 10min, sucking out supernatant, and gently flicking the bottom of the centrifuge tube to loosen cell precipitate slightly; slowly adding 45% of PEG1450 solution preheated to 37 deg.C 1mL within 90 s; and continuously and lightly shaking the centrifuge tube; the tubes were placed in a37 ℃ water bath throughout the process.

Then, DMEM medium was gradually added to the cell mixture, the lmL was dropwise added for the first minute, 2mL for the second minute, 3mL for 3min, 4mL for 4min, 5mL for 5min, and the mixture was shaken in a water bath at 37 ℃. Then incubated at 37 ℃ for 15min, centrifuged at 500g for 5min, and the supernatant was removed.

5mL of DMEM medium containing HAT was added, the cells were pelleted by gentle suspension, and finally DMED medium containing HAT was added to about 100 mL. Subpackaging in macrophage-plated 96-well cell culture plates at 100 uL/well, and subjecting the plates to 37 ℃ C., 5% CO ₂ And (5) culturing in an incubator.

Step three, ELISA detection of positive hybridoma cells

1. ELISA plates were coated with ENO2 (ENO 2-GST) protein expressed in pGEX4t-1 vector at 100 ng/well overnight at 4 ℃.

2. Observing the growth condition of the hybridoma cells, and after the cell culture supernatant turns yellow seven days later, sucking a proper amount of cell supernatant to detect the antibody by ELISA.

3. According to the ELISA result, selecting the clone with high OD value, wherein the OD value is more than twice of that of the negative control, paving a 96-well plate, and carrying out primary subclone screening.

4. Performing ELISA detection on the antibody after seven to ten days, selecting a clone with a high OD value, laying the clone on a 96-well plate, and performing secondary subclone screening to ensure that each well has about 1 cell;

5. after culturing for seven to ten days, performing ELISA to detect the antibody, selecting the clone with high OD value, laying the clone on a 96-well plate, and performing third subclone screening to ensure that each well has about 1 cell. Several clones with high OD values were selected for further validation (ELISA for antibody detection). Finally screening 1 positive clone cells capable of recognizing three antigens of ENO1, ENO2 and ENO3, and naming the positive clone cells as follows: 12D11.

Example 3

Preparation of monoclonal antibody from ascites and purification of monoclonal antibody

Step one, preparing monoclonal antibody from ascites

1. 300ul ascites adjuvant was injected into the abdominal cavity of 12 week old Balb/c mice.

2. Two weeks later, the hybridoma cells were cultured to optimize cell viability and the cell number was adjusted to about 1X 10 ⁶ Every 100ul, 100ul hybridoma cells were inoculated into the abdominal cavity of the mice injected with ascites adjuvant.

Ascites was collected after 3.7-10 days.

Step two, monoclonal antibody purification

1. The ascites fluid was diluted with PBS pH7.4, centrifuged to take the supernatant, and purified by protein G affinity chromatography.

2. Balancing: equilibrating the column with 0.4M PB buffer (pH 7.0);

3. and (3) column mounting: slowly passing the diluted ascites supernatant through a column to ensure that the antibody is better combined on a protein G column;

4. washing: washing the column with equilibration buffer;

5. and (3) elution: the antibody bound to the column was eluted with 0.1M glycine buffer (pH 2.7) and glycine was neutralized by adding 1M Tris-HCl (pH 8.0) to maintain pH at neutrality suitable for antibody preservation.

The purified monoclonal antibody and the control serum were subjected to SDS-PAGE, and the results are shown in FIG. 2, in which the heavy chain and the light chain of the antibody were clearly seen.

Example 4

Typing and identification of monoclonal antibody

1. ELISA plates, 100 ng/well, were coated overnight with ENO2 expressed from pGEX4t-1 vector.

2. The coated ELISA plates were washed 3 times with PBST and blocked for 1 hour with 2% BSA full wells;

3. adding hybridoma cell supernatant 100ul, and incubating at 37 ℃ for 1h; washed 3 times with PBST;

4. HRP-labeled secondary antibodies (IgG 1, igG2a, igG2b, igG2c, igG3, igM, igGkappa chain) were incubated at 37 ℃ for 30min.

PBST was washed 3 times, TMB was developed, and absorbance was measured at 450 nm.

The results of the experiment are shown in fig. 3 and table 2, where the 12D11 monoclonal antibody has a heavy chain of IgG1 and a light chain of κ.

TABLE 2 monoclonal antibody typing identification

Example 5

Sequencing of monoclonal antibodies

1. Extracting RNA from the cultured hybridoma cells, and performing reverse transcription to obtain cDNA;

2. amplifying antibody heavy chain and light chain variable region fragments by a 5' RACE method;

3. connecting the amplified fragment to a pEASY-Blunt vector, extracting a plasmid, and sequencing; the antibody sequencing results are shown in appendix 4;

4. the CDR regions of the antibody amino acid sequence were labeled using Kabat method.

Example 6

Effect of monoclonal antibody application

Western verification of the specificity of monoclonal antibodies

1) Adding a flag tag at the C terminal of ENO2, and connecting to a eukaryotic expression vector pcDNA3.1;

2) Transfecting the constructed ENO2flag-pcDNA3.1 and the empty vector control pcDNA3.1 to a 10cm cell culture dish;

3) After 48h, the cells were harvested, sonicated, and centrifuged at 15000rpm for 20min to collect the supernatant albumin.

4) Collecting 50ul protein supernatant, adding 10ul 6 × protein Loading, and metal bath boiling at 100 deg.C for 10min;

5) Preparing SDS-PAGE gel, loading the boiled protein at 20ul, and applying 120V voltage to blue loading to the bottom of the gel;

6) Sealing the PVDF film by using 5 percent skim milk powder for 2 hours;

7) Incubating hybridoma cell supernatant for 2h, washing for 3 times by TBST, and incubating by HRP-labeled secondary antibody for 1h;

8) TBST was washed 3 times and ECL developed exposure. The results are shown in FIG. 4: ENO2 mab was a commercial antibody purchased as a positive control, and normal mouse blood as a negative control. The monoclonal antibody 12D11 can recognize ENO2 antigen and has high specificity in 293T cells and human serum.

2. Immunofluorescence for verifying effect of monoclonal antibody

1) Construction of vectors

Respectively constructing ENO2 (ENO 2 flag), an ENO2 front half section (ENO 21 flag) and an ENO2 rear half section (ENO 22 flag) on a eukaryotic expression vector pcDNA3.1, and using large upgraded particles for later use;

2) Spreading the slide in a 10cm cell culture dish, treating with polylysine, uniformly seeding 293T cells in the dish, standing at 37 deg.C and 5% CO ₂ Culturing in a cell culture box overnight;

3) Transfecting ENO2flag, ENO21flag and ENO22flag to the prepared 293T cells;

4) After transfecting the cells for 48 hours, removing the culture solution, fixing the cells for 30min by using acetone, and drying the cells in an incubator for later use;

5) Incubating the tablets with ascites generated by mice injected with immune mouse eyeball blood and positive hybridoma cells respectively at room temperature for 1h;

6) PBST washing 3 times, alexaFluor 594 labeled secondary antibody incubation for 30min;

7) PBST was washed 3 times and observed under microscope.

The experimental results are shown in FIG. 5, and the immunofluorescence results show that the 12D11 monoclonal antibody can recognize the ENO2 antigen.

ELISA verification of Effect of monoclonal antibodies

1) Construction of vectors

Respectively constructing ENO1 (SEQ ID NO. 12) and ENO3 (SEQ ID NO. 13) in pGEX4t-1

On a carrier; ENO1 serial number is NM _001428.5, ENO3 serial number is NM _001374524.1;

2) Transforming the constructed plasmid into BL21 (DE 3) expression competence, shaking bacteria and ITPG induction expression;

3) Collecting thalli, centrifuging after carrying out ultrasonic disruption, collecting supernatant, and purifying by using a GST column;

4) Marking the obtained ENO1 and ENO3 proteins as ENO1-GST and ENO3-GST, dialyzing and concentrating the proteins, and measuring the protein concentration;

5) Coating ELISA plates with ENO1-GST, ENO2-GST and ENO3-GST at 100 ng/hole overnight at 4 ℃;

6) Washing the coated ELISA plates 3 times with PBST, then blocking with 2% BSA full plates for 1 hour;

7) Washing with PBST for 3 times, and drying each time;

8) Adding hybridoma cell supernatant 100ul, and incubating at 37 ℃ for 1h; washing with PBST for 3 times, and drying each time;

9) HRP-labeled secondary antibodies were incubated at 37 ℃ for 30min.

10 Wash 3 times with PBST, pat dry each time;

11 10min of color development of TMB, 2M H ₂ SO ₄ The reaction was terminated and absorbance at 450nm was measured.

The experimental results are shown in table 3 and fig. 6, and the 12D11 monoclonal antibody can identify three antigens, namely ENO1, ENO2 and ENO3, and can be applied to the detection of any isoenzyme of enolase.

TABLE 3 ELISA test of the Effect of monoclonal antibodies

Example 7

Recombinant 12D11 monoclonal antibody and effect thereof

Step one, constructing an antibody expression vector

1. Respectively connecting the sequenced antibody heavy chain and light chain variable regions to an antibody expression vector

And pFUSE-CHIg-mG1 and pFUSE2ss-CLIg-mk are marked as 12D11mG1 and 12D11mk.

2. After the sequencing is correct, a large amount of plasmids are extracted and used for preparing the antibody by cell transfection.

Step two, ELISA verification of the effect of the recombinant monoclonal antibody

1. Spreading the slide in 10cm cell culture dish, treating with polylysine, uniformly seeding 293T cells in the dish, standing at 37 deg.C and 5% CO ₂ Culturing in a cell culture box overnight;

2. cell transfection: the constructed recombinant antibody expression plasmids 12D11mG1, 12D11mk,

12D1mG1 + 12D1mk, mG1+ mk into the 293T cells prepared above, mG1+ mk as empty vector control, 37 ℃,5% CO ₂ Culturing for 48h.

3. Coating the purified ENO2 protein on an ELISA plate at 100 ng/well and overnight at 4 ℃;

4. plate-full blocking with 2% BSA37 ℃ for 1 hour, PBST washing 3 times;

5. collecting the cultured cell supernatant, centrifuging to obtain the supernatant, adding 100ul of the supernatant into a well-coated ELISA plate hole, taking the hybridoma cell supernatant as a positive control, and incubating for 1h at 37 ℃; washing with PBST for 3 times, and drying each time;

6. HRP-labeled anti-heavy chain secondary IgG and anti-light chain secondary IgG Kappa chain were incubated, and the incubation was performed at 37 ℃ for 30min.

7. Washing with PBST for 3 times, and drying each time;

8.20M H, color development of TMB (1: 10 min) ₂ SO ₄ The reaction was terminated and absorbance at 450nm was measured.

The experimental results are shown in table 4, when recombinant 12D11 antibody heavy chain 12D11mG1 and light chain 12D11mk are transfected separately, no signal can be detected from the cell supernatant; heavy chain 12D11mG1 and light chain 12D11mk were co-transfected and anti-ENO 2 antibodies were detected in the cell supernatant. The 12D11 monoclonal antibody was successfully expressed in 293T cells and secreted into the cell supernatant. The recombinant monoclonal antibody can identify ENO2 protein and has biological activity. The recombinant monoclonal antibody can also recognize ENO1 (table 5) and ENO3 (table 6) proteins.

Table 4 ELISA validation of Effect of recombinant 12D11 monoclonal antibody (recognition of ENO 2)

Table 5 ELISA verification of Effect of recombinant 12D11 monoclonal antibody (recognition of ENO 1)

Table 6 ELISA verification of Effect of recombinant 12D11 monoclonal antibody (recognition of ENO 3)

Step three, verifying the expression condition of the recombinant monoclonal antibody in the cell by immunofluorescence

1. Fixing the cells in the step two with acetone for 30min, and drying the cells in an incubator for later use;

2. respectively incubating the prepared slices with anti-heavy chain secondary antibody IgG and anti-light chain secondary antibody Kappa chain marked by AlexaFluor 594 for 30min at room temperature;

PBST wash 3 times, alexaFluor 594 labeled secondary antibody incubate 30min;

PBST was washed 3 times and observed by microscope.

The experimental results are shown in FIG. 7, the heavy chain 12D11mG1 and the light chain 12D11mk can be expressed in 293T cells, and after co-transfection, the expression signals are stronger than those of single transfection; in combination with the above ELISA results, the recombinant 12D11 monoclonal antibody was successfully expressed in 293T cells and successfully secreted into the cell supernatant. The recombinant monoclonal antibody can identify ENO1, ENO2 and ENO3 proteins and has biological activity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Sequence listing

<110> Shanxi vessel Biotechnology GmbH

<120> enolase monoclonal antibody, preparation method and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr

1 5 10 15

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Arg Met Ser Asn Leu Ala Ser

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Gln His Leu Glu Tyr Pro Phe Thr

1 5

<210> 4

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ser Tyr Asn Met His

1 5

<210> 5

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ala Ile Ser Pro Gly Asn Gly Asp Thr Ser Phe Asn Gln Lys Phe Arg

1 5 10 15

Gly

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Trp Gly Leu Phe Gly Glu Ala Trp Phe Ala Tyr

1 5 10

<210> 7

<211> 396

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgaggtgcc tagctgagtt cctggggctg cttgtgctct ggatccctgg agccattggg 60

gatattgtga tgactcaggc tgcaccctct gtacctgtca ctcctggaga gtcagtatcc 120

atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 180

ttcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc 240

tcaggagtcc cagacaggtt cagtggcagt gggtcaggaa ctgctttcac actgagaatc 300

agtagagtgg aggctgagga tgtgggtgtt tattactgta tgcaacatct agaatatcca 360

ttcacgttcg gctcggggac aaagttggaa ataaaa 396

<210> 8

<211> 417

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60

gtgcaactgc agcagcctgg ggctgagctg gtgaagcctg gggcctcagt gaagatgtcc 120

tgcaaggctt ctggttacac atttaccagt tacaatatgc actgggtaaa gcagacacct 180

ggacagggcc tggaatggat tggagctatt tctccaggaa atggtgatac ttccttcaat 240

cagaagttca gaggcaaggc cacattgact gcagacaaat cctccagcac agcctacatg 300

cagctcagca gcctgacatc tgcagactct gcggtctatt actgtgcaag atggggatta 360

ttcggggagg cctggtttgc ttattggggc caagggactc tggtcactgt ctctgca 417

<210> 9

<211> 132

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Leu Trp Ile Pro

1 5 10 15

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

20 25 30

Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser

35 40 45

Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe

85 90 95

Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys

130

<210> 10

<211> 139

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

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

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Ser Pro Gly Asn Gly Asp Thr Ser Phe Asn

65 70 75 80

Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Trp Gly Leu Phe Gly Glu Ala Trp Phe Ala Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

130 135

<210> 11

<211> 1305

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtccatag agaagatctg ggcccgggag atcctggact cccgcgggaa ccccacagtg 60

gaggtggatc tctatactgc caaaggtctt ttccgggctg cagtgcccag tggagcctct 120

acgggcatct atgaggccct ggagctgagg gatggagaca aacagcgtta cttaggcaaa 180

ggtgtcctga aggcagtgga ccacatcaac tccaccatcg cgccagccct catcagctca 240

ggtctctctg tggtggagca agagaaactg gacaacctga tgctggagtt ggatgggact 300

gagaacaaat ccaagtttgg ggccaatgcc atcctgggtg tgtctctggc cgtgtgtaag 360

gcaggggcag ctgagcggga actgcccctg tatcgccaca ttgctcagct ggccgggaac 420

tcagacctca tcctgcctgt gccggccttc aacgtgatca atggtggctc tcatgctggc 480

aacaagctgg ccatgcagga gttcatgatc ctcccagtgg gagctgagag ctttcgggat 540

gccatgcgac taggtgcaga ggtctaccat acactcaagg gagtcatcaa ggacaaatac 600

ggcaaggatg ccaccaatgt gggggatgaa ggtggctttg cccccaatat cctggagaac 660

agtgaagcct tggagctggt gaaggaagcc atcgacaagg ctggctacac ggaaaagatc 720

gttattggca tggatgttgc tgcctcagag ttttatcgtg atggcaaata tgacttggac 780

ttcaagtctc ccactgatcc ttcccgatac atcactgggg accagctggg ggcactctac 840

caggactttg tcagggacta tcctgtggtc tccattgagg acccatttga ccaggatgat 900

tgggctgcct ggtccaagtt cacagccaat gtagggatcc agattgtggg tgatgacctg 960

acagtgacca acccaaaacg tattgagcgg gcagtggaag aaaaggcctg caactgtctg 1020

ctgctcaagg tcaaccagat cggctcggtc actgaagcca tccaagcgtg caagctggcc 1080

caggagaatg gctggggggt catggtgagt catcgctcag gagagactga ggacacattc 1140

attgctgacc tggtggtggg gctgtgcaca ggccagatca agactggtgc cccgtgccgt 1200

tctgaacgtc tggctaaata caaccagctc atgagaattg aggaagagct gggggatgaa 1260

gctcgctttg ccggacataa cttccgtaat cccagtgtgc tgtga 1305

<210> 12

<211> 1305

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgtctattc tcaagatcca tgccagggag atctttgact ctcgcgggaa tcccactgtt 60

gaggttgatc tcttcacctc aaaaggtctc ttcagagctg ctgtgcccag tggtgcttca 120

actggtatct atgaggccct agagctccgg gacaatgata agactcgcta tatggggaag 180

ggtgtctcaa aggctgttga gcacatcaat aaaactattg cgcctgccct ggttagcaag 240

aaactgaacg tcacagaaca agagaagatt gacaaactga tgatcgagat ggatggaaca 300

gaaaataaat ctaagtttgg tgcgaacgcc attctggggg tgtcccttgc cgtctgcaaa 360

gctggtgccg ttgagaaggg ggtccccctg taccgccaca tcgctgactt ggctggcaac 420

tctgaagtca tcctgccagt cccggcgttc aatgtcatca atggcggttc tcatgctggc 480

aacaagctgg ccatgcagga gttcatgatc ctcccagtcg gtgcagcaaa cttcagggaa 540

gccatgcgca ttggagcaga ggtttaccac aacctgaaga atgtcatcaa ggagaaatat 600

gggaaagatg ccaccaatgt gggggatgaa ggcgggtttg ctcccaacat cctggagaat 660

aaagaaggcc tggagctgct gaagactgct attgggaaag ctggctacac tgataaggtg 720

gtcatcggca tggacgtagc ggcctccgag ttcttcaggt ctgggaagta tgacctggac 780

ttcaagtctc ccgatgaccc cagcaggtac atctcgcctg accagctggc tgacctgtac 840

aagtccttca tcaaggacta cccagtggtg tctatcgaag atccctttga ccaggatgac 900

tggggagctt ggcagaagtt cacagccagt gcaggaatcc aggtagtggg ggatgatctc 960

acagtgacca acccaaagag gatcgccaag gccgtgaacg agaagtcctg caactgcctc 1020

ctgctcaaag tcaaccagat tggctccgtg accgagtctc ttcaggcgtg caagctggcc 1080

caggccaatg gttggggcgt catggtgtct catcgttcgg gggagactga agataccttc 1140

atcgctgacc tggttgtggg gctgtgcact gggcagatca agactggtgc cccttgccga 1200

tctgagcgct tggccaagta caaccagctc ctcagaattg aagaggagct gggcagcaag 1260

gctaagtttg ccggcaggaa cttcagaaac cccttggcca agtaa 1305

<210> 13

<211> 1332

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggccgtta tgaggaccct aagagccatg gccatgcaga aaatctttgc ccgggaaatc 60

ttggactcca ggggcaaccc cacggtggag gtggacctgc acacggccaa gggccgattc 120

cgagcagctg tgcccagtgg ggcttccacg ggtatctatg aggctctgga actaagagac 180

ggagacaaag gccgctacct ggggaaagga gtcctgaagg ctgtggagaa catcaacaat 240

actctgggcc ctgctctgct gcaaaagaaa ctaagcgttg tggatcaaga aaaagttgac 300

aaatttatga ttgagctaga tgggaccgag aataagtcca agtttggggc caatgccatc 360

ctgggcgtgt ccttggccgt gtgtaaggcg ggagcagctg agaagggggt ccccctgtac 420

cgccacatcg cagatctcgc tgggaaccct gacctcatac tcccagtgcc agccttcaat 480

gtgatcaacg ggggctccca tgctggaaac aagctggcca tgcaggagtt catgattctg 540

cctgtgggag ccagctcctt caaggaagcc atgcgcattg gcgccgaggt ctaccaccac 600

ctcaaggggg tcatcaaggc caagtatggg aaggatgcca ccaatgtggg tgatgaaggt 660

ggcttcgcac ccaacatcct ggagaacaat gaggccctgg agctgctgaa gacggccatc 720

caggcggctg gttacccaga caaggtggtg atcggcatgg atgtggcagc atctgagttc 780

tatcgcaatg ggaagtacga tcttgacttc aagtcgcctg atgatcccgc acggcacatc 840

actggggaga agctcggaga gctgtataag agctttatca agaactatcc tgtggtctcc 900

atcgaagacc cctttgacca ggatgactgg gccacttgga cctccttcct ctcgggggtg 960

aacatccaga ttgtggggga tgacttgaca gtcaccaacc ccaagaggat tgcccaggcc 1020

gttgagaaga aggcctgcaa ctgtctgctg ctgaaggtca accagatcgg ctcggtgacc 1080

gaatcgatcc aggcgtgcaa actggctcag tctaatggct ggggggtgat ggtgagccac 1140

cgctctgggg agactgagga cacattcatt gctgaccttg tggtggggct ctgcacagga 1200

cagatcaaga ctggcgcccc ctgccgctcg gagcgtctgg ccaaatacaa ccaactcatg 1260

aggatcgagg aggctcttgg ggacaaggca atctttgctg gacgcaagtt ccgtaacccg 1320

aaggccaagt ga 1332

Claims

1. An anti-human enolase monoclonal antibody 12D11, wherein said monoclonal antibody 12D11 comprises a light chain and a heavy chain, said light chain being of kappa, said heavy chain being of IgG1, the amino acid sequences of the 3 complementarity determining regions of the variable region of said light chain being represented by SEQ ID Nos. 1 to 3, respectively, and the amino acid sequences of the 3 complementarity determining regions of the variable region of said heavy chain being represented by SEQ ID Nos. 4 to 6, respectively.

2. The monoclonal antibody 12D11 of claim 1, wherein the amino acid sequence of the variable region of the light chain of monoclonal antibody 12D11 is represented by SEQ ID No.9, and the amino acid sequence of the variable region of the heavy chain of monoclonal antibody 12D11 is represented by SEQ ID No. 10.

3. The monoclonal antibody 12D11 of claim 2, wherein the nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 12D11 is represented by seq id No.7, and the nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 12D11 is represented by seq id No. 8.

4. An expression vector for the monoclonal antibody 12D11 according to any one of claims 1 to 3.

5. The expression vector of claim 4, wherein the backbone vector of the expression vector comprises pFUSE-CHIg-mG1 and pFUSE2ss-CLIg-mk.

6. A cell overexpressing the monoclonal antibody 12D11 according to any one of claims 1 to 3.

7. The cell of claim 6, wherein the cell is a eukaryotic cell.

8. The cell of claim 7, wherein the eukaryotic cell is a 293T cell or a CHO cell.

9. A method for producing the monoclonal antibody 12D11 according to any one of claims 1 to 3, comprising the steps of: the heavy chain and light chain sequences of monoclonal antibody 12D11 were cloned into expression vectors, transfected into cells, and expressed to obtain monoclonal antibody 12D11.

10. Use of the monoclonal antibody 12D11 according to any one of claims 1 to 3 for the preparation of a kit for the specific detection of enolase.

11. The use according to claim 10, wherein the enolase comprises a neuron-specific enolase.

12. The use according to claim 10, wherein the enolase comprises a non-neuronal enolase.

13. Use according to claim 10, wherein the enolase enzyme comprises a muscle-specific enolase enzyme.