CN114426582A

CN114426582A - Nerve specific enolase monoclonal antibody and preparation method and application thereof

Info

Publication number: CN114426582A
Application number: CN202210011887.9A
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: 2022-05-03
Anticipated expiration: 2042-01-07
Also published as: CN114426582B

Abstract

The invention relates to a nerve specific enolase monoclonal antibody and a preparation method and application thereof, belonging to the technical field of biology. The monoclonal antibody 4C11 for resisting the nerve specific enolase comprises a light chain and a heavy chain, wherein the light chain belongs to kappa, the heavy chain belongs to IgM, 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. The nerve specific enolase monoclonal antibody provided by the invention has good specificity and high titer.

Description

Nerve specific enolase monoclonal antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a nerve specific enolase monoclonal antibody and a preparation method and application thereof.

Background

Enolase is a key enzyme in the glycolysis process in organisms, and in vertebrates, 3 isoenzymes, alpha, beta and gamma, exist for enolase (enolase). α -enolase (ENO1), also known as non-neuronal enolase (non-neuronal enolase, NNE), is present in many tissues; beta-enolase (ENO3), also known as muscle-specific enolase (MSE), skeletal muscle enolase (skeletalslemuscelenolase), is found almost exclusively in muscle tissue; γ -enolase (ENO2), also known as neuroenolase (neuroenolase), neuron-specific enolase (NSE), is found primarily in neurons and neuroendocrine tissues. Enolase is a dimer in its active form, 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.

Enolase 2(ENO2), also known as Neuron Specific Enolase (NSE), NSE, is mainly present 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 serum NSE level 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.

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 a nerve-specific enolase monoclonal antibody and a preparation method and application thereof. The nerve specific enolase monoclonal antibody provided by the invention has good specificity and high titer.

The invention provides a monoclonal antibody 4C11 for resisting nerve specific enolase, wherein the monoclonal antibody 4C11 comprises a light chain and a heavy chain, the light chain belongs to kappa, the heavy chain belongs to IgM, 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 monoclonal antibody 4C11 is shown as SEQ ID NO.7, and the nucleotide sequence of the variable region of the heavy chain of monoclonal antibody 4C11 is shown as SEQ ID NO. 8.

Preferably, the amino acid sequence of the variable region of the light chain of monoclonal antibody 4C11 is shown in SEQ ID NO.9, and the amino acid sequence of the variable region of the heavy chain of monoclonal antibody 4C11 is shown in SEQ ID NO. 10.

The invention also provides an expression vector of the monoclonal antibody 4C11 in the technical scheme.

Preferably, the backbone vector of the expression vector comprises pFUSE-CHIg-mG1 and pFUSE2 ss-CLIg-mk.

The invention also provides a cell over-expressing the monoclonal antibody 4C 11.

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

The invention also provides a preparation method of the monoclonal antibody 4C11, which comprises the following steps: the heavy chain and light chain sequences of monoclonal antibody 4C11 were cloned into expression vectors, transfected into cells, and expressed to give monoclonal antibody 4C 11.

The invention also provides application of the monoclonal antibody 4C11 in the technical scheme in preparation of a kit for specifically detecting the nerve specific enolase.

The invention provides a nerve specific enolase monoclonal antibody. The invention provides an ENO2 monoclonal antibody with good specificity and high titer, and a recombinant antibody expression vector and cells are obtained through 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 ENO2 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;

FIG. 5 shows the effect of monoclonal antibody tested by ELISA provided by the present invention;

FIG. 6 shows the effect of the monoclonal antibody of 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 4C11 for resisting nerve specific enolase, wherein the monoclonal antibody 4C11 comprises a light chain and a heavy chain, the light chain belongs to kappa, the heavy chain belongs to IgM, 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 (CDR 1: Arg-Ala-Ser-Gln-Asp-Ile-Ser-Asn-Tyr-Leu-Asn (SEQ ID No. 1); CDR 2: Tyr-Thr-Ser-Arg-Leu-His-Ser (SEQ ID No. 2); CDR 3: Gln-Gln-Gly-Asn-Thr-Leu-Pro-Tyr-Thr (SEQ ID No.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 (CDR 1: Asp-Tyr-Tyr-Met-Ser (SEQ ID No. 4); CDR 2: Phe-Ile-Arg-Asn-Lys-Ala-Asn-Gly-Tyr-Thr-Thr-Glu-Tyr-Ser-Ala-Ser-Val-Lys-Gly (SEQ ID NO. 5); CDR 3: Asp-Gly-Asp-Gly-Ser-Ala-Arg (SEQ ID NO. 6)). The antibody is obtained by carrying out 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 of the invention has good specificity and high titer.

In the invention, the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 4C11 is shown as SEQ ID NO.7, and the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody 4C11 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 4C11 is shown as SEQ ID NO.9, and the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 4C11 is shown as SEQ ID NO. 10.

The invention also provides an expression vector of the monoclonal antibody 4C11 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 construction of a heavy chain expression vector, and pFUSE2ss-CLIg-mk is used for construction of 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 over-expressing the monoclonal antibody 4C 11. In the present invention, the cell is preferably obtained by transfection using the above-mentioned expression vector. In the present invention, the cells preferably comprise eukaryotic cells, which preferably comprise 293T cells or CHO cells.

The present invention provides a neural specific enolase monoclonal antibody, a preparation method and applications thereof, which are described in detail below with reference to specific examples, but the technical scheme 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. The human ENO2 gene sequence (SEQ ID NO.11) with the sequence number of NM-001975.3 is searched from a GenBank sequence database, the gene sequence is synthesized to pET-32a vector, and then the target gene ENO2 is connected to pGEX 4t-1 vector. ENO2 is synthesized on a pET-32a vector, which is marked as ENO2-32a, and the selected enzyme cutting sites are NdeI and NotI; ENO2 is connected to a pGEX 4t-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 single clone to a corresponding resistant LB culture medium, and shaking for 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 coating ELISA plate.

Step two, ENO2 protein expression and purification

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

2. selecting a monoclonal antibody, inoculating the monoclonal antibody into 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 the strain 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 ENO 2-his; the ENO2 purified protein expressed by pGEX 4t-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 the ENO2 antibody.

Example 2

Obtaining hybridoma cells

Step one, immunizing a mouse by using ENO2 antigen

1. Mixing 40ug of purified ENO2-his antigen with complete perfluor adjuvant 1: 1;

2. taking 5 female Balb/c mice of 6-8 weeks old, injecting 100ul (40ug antigen) of the mixed antigen into the left rear calf muscle; three weeks later, the second immunization is carried out, 40ug of antigen is mixed with incomplete Freund's adjuvant 1:1, and 100ul (40ug of antigen) of mixed antigen is injected into the muscle of the lower leg at the right side;

step two, ELISA detection of antibody production of mice

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 ℃,

coating liquid: 25mL carbonate buffer pH 9.6 (Na)₂CO₃ 0.03975 g，NaHCO₃ 0.07325g，KH₂PO₄0.00625 g)；

PBST washing 3 times, each time for 3min, each time drying;

3.2% BSA was blocked in full wells and incubated at 37 ℃ for 1 h;

PBST washing 3 times, each time for 3min, each time drying;

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: diluting with 64 ten thousand, 1:128 thousand, 1:256 thousand and 1:512 ten thousand, adding 100ul of each well into the plate, incubating for 1h at 37 ℃, washing 3 times with PBST, 3min each time, and drying each time;

6. goat anti-mouse IgG-HRP 1:5000 dilution and incubation at 37 ℃ for 30 min.

PBST 3 times washing, after patting dry, TMB color development 10min, 2M H₂SO₄The reaction was terminated and absorbance was measured at 450 nm. The results are shown in figure 1, and the mouse antibody titers No. 33 and No. 44 are more than 1024 ten thousand and more than 500 ten thousand (No. 33 and No. 44 are numbers of two of 5 mice) relative to the non-immunized normal mice. The results show that the antibody titers of the No. 33 mouse and the No. 44 mouse are high, and the antibodies can be used for subsequent experiments.

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 are selected.

2. Preparation of splenocytes

Taking No. 44 mouse with good immune effect, removing eyeball, collecting blood, separating serum, and killing mouse by dislocation of neck, soaking in 75% alcohol for 5min for sterilization; fixing the mouse in a super clean bench, taking out spleen, and removing adipose tissue and connective tissue of adherent cells with 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

A water bath kettle at 37 ℃ is prepared in a super clean bench, and SP2/0 cells and splenocytes are added into a 50ml centrifuge tube according to the proportion of 1:10 and are mixed evenly. Centrifuging for 10min at 500g, sucking out supernatant, and flicking the bottom of the centrifuge tube to slightly loosen cell precipitate; slowly dropping 1mL of 45% PEG1450 solution preheated to 37 ℃ 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, lmL was added dropwise 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 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 96-well cell culture plate with macrophage cells, 100 uL/well, placing the culture plate at 37 deg.C and 5% CO₂Culturing in an incubator.

Step three, ELISA detection of positive hybridoma cells

1. ELISA plates were coated with ENO2(ENO2-GST) protein expressed in pGEX 4t-1 vector, 100 ng/well and coated overnight at 4 ℃.

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

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. Finally, 1 positive clone cell was selected and named: 4C 11.

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 10⁶Every 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. 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 were coated with ENO2 expressed from pGEX 4t-1 vector at 100 ng/well overnight.

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

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

4. HRP-labeled secondary antibodies (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, IgG kappa chain) were incubated and incubated at 37 ℃ for 30 min.

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

The results are shown in FIG. 3, and based on the negative control, 4C11 showed significantly higher IgM and kappa than the negative control mice. Thus, the 4C11 monoclonal antibody has an IgM for the heavy chain and a kappa for the light chain.

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 using a 5' RACE method;

3. connecting the amplified fragment to a pEASY-Blunt vector, extracting a plasmid, and sequencing; antibody sequencing results were obtained:

4C11 antibody light chain variable region gene sequence (SEQ ID NO. 7):

ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAACAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGGGGGGGGACCAGGCTGGAAATAAGA。

4C11 light chain variable region amino acid sequence (SEQ ID NO. 9):

MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTRLEIR。

4C11 heavy chain variable region gene sequence (SEQ ID NO. 8):

ATGAAGTTGTGGCTGAACTGGATTTTCCTTGTAACACTTTTAAATGGTATCCAGTGTGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCACTGATTACTACATGAGCTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGTTTTATTAGAAACAAAGCTAATGGTTACACAACAGAGTACAGTGCATCTGTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTTCAAATGAACACCCTGAGAGCTGAGGACAGTGCCACTTATTACTGTGCAAGAGATGGGGACGGTAGTGCCCGCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA。

4C11 heavy chain variable region amino acid sequence (SEQ ID No.10):

MKLWLNWIFLVTLLNGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDGDGSARWGQGTLVTVSA。

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 in which 293T cells are planted in advance;

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

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

5) preparing SDS-PAGE gel, loading the boiled protein with 20ul, and running to the bottom of the gel when the voltage of 120V is changed to blue loading;

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 1 h;

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 (4-2 in FIG. 4) and normal murine blood as a negative control (4-3 in FIG. 4). The monoclonal antibody 4C11 can specifically recognize ENO2 antigen (4-1 in figure 4), and has high specificity in 293T cells and human serum.

ELISA verification of Effect of monoclonal antibodies

1) Construction of vectors

Respectively constructing ENO1(SEQ ID NO.12) and ENO3(SEQ ID NO.13) on a pGEX 4t-1 vector; ENO1 has sequence number NM _001428.5, ENO3 has sequence number NM _ 001374524.1;

2) the constructed plasmid is transformed to BL21(DE3) to express competence, shake bacteria and ITPG to induce expression;

3) collecting thalli, centrifuging after ultrasonication, collecting supernatant, and purifying by a GST column;

4) marking the obtained ENO1 and ENO3 proteins as ENO1-GST and ENO3-GST, dialyzing the proteins, concentrating, 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) the coated ELISA plate was washed 3 times with PBST and blocked for 1 hour with 2% BSA full plate;

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

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

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

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

11) TMB color development 10min, 2M H₂SO₄The reaction was terminated and absorbance was measured at 450 nm.

The experimental results are shown in Table 1 and FIG. 5, and the 4C11 monoclonal antibody can specifically recognize the ENO2 antigen.

Table 1 ELISA test of the Effect of monoclonal antibodies

3. Immunofluorescence verification of effect of monoclonal antibody

1) Construction of vectors

Respectively constructing ENO2(ENO2flag), an ENO2 front half segment (ENO21flag) and an ENO2 rear half segment (ENO22flag) on a eukaryotic expression vector pcDNA3.1, and using large upgraded particles for later use;

2) spreading the slide in 10cm cell culture dish, treating with polylysine, uniformly inoculating 293T cells in the dish, placing at 37 deg.C and 5% CO₂Culturing the cells 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 the eyeball blood of an immunized mouse and the ascites generated by injecting the mouse with positive hybridoma cells respectively at room temperature for 1 h;

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

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

The experimental results are shown in FIG. 6, and the immunofluorescence results show that the 4C11 monoclonal antibody can recognize the ENO2 antigen.

Example 7

Recombinant 4C11 monoclonal antibody and action effect thereof

Step one, constructing an antibody expression vector

1. The sequenced antibody heavy chain and light chain variable regions are respectively connected to antibody expression vectors pFUSE-CHIg-mG1 and pFUSE2ss-CLIg-mk which are marked as 4C11mG1 and 4C11 mk.

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 inoculating 293T cells in the dish, placing at 37 deg.C and 5% CO₂Culturing the cells in a cell culture box overnight;

2. cell transfection: the constructed recombinant antibody expression plasmid 4C11mG1, 4C11mk, 4C11mG1+4C11mk and mG1+ mk were transferred to the prepared 293T cells, mG1+ mk was an empty vector control, 37 ℃, 5% CO₂Culturing for 48 h.

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

4. blocking with 2% BSA37 ℃ in full plate for 1 hour, and washing with PBST 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 30 min.

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

TMB development for 10min, 2M H₂SO₄The reaction was terminated and absorbance was measured at 450 nm.

The experimental results are shown in table 2, the recombinant 4C11 antibody, namely the heavy chain 4C11mG1 and the light chain 4C11mk, are transfected alone and cannot recognize the antigen ENO 2; by co-transfecting the heavy and light chains, significant signal detection was possible. The recombinant 4C11 monoclonal antibody was successfully expressed in 293T cells and secreted into the cell supernatant. The recombinant monoclonal antibody can recognize ENO2 protein and has biological activity.

Table 2 ELISA verification of recombinant 4C11 monoclonal antibody effect

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 Alexa Fluor 594 for 30min at room temperature;

PBST 3 times washing, Alexa Fluor 594 labeled secondary antibody incubation for 30 min;

PBST was washed 3 times and observed by microscope.

The experimental results are shown in FIG. 7, the heavy chain 4C11mG1 and the light chain 4C11mk 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 4C11 monoclonal antibody was successfully expressed in 293T cells and successfully secreted into the cell supernatant. The recombinant monoclonal antibody can recognize ENO2 protein 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 decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asp Gly Asp Gly Ser Ala Arg

1 5

<210> 7

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg taccagatgt 60

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 120

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 180

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 240

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggaacaa 300

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcgggggg 360

gggaccaggc tggaaataag a 381

<210> 8

<211> 411

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgaagttgt ggctgaactg gattttcctt gtaacacttt taaatggtat ccagtgtgag 60

gtgaagctgg tggagtctgg aggaggcttg gtacagcctg ggggttctct gagactctcc 120

tgtgcaactt ctgggttcac cttcactgat tactacatga gctgggtccg ccagcctcca 180

ggaaaggcac ttgagtggtt gggttttatt agaaacaaag ctaatggtta cacaacagag 240

tacagtgcat ctgtgaaggg tcggttcacc atctccagag ataattccca aagcatcctc 300

tatcttcaaa tgaacaccct gagagctgag gacagtgcca cttattactg tgcaagagat 360

ggggacggta gtgcccgctg gggccaaggg actctggtca ctgtctctgc a 411

<210> 9

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Arg

115 120 125

<210> 10

<211> 137

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Lys Leu Trp Leu Asn Trp Ile Phe Leu Val Thr Leu Leu Asn Gly

1 5 10 15

Ile Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe

35 40 45

Thr Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu

50 55 60

Glu Trp Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu

65 70 75 80

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

85 90 95

Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser

100 105 110

Ala Thr Tyr Tyr Cys Ala Arg Asp Gly Asp Gly Ser Ala Arg Trp Gly

115 120 125

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. The monoclonal antibody 4C11 for resisting the neural specific enolase is characterized in that the monoclonal antibody 4C11 comprises a light chain and a heavy chain, the light chain belongs to kappa, the heavy chain belongs to IgM, 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.

2. The monoclonal antibody 4C11, wherein the nucleotide sequence of the variable region of the light chain of monoclonal antibody 4C11 is shown in SEQ ID No.7 and the nucleotide sequence of the variable region of the heavy chain of monoclonal antibody 4C11 is shown in SEQ ID No. 8.

3. The monoclonal antibody 4C11, wherein the amino acid sequence of the variable region of the light chain of monoclonal antibody 4C11 is shown in SEQ ID No.9 and the amino acid sequence of the variable region of the heavy chain of monoclonal antibody 4C11 is shown in SEQ ID No. 10.

4. An expression vector for the monoclonal antibody 4C11 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 pFUSE2 ss-CLIg-mk.

6. A cell overexpressing the monoclonal antibody 4C11 according to any one of claims 1 to 3.

7. The cell of claim 6, wherein the cell type comprises a eukaryotic cell comprising a 293T cell or a CHO cell.

8. A method for preparing the monoclonal antibody 4C11 of any one of claims 1-3, comprising the steps of: the heavy chain and light chain sequences of monoclonal antibody 4C11 were cloned into expression vectors, transfected into cells, and expressed to give monoclonal antibody 4C 11.

9. Use of the monoclonal antibody 4C11 of any one of claims 1-3 in the preparation of a kit for the specific detection of a neural specific enolase.