Recombinant human VASN protein LRR structural domain protein and monoclonal antibody thereof
1. A gene of recombinant human VASN protein LRR structural domain protein is characterized in that the nucleotide sequence is shown as SEQ ID No. 1.
2. A recombinant human VASN protein LRR structural domain protein is characterized in that the amino acid sequence is shown as SEQ ID No. 2.
3. A recombinant vector comprising a gene of the recombinant human VASN protein LRR domain protein of claim 1, wherein the recombinant vector is pET30a (+) vector.
4. A recombinant engineered strain comprising a gene of the recombinant human VASN protein LRR domain protein of claim 1 or the recombinant vector of claim 3.
5. The recombinant engineered strain of claim 4, wherein the recombinant working strain is Escherichia coli BL21(DE 3).
6. Use of a gene of the recombinant human VASN protein LRR domain protein of claim 1, the recombinant vector of claim 3, or the recombinant engineered strain of any one of claims 4-5 for expressing the recombinant human VASN protein LRR domain protein.
7. A preparation method of a VASN hybridoma cell strain is characterized by comprising the following steps:
a. immunizing an animal with the VASN protein LRR domain protein of claim 2;
b. fusing spleen cells or lymphocytes of the animal with myeloma cells;
c. screening to obtain the VASN hybridoma cell strain.
8. The method of claim 7, wherein the animal is a mouse, the VASN protein LRR domain protein is mixed with an adjuvant, and the animal is immunized by subcutaneous injection, and the volume ratio of the VASN protein LRR domain protein to the adjuvant is 1: 1.
9. A VASN hybridoma cell line produced by the production method according to any one of claims 7 to 8.
10. A VASN monoclonal antibody secreted from the VASN hybridoma cell line of claim 9.
Background
Human Vasorin (VASN), also known as SLIT-like2, was found to participate in the response of injured blood vessels, mainly through down-regulation of TGF- β, in mechanism studies of VASN, ultimately affecting the formation of blood vessel walls. By binding TGF-beta, the signal transduction of TGF-beta is weakened, the migration of vascular endothelial cells is attracted and induced to form a vascular-like structure, and the migration of leukocytes is inhibited. TGF-beta is closely related to tumor occurrence and development, and has important functions in regulating proliferation and differentiation of tumor cells, inhibiting immune function of organisms, increasing angiogenesis, inducing production of extracellular matrix to promote invasion and metastasis of tumors. During the development of tumors, tumor-induced neovascularization, "communication" between tumor cells and endothelial cells has been considered to be of paramount importance. Therefore, the relationship between VASN and tumor is very close. However, antibodies with high specificity against VASN have been lacking to date.
Disclosure of Invention
The present invention has been made in an effort to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a recombinant human VASN protein LRR domain protein and a monoclonal antibody thereof, which can bind VASN antigen existing in a human body with high specificity.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a gene of a recombinant human VASN protein LRR structural domain protein, and the nucleotide sequence of the gene is shown in SEQ ID No. 1.
The invention also provides a recombinant human VASN protein LRR structural domain protein, and the amino acid sequence of the recombinant human VASN protein LRR structural domain protein is shown in SEQ ID No. 2.
The invention also provides a recombinant vector, which comprises the gene of the LRR domain protein of the recombinant human VASN protein, and the recombinant vector is a pET30a (+) vector.
The invention also provides a recombinant engineering strain, which comprises the gene of the recombinant human VASN protein LRR domain protein or the recombinant vector.
Further, the recombinant working strain was Escherichia coli BL21(DE 3).
The invention also provides a gene of the recombinant human VASN protein LRR structural domain protein, the recombinant vector or the recombinant engineering strain, and application of the gene, the recombinant vector or the recombinant engineering strain in expression of the recombinant human VASN protein LRR structural domain protein.
The invention also provides a preparation method of the VASN hybridoma cell strain, which comprises the following steps:
a. immunizing an animal with the VASN protein LRR structural domain protein;
b. fusing spleen cells or lymphocytes of the animal with myeloma cells;
c. screening to obtain the VASN hybridoma cell strain.
Further, the animal is a mouse, the VASN protein LRR domain protein is mixed with an adjuvant and then is immunized in a subcutaneous injection mode, and the volume ratio of the VASN protein LRR domain protein to the adjuvant is 1: 1.
The invention also provides a VASN hybridoma cell strain prepared by the preparation method.
The invention also provides a VASN monoclonal antibody secreted by the VASN hybridoma cell strain.
The invention discloses the following technical effects:
the target protein expression and purification method is simple and easy, the cost is low, and the recombinant protein with high yield and high purity can be finally obtained through the purification of the Ni agarose gel chromatographic column.
The mouse monoclonal antibody of the LRR structural domain protein of the anti-human VASN protein can sensitively recognize natural VASN protein in a human body, and has high specificity. The monoclonal antibody is specifically combined with IgG2b subtype, and the result shows that the monoclonal antibody belongs to IgG2b subtype. The indirect ELISA titer determination of the antibody of the purified monoclonal hybridoma cell strain can reach 1:2187000, which shows that the antibody has higher titer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a diagram of target gene amplification, in which M: nucleic acid Marker, 1, 2: the target gene LRR.
FIG. 2 is an electrophoresis picture of human VASN protein LRR domain protein induced expression, wherein M: protein Marker, 1: recombinant protein supernatant was induced with 1mM IPTG, 2: recombinant protein supernatant was not induced, 3: pET30a (+) control supernatant was induced with 1mM IPTG, 4: pET30a (+) control supernatant was not induced, 5: precipitation of recombinant protein was not induced, 6: precipitation of recombinant protein induced by 1mM IPTG, 7: pET30a (+) control precipitation was not induced, 8: pET30a (+) control precipitation was induced with 1mM IPTG.
Fig. 3 is an elution electrophoretogram of recombinant human VASN protein LRR domain protein, wherein M: protein Marker, 1: column-passing liquid, 2: column wash, 3-8:150mM imidazole eluate protein.
FIG. 4 is the result of immunoblotting detection of recombinant human VASN protein LRR domain protein with anti-His tag antibody.
FIG. 5 shows the subtype identification of the VASN monoclonal antibody secreted by the VASN hybridoma cell strain.
FIG. 6 shows the detection of VASN monoclonal antibody titer secreted by VASN hybridoma cell strain.
FIG. 7 shows the result of immunoblotting detection of VASN protein in normal liver cells and liver cancer cells using VASN monoclonal antibody secreted from VASN hybridoma cell strain.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
On the basis of research that VASN is closely related to the generation and development process of tumor cells, the invention prepares the VASN monoclonal antibody, lays a foundation for further research that VASN can become an effective target for biological treatment of liver cancer, promotes the research processes of using VASN as a tumor marker for screening diseases, treating, judging prognosis and the like, and develops a series of research and exploration around how to convert disease markers of the basic research into products which can be clinically used in the future.
Example 1
Construction of expression vector pET30a (+) -LRR
1.1PCR obtaining LRR
7702 human liver cell Total RNA is extracted by RNA extraction kit of TaKaRa company, cDNA obtained by reverse transcription is taken as a template to be amplified to obtain a target segment, and a PCR reaction system comprises: upstream and downstream primers 0.4. mu.L each, template 2.0. mu.L, PCR Pfu Supermix 10. mu.L, ddH2O7.2 mu L; pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 60s, extension at 72 ℃ for 60s and 38 cycles, extension at 72 ℃ for 5min, and identification of PCR products by 1.0% agarose gel electrophoresis. As can be seen from FIG. 1, there is a clear bright band at approximately 1032bp, consistent with the expected size.
Wherein, the upstream primer LRR-F (B) (SEQ ID No. 3): AAAGGATCCTGCCCATCCGGCTGCCAGTGCA, downstream primer RSF-LRR-R (H) (SEQ ID No. 4): GGGAAGCTTTTAGGTGGGCACTGTGGCTGTGG are provided.
The nucleotide sequence of the target fragment, namely the coding gene of the LRR structural domain protein of the VASN protein is shown as SEQ ID No. 1:
TGCCCATCCGGCTGCCAGTGCAGCCAGCCACAGACAGTCTTCTGCACTGCCCGCCAGGGGACCACGGTGCCCCGAGACGTGCCACCCGACACGGTGGGGCTGTACGTCTTTGAGAACGGCATCACCATGCTCGACGCAGGCAGCTTTGCCGGCCTGCCGGGCCTGCAGCTCCTGGACCTGTCACAGAACCAGATCGCCAGCCTGCCCAGCGGGGTCTTCCAGCCACTCGCCAACCTCAGCAACCTGGACCTGACAGCCAACAGGCTGCATGAAATCACCAATGAGACCTTCCGTGGCCTGCGGCGCCTCGAGCGCCTCTACCTGGGCAAGAACCGCATCCGCCACATCCAGCCTGGTGCCTTCGACACGCTCGACCGCCTCCTGGAGCTCAAGCTGCAGGACAACGAGCTGCGGGCACTGCCCCCGCTGCGCCTGCCCCGCCTGCTGCTGCTGGACCTCAGCCACAACAGCCTCCTGGCCCTGGAGCCCGGCATCCTGGACACTGCCAACGTGGAGGCGCTGCGGCTGGCTGGTCTGGGGCTGCAGCAGCTGGACGAGGGGCTCTTCAGCCGCTTGCGCAACCTCCACGACCTGGATGTGTCCGACAACCAGCTGGAGCGAGTGCCACCTGTGATCCGAGGCCTCCGGGGCCTGACGCGCCTGCGGCTGGCCGGCAACACCCGCATTGCCCAGCTGCGGCCCGAGGACCTGGCCGGCCTGGCTGCCCTGCAGGAGCTGGATGTGAGCAACCTAAGCCTGCAGGCCCTGCCTGGCGACCTCTCGGGCCTCTTCCCCCGCCTGCGGCTGCTGGCAGCTGCCCGCAACCCCTTCAACTGCGTGTGCCCCCTGAGCTGGTTTGGCCCCTGGGTGCGCGAGAGCCACGTCACACTGGCCAGCCCTGAGGAGACGCGCTGCCACTTCCCGCCCAAGAACGCTGGCCGGCTGCTCCTGGAGCTTGACTACGCCGACTTTGGCTGCCCAGCCACCACCACCACAGCCACAGTGCCCACC。
1.2 construction of the pET30a (+) -LRR expression vector
(1) Enzyme digestion: after the PCR product is purified by a purification kit, enzyme BamH I and Hind III are adopted to carry out double enzyme digestion of pET30a (+) and the purified PCR product, the reaction is carried out for 4h at 37 ℃, and the purification is carried out by applying a DNA purification kit of an OMEGA company.
(2) Connecting: mu.L of the target fragment, 2. mu.L of plasmid, 1. mu.L of T4Buffer, 1. mu.L of T4 ligase, and ligation at room temperature for 4 h.
(3) And (3) transformation: the ligation products were transformed into 50. mu.L DH 5. alpha. competent cells, ice-bathed for 30min, heat-shocked at 42 ℃ for 45S, ice-bathed for 2min, added with 800. mu.L LB medium, pre-cultured at 37 ℃ with shaking at 200rpm for 1h, finally spread on a kanamycin-resistant LB plate, and cultured overnight at 37 ℃.
(4) And (3) verification: and selecting a single colony for PCR verification, screening positive clones, sending the positive clones to a sequencing company for sequencing identification, and naming the recombinant plasmid with correct sequencing as pET30a (+) -LRR.
Example 2
Prokaryotic expression of human recombinant VASN protein LRR structural domain protein
2.1 transforming the pET30a (+) -LRR recombinant plasmid into expression host cell Escherichia coli BL21(DE3) to obtain recombinant engineering strain BL21-pET30a (+) -LRR.
2.2 Induction of expression: the recombinant engineered strain BL21-pET30a (+) -LRR was picked and inoculated into 10mL LB liquid medium (containing 25. mu.g/mL kanamycin), at 37 ℃, 200rpm overnight. The next day, 1mL of BL21-pET30a (+) -LRR bacterial solution was added to a new 10mL of LB liquid medium (containing 25. mu.g/mL kanamycin), cultured with shaking at 37 ℃ and 200rpm for about 3 hours, and induced by adding IPTG (1 mM final concentration) while performing plasmid pET30a (+) negative control and induced at 28 ℃ and 200rpm for 4 hours. The cells were centrifuged at 12000g for 1min at 4 ℃ and the lower layer of cells was collected. The cells were resuspended in an appropriate amount of PBS buffer and subjected to 12% conventional SDS-PAGE and identified by Coomassie Brilliant blue, as shown in FIG. 2.
Example 3
Purification and identification of LRR structural domain protein of human recombinant VASN protein
3.1 Inclusion body treatment: 150mL of the cells were induced, and the specific method was as described in example 2, and the cells were resuspended in an appropriate amount of precooled PBS buffer, sonicated in ice bath (380W, 60min, 5s on, 2s off), centrifuged at 12000rpm for 10min at 4 ℃ and the precipitated inclusion bodies were collected. The inclusion bodies were washed with an appropriate amount of washing buffer (20mM Tris-HCl, pH 7.9, 500mM NaCl, 10mM imidazole, 1M urea), centrifuged at 12000rpm for 10min at 4 ℃ for several times until the inclusion bodies were washed clean. The inclusion bodies were dissolved in equilibration buffer (20mM Tris-HCl, pH 7.9, 500mM NaCl, 10mM imidazole, 6M urea), centrifuged at 10000g for 20min after ice bath for 1 hour, and the supernatant was collected.
3.2 passing through a chromatographic column: the supernatant protein solution was loaded onto a Ni Sepharose chromatography column and adsorbed in a shaker at 4 ℃ for 2 hours. The column was washed with 15 column volumes of equilibration buffer (20mM Tris-HCl, pH 7.9, 500mM NaCl, 10mM imidazole, 6M Urea), followed by elution of the protein of interest with 6 column volumes of an eluate containing 150mM imidazole (150mM imidazole eluate (20mM Tris-HCl, pH 7.9, 500mM NaCl, 150mM imidazole, 6M Urea), followed by collection of the eluate, identification by 12% SDS-PAGE and Coomassie Brilliant blue method, and the results are shown in FIG. 3.
3.3 identification of LRR domain proteins: the purified LRR structural domain protein is subjected to 12% SDS-PAGE electrophoresis according to 100ng, 50ng and 20ng respectively, the protein is transferred on a PVDF membrane by a wet transfer method, 5% skimmed milk powder is sealed at room temperature for 2h, primary antibody is incubated overnight at 4 ℃ by using an anti-His tag antibody (1:2000), TBST is washed for three times, horse radish peroxidase-labeled goat anti-mouse IgG (1:10000) is added, the incubation is carried out at room temperature for 1h, and the washing is carried out for three times by using ECL for color development. The results are shown in FIG. 4.
Wherein, the amino acid sequence of the VASN protein LRR structural domain protein is shown as SEQ ID No. 2:
CPSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVFENGITMLDAGSFAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNETFRGLRRLERLYLGKNRIRHIQPGAFDTLDRLLELKLQDNELRALPPLRLPRLLLLDLSHNSLLALEPGILDTANVEALRLAGLGLQQLDEGLFSRLRNLHDLDVSDNQLERVPPVIRGLRGLTRLRLAGNTRIAQLRPEDLAGLAALQELDVSNLSLQALPGDLSGLFPRLRLLAAARNPFNCVCPLSWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYADFGCP。
example 4
Preparation of VASN monoclonal antibody
4.1 immunization of BALB/c mice: the purchased female BALB/c mice (4 weeks old) are firstly raised for one week in the center of experimental animals to ensure the stable growth of the mice, after the mice are adapted to a new environment, the purified VASN-LRR recombinant protein with the concentration of 1 mu g/mu L is used as immunogen to immunize 6 BALB/c female mice, and another female BALB/c female mouse is taken to be injected with PBS solution with the same volume as the negative control. First immunization: the injection is injected at 2-3 points under the abdomen subcutaneous tissue after being fully mixed with the Freund's complete adjuvant with the same volume according to the dosage of 150 mu L per patient, and the interval is 14 days. And (3) second immunization: the injection is injected at 2-3 points under the abdomen subcutaneous tissue after being mixed with Freund's incomplete adjuvant with the same volume according to the dose of 80 mu L per patient, and the interval is 14 days. Third and fourth immunizations: the route and dose of immunization of mice was consistent with the second immunization, with 14 days intervals. After 7 days of the fourth immunization, tail vein blood of a negative control mouse injected with PBS solution and a positive mouse immunized by VASN-LRR recombinant protein is taken, centrifuged at 12000 Xg for 5min, and upper serum is reserved and placed in a refrigerator at 4 ℃.
4.2 Indirect ELISA assay for serum titers: the VASN-LRR recombinant protein with the concentration of 2 mug/mL is taken and coated on an enzyme label plate according to 100 mug/hole, and the enzyme label plate is incubated overnight at 4 ℃. The next day, 300. mu.L/well of PBST wash was added and the wash was repeated 3 times. 1% BSA blocking solution was added at 100. mu.L/well and incubated at 37 ℃ for 30 min. The positive serum and the negative serum samples were diluted 3-fold, from 1:1000 to 1:729000, while 0.1% BSA solution was added as a blank well at 100. mu.L/well, and incubated at 37 ℃ for 60 min. A goat anti-mouse HRP-IgG secondary antibody at a ratio of 1:10000 was added to 100. mu.L/well, and the mixture was incubated at 37 ℃ for 30 min. After the end of each incubation period, the cells were washed 3 times with PBST. Adding substrate color developing solution at 100 μ L/well in dark condition, incubating at 37 deg.C for 15min, adding 2M sulfuric acid at 50 μ L/well to terminate reaction, and detecting OD450The value is obtained. Taking the serum dilution with the P/N value more than or equal to 2.1 as the serum titer of the immune mice, and selecting the immune mice with the serum titer more than 1:10000 for the next cell fusion, wherein P is positive serum OD450Value, N is negative serum OD450The value is obtained.
4.3 culture of SP2/0 myeloma cells: the SP2/0 myeloma cells purchased from the Shanghai institute of biological sciences cell preservation center are subjected to passage and frozen preservation until 5-6 generations are reached, the SP2/0 myeloma cells are stably proliferated and in a good state, and cell fusion operation can be performed. SP2/0 myeloma cells were cultured in 9 flasks and the cells were plated off the day before cell fusion. Before cell fusion, the culture flask from which the culture supernatant was discarded was gently washed with PBS, an appropriate amount of the culture medium was added, the cells were gently pipetted with a bus pipette to exfoliate the cells, the cells were collected in multiple tubes into 15mL centrifuge tubes, centrifuged for 3min (1000rpm), and finally the SP2/0 myeloma cells were collected in their entirety and cultured for 1h in 10mL RPMI1640 medium containing 30% serum in 15mL centrifuge tubes.
4.4 spleen cell preparation: and (2) carrying out cell fusion on the mouse with the serum titer larger than 1:10000, before cell fusion, carrying out intraperitoneal injection on the mouse for boosting immunity, injecting 100-200 mu L, after 3 days, taking the eyeball of the mouse, discharging blood, collecting blood, standing at room temperature for a period of time, centrifuging for 5min at 12000g, and keeping the upper layer of serum as positive control serum for later use at-80 ℃. The immunized mice are killed by means of spondylolisthesis, and then soaked in 75% alcohol for 6-10 min. In a clean bench that had been UV-sterilized for half an hour, the mice were mounted on a flat plate with the abdomen facing up, and the skin was cut open with surgical scissors while the epidermis was stretched with forceps to expose the peritoneum. The peritoneum was scrubbed and disinfected with an alcohol cotton ball, then the peritoneum was cut open with a new pair of surgical scissors, the abdominal viscera were exposed, the spleen on the left side of the abdomen was found, and the spleen was peeled off from the other viscera with forceps. The cells were plated in a petri dish containing 2mL of serum-free RPMI1640 medium, surrounding fat and connective tissue were removed, the spleen was placed on a sieve, the spleen was pressed hard through the sieve with a plunger of a 10mL syringe to disperse the spleen into individual spleen cells, and the cells were rinsed with 10mL of serum-free RPMI1640 medium and collected in a 15mL centrifuge tube.
4.5 cell fusion: the collected splenocytes and SP2/0 myeloma cells were washed with 7mL serum-free RPMI1640 medium, centrifuged at 1050rpm for 4min, discarded, and washed repeatedly three times. 1/5 serum RPMI1640-HAT medium and 50% PEG solution were pre-warmed in a 37 ℃ water bath prior to fusion. 1mL of serum-free RPMI1640 medium was added to SP2/0 myeloma cells, the mixture was transferred to a centrifuge tube containing spleen cells after thoroughly mixing, the mixture was mixed (the ratio of myeloma cells to spleen cells was 1: 2-10), centrifuged at 1050rpm for 4min, the supernatant was discarded, and 1mL of serum-free RPMI1640 medium was added thereto to repeat washing twice. Gently beating the tube bottom of the centrifuge tube to loosen the cells, placing the tube bottom of the centrifuge tube in a 37 ℃ water bath, slowly adding a 50% PEG solution along the tube wall in the first 30s, then gently mixing the solution while dripping the solution for the second 30s, after dripping 1mL of 50% PEG solution, performing the 37 ℃ water bath for 1min, then slowly adding 2mL of a serum-free culture medium along the tube wall, turning the tube upside down and mixing the solution uniformly, continuously adding 4mL of the serum-free culture medium, and placing the centrifuge tube in the 37 ℃ water bath for 10 min. Centrifuging at 1050rpm for 4min, discarding the supernatant, adding 3mL of preheated 1/5 serum RPMI1640-HAT culture medium, mixing, transferring into 50mL of 1/5 serum RPMI1640-HAT culture medium, mixing, spreading into 10 96-well plates with PBS dropped into the outermost edge of the well, and culturing in 37 deg.C cell culture box. After 24 hours, a drop (about 50. mu.L) of 20% HAT medium was added dropwise with a bus pipette, and after 8 to 11 days when hybridoma cells reached 1/5 around the bottom of the well, the cells were cultured in a 20% HT medium complete exchange, and positive wells were screened by indirect ELISA.
4.6 screening positive clones: cell supernatants from each well were collected and screened for positive clones by indirect ELISA as described above. And judging that the hole contains a positive strain according to the P/N ratio of more than 2.1, transferring the positive strain to 24 holes for amplification culture, and continuously re-screening by using an indirect ELISA (enzyme-Linked immuno sorbent assay) once, so that a positive clone strain can be accurately judged, and a false positive strain is avoided being screened.
4.7 subcloning of positive clones: and selecting a positive clone strain with stronger positive for subcloning by adopting a limiting dilution method. During subcloning, the cells to be cloned are gently blown evenly, the cells are counted by a cell counter, and then the positive clone cells are diluted to 40/mL. According to the gradient dilution method, 100 μ L of culture medium is added into 4 consecutive wells in advance, then 100 μ L of positive clone cells are added into the first well, after fully mixing, 100 μ L of mixed solution is added into the second well, after the gradient dilution is carried out in sequence, 200 μ L of culture medium is filled into each well, and the number of cells in each well is 2, 1, 0.5 and 0.5. And (4) performing indirect ELISA detection when the cloned cells grow to the bottom of the hole 1/5 in about 8-10 days. And then, carrying out subcloning on the two subclones by selecting a positive clone strain with good growth state and strong positive in the last time, observing only one cell mass under a mirror after 3 times of subcloning, and determining that the cell is a monoclonal hybridoma when 100% of cell supernatant in each hole is positive by indirect ELISA (enzyme-linked immunosorbent assay). Then, the monoclonal hybridoma cells are expanded and stored for multiple times of freezing, and are simultaneously used for preparing a large amount of monoclonal antibodies.
4.8 Mass preparation and purification of monoclonal antibodies: selecting female BALB/C mice (5 weeks old), injecting 500 microlitre paraffin oil into abdominal cavity, collecting monoclonal hybridoma cells 0.5-1 × 10 after 7 days7Separately, the cells were dissolved in 500. mu.L PBS and injected into the abdominal cavity, and the abdomen of the mouse was massaged with hands to disperse the cells. Then, the abdomen condition and the activity state of the mouse are observed regularly, ascites is collected by a 1mL injector after the mouse has retarded action and the abdomen is swelled, the ascites is centrifuged at 10000 Xg for 6min at 4 ℃, upper oil is discarded, the ascites is kept in a refrigerator at-80 ℃, the ascites is purified according to the method of the rProtein G kit, and then the titer of the purified antibody is detected by adopting an indirect ELISA method.
Example 5
VASN monoclonal antibody availability evaluation
5.1 monoclonal antibody subtype identification: and (4) taking the antibody of the purified monoclonal hybridoma cell strain to perform subclass identification according to an experimental method of the kit. The result shows that the obtained monoclonal antibody is specifically combined with the IgG2b subtype, and the monoclonal antibody is shown to belong to the IgG2b subtype. The results are shown in FIG. 5.
5.2 monoclonal antibody titer identification: the ELISA titer determination of the antibody of the purified monoclonal hybridoma cell strain by an indirect method can reach 1:2187000, which shows that the antibody has higher titer. The results are shown in FIG. 6.
5.3 monoclonal antibody specificity identification: extracting total proteins of normal liver cells and liver cancer cell strains, carrying out SDS-PAGE electrophoresis, transferring the proteins on a PVDF membrane by a wet transfer method, sealing 5% skimmed milk powder at room temperature for 2h, incubating the supernatant of a hybridoma cell secreting VASN monoclonal antibody at 4 ℃ overnight, washing with TBST for three times, adding horseradish peroxidase-labeled goat anti-mouse IgG (1:10000), incubating at room temperature for 1h, washing with TBST for three times, and developing with ECL. The results are shown in FIG. 7.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> Guangxi university of medical science
<120> a recombinant human VASN protein LRR structural domain protein and monoclonal antibody thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1011
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgcccatccg gctgccagtg cagccagcca cagacagtct tctgcactgc ccgccagggg 60
accacggtgc cccgagacgt gccacccgac acggtggggc tgtacgtctt tgagaacggc 120
atcaccatgc tcgacgcagg cagctttgcc ggcctgccgg gcctgcagct cctggacctg 180
tcacagaacc agatcgccag cctgcccagc ggggtcttcc agccactcgc caacctcagc 240
aacctggacc tgacagccaa caggctgcat gaaatcacca atgagacctt ccgtggcctg 300
cggcgcctcg agcgcctcta cctgggcaag aaccgcatcc gccacatcca gcctggtgcc 360
ttcgacacgc tcgaccgcct cctggagctc aagctgcagg acaacgagct gcgggcactg 420
cccccgctgc gcctgccccg cctgctgctg ctggacctca gccacaacag cctcctggcc 480
ctggagcccg gcatcctgga cactgccaac gtggaggcgc tgcggctggc tggtctgggg 540
ctgcagcagc tggacgaggg gctcttcagc cgcttgcgca acctccacga cctggatgtg 600
tccgacaacc agctggagcg agtgccacct gtgatccgag gcctccgggg cctgacgcgc 660
ctgcggctgg ccggcaacac ccgcattgcc cagctgcggc ccgaggacct ggccggcctg 720
gctgccctgc aggagctgga tgtgagcaac ctaagcctgc aggccctgcc tggcgacctc 780
tcgggcctct tcccccgcct gcggctgctg gcagctgccc gcaacccctt caactgcgtg 840
tgccccctga gctggtttgg cccctgggtg cgcgagagcc acgtcacact ggccagccct 900
gaggagacgc gctgccactt cccgcccaag aacgctggcc ggctgctcct ggagcttgac 960
tacgccgact ttggctgccc agccaccacc accacagcca cagtgcccac c 1011
<210> 2
<211> 327
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Cys Pro Ser Gly Cys Gln Cys Ser Gln Pro Gln Thr Val Phe Cys Thr
1 5 10 15
Ala Arg Gln Gly Thr Thr Val Pro Arg Asp Val Pro Pro Asp Thr Val
20 25 30
Gly Leu Tyr Val Phe Glu Asn Gly Ile Thr Met Leu Asp Ala Gly Ser
35 40 45
Phe Ala Gly Leu Pro Gly Leu Gln Leu Leu Asp Leu Ser Gln Asn Gln
50 55 60
Ile Ala Ser Leu Pro Ser Gly Val Phe Gln Pro Leu Ala Asn Leu Ser
65 70 75 80
Asn Leu Asp Leu Thr Ala Asn Arg Leu His Glu Ile Thr Asn Glu Thr
85 90 95
Phe Arg Gly Leu Arg Arg Leu Glu Arg Leu Tyr Leu Gly Lys Asn Arg
100 105 110
Ile Arg His Ile Gln Pro Gly Ala Phe Asp Thr Leu Asp Arg Leu Leu
115 120 125
Glu Leu Lys Leu Gln Asp Asn Glu Leu Arg Ala Leu Pro Pro Leu Arg
130 135 140
Leu Pro Arg Leu Leu Leu Leu Asp Leu Ser His Asn Ser Leu Leu Ala
145 150 155 160
Leu Glu Pro Gly Ile Leu Asp Thr Ala Asn Val Glu Ala Leu Arg Leu
165 170 175
Ala Gly Leu Gly Leu Gln Gln Leu Asp Glu Gly Leu Phe Ser Arg Leu
180 185 190
Arg Asn Leu His Asp Leu Asp Val Ser Asp Asn Gln Leu Glu Arg Val
195 200 205
Pro Pro Val Ile Arg Gly Leu Arg Gly Leu Thr Arg Leu Arg Leu Ala
210 215 220
Gly Asn Thr Arg Ile Ala Gln Leu Arg Pro Glu Asp Leu Ala Gly Leu
225 230 235 240
Ala Ala Leu Gln Glu Leu Asp Val Ser Asn Leu Ser Leu Gln Ala Leu
245 250 255
Pro Gly Asp Leu Ser Gly Leu Phe Pro Arg Leu Arg Leu Leu Ala Ala
260 265 270
Ala Arg Asn Pro Phe Asn Cys Val Cys Pro Leu Ser Trp Phe Gly Pro
275 280 285
Trp Val Arg Glu Ser His Val Thr Leu Ala Ser Pro Glu Glu Thr Arg
290 295 300
Cys His Phe Pro Pro Lys Asn Ala Gly Arg Leu Leu Leu Glu Leu Asp
305 310 315 320
Tyr Ala Asp Phe Gly Cys Pro
325
<210> 3
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaaggatcct gcccatccgg ctgccagtgc a 31
<210> 4
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gggaagcttt taggtgggca ctgtggctgt gg 32
