CN111499740A

CN111499740A - Tartary buckwheat metallothionein FtMT (FtMT) resistant monoclonal antibody as well as preparation method and application thereof

Info

Publication number: CN111499740A
Application number: CN202010248200.4A
Authority: CN
Inventors: 何永吉; 李云龙; 卢晓霞; 李红梅; 胡俊君; 程哲; 郭洪; 李琪
Original assignee: Research Institute of Agro Products Processing of Shanxi Academy of Agricultural Sciences
Current assignee: Research Institute of Agro Products Processing of Shanxi Academy of Agricultural Sciences
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2020-08-07

Abstract

The invention belongs to the technical field of monoclonal antibody preparation, and provides a tartary buckwheat metallothionein FtMT monoclonal antibody, and a preparation method and application thereof. The antibody is eluted by a polyhydroxy compound and a non-chaotropic salt to realize the tartary buckwheat metallothionein monoclonal antibody dissociated from an antigen FtMT, namely the anti-FtMT PR-mAb. Dissociation from the antigen FtMT can be achieved under mild conditions other than strong acid, non-high salt in the presence of polyhydroxy compounds such as ethylene glycol, propylene glycol and butylene glycol and non-chaotropic salts such as ammonium sulfate, sodium chloride and the like. The ligand is used as a good immunoaffinity ligand in FtMT immunoaffinity chromatography, can ensure the characteristics and the activity of target protein, can reduce the damage to a monoclonal antibody, prolongs the service life of an immunoaffinity column, more importantly ensures the existence form of an MT/metal ion compound of FtMT in the purification process, and realizes the high-efficiency separation and purification of non-fused MT with natural structure and function.

Description

Tartary buckwheat metallothionein FtMT (FtMT) resistant monoclonal antibody as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a tartary buckwheat metallothionein (FtMT) resistant monoclonal antibody, and a preparation method and application thereof.

Background

MT (Metalothionein, MT) is a heat-stable intracellular protein which is widely present in the biological world, has low molecular weight (6-7 kDa), is rich in cysteine, lacks aromatic amino acids and histidine and can be combined with various heavy metals. Studies have demonstrated the presence of more than 1 MT subtype (isoform) with different amino acid composition and properties in most species. The wide, polymorphic, highly inducible and conserved existence of the MT protein suggests that the MT protein has extremely important physiological functions in organisms. The existing research results prove that MT is mainly responsible for removing heavy metal toxicity in organisms; participating in the balance of essential metal elements in the body; scavenging free radicals and antagonizing ionizing radiation; participate in the regulation of hormone and development process, and enhance the response of organisms to various stresses; inhibiting cell apoptosis plays an important role in the generation and development of tumors, and is closely related to the proliferation, differentiation and apoptosis of cells. However, it has not yet been elucidated as to what specific biological functions the MT8 of different origins performs in physiological processes in vivo.

As a functional protein capable of specifically binding metal ions, the metal binding ability of MT is the basis for its many biological functions; MT or MT subtypes of different origin have specific metal binding properties or preferences, which are determinants of their ability to perform specific physiological functions in vivo. The large-scale preparation of MT with natural structure and function is the premise of function research and application development.

However, MT must be fused with solubilizing labels such as GST, MBP, SUMO and the like to prevent intracellular degradation and inclusion body formation, or fused and expressed with outer membrane protein L amB, OmpA or transmembrane protein PA L to the cell outer membrane to reduce intracellular toxicity and improve solubility, so that recombinant expression can be realized.

Patent Z L201310430331.4 discloses a method for preparing soluble recombinant Huaxi metallothionein (rcMT) by using phoA plasmid containing phoA promoter and signal peptide sequence capable of being secreted to cell periplasm as expression vector, which better solves the technical problem of soluble expression of small molecule MT protein, but still needs to fuse 6His label to achieve the purpose of rapid purification, however, even the small peptide purification label of 6His will have great influence on the metal binding property of MT (Tarasa, K., Johannsen, S., Freesing, E. Solution structure of the circulating gamma-domain analogue from the tungsten metallothionein E (c) -1. Molecules,2013, 18(11): 14414 and 14429).

The non-fused MT without any purification label needs to be separated and purified by a series of complicated operations of acetone gradient crude extraction, agarose gel separation and ion exchange chromatography. Too complicated operation not onlyTime and labor consuming, and MT is susceptible to oxidative damage during handling (pirez-Rafael, S, Mezger, a, L ieb, B,et al. The metalbinding abilities ofMegathura crenulatametallothionein (McMT) in the frameof Gastropoda MTs. J Inorg Biochem, 2012, 108:84-90）。

the monoclonal antibody is used for establishing an immunoaffinity purification method of the recombinant MT, and is an optimal choice for separating and purifying the non-fusion MT protein. However, the immunoaffinity chromatography purification method mostly adopts strong acid and high salt conditions to realize antigen-antibody separation, and easily influences the existence form of MT/metal ion complex in the purification process and influences the analysis of metal binding characteristics.

Tartar buckwheat (Tartar buckwheat) belongs to Polygonaceae (Polygonaceae), buckwheat (Fagopyrum Mill) is a food and economic crop which originates from China and is widely distributed and planted in high and cold areas such as northeast Asia and east Europe. As a 'food and drug homologous' crop with good stress resistance performance, the tartary buckwheat metallothionein (FtMT) has wide application prospect in the fields of health care, medicine, cosmetics and the like. The problem of large-scale preparation of non-fused FtMT with a natural structure and functions cannot be solved in the prior art for the reasons.

Disclosure of Invention

The invention aims to provide an anti-tartary buckwheat metallothionein FtMT monoclonal antibody, which can realize the dissociation with antigen FtMT under the mild conditions of non-strong acid and non-high salt in the presence of polyhydroxy compounds (polyol for short) such as ethylene glycol, propylene glycol and butanediol and non-chaotropic salts (non-chaotropic salts) such as ammonium sulfate, sodium chloride and the like, has the characteristic of so-called "polyol-response", and is called as anti-FtMT PR-mAb.

The invention also aims to provide the application of the monoclonal antibody against tartary buckwheat metallothionein (FtMT) in FtMT immunoaffinity chromatography. The antibody is used as a good immunoaffinity ligand to be applied to FtMT immunoaffinity chromatography, so that the characteristics and the activity of target protein can be ensured, the damage to a monoclonal antibody can be reduced, the service life of an immunoaffinity column can be prolonged, more importantly, the existence form of an MT/metal ion compound of FtMT in the purification process is ensured, and the high-efficiency separation and purification of non-fused MT with a natural structure and functions are realized.

The invention is realized by the following technical scheme: a preparation method of an anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMTPR-mAb comprises the following steps:

(1) preparing an antigen: KY643823.1 of a tartary buckwheat metallothionein FtMT coding region gene access is subcloned into phoA expression plasmid, the phoA promoter and a secretion signal sequence of the plasmid are utilized to be secreted to periplasm under the induction of a low-phosphorus culture medium, soluble FtMT-6His fusion protein is expressed, and the FtMT-6His fusion protein is obtained by nickel column affinity chromatography purification;

(2) animal immunization, namely, rapidly mixing recombinant FtMT-6His as an immune protein with Quick Antibody-Mouse5W water-soluble immunologic adjuvant 1:1, injecting the mixture into the calf muscle of a Mouse according to the immunization dose of 25 mu g/100 mu L, immunizing a female BA L B/c Mouse with the age of 5-6 weeks, enhancing the immunization for 1 needle in the same way after 3 weeks, taking tail blood for indirect E L ISA detection after 5 weeks, and when the serum titer reaches 1:10⁴~1:10⁷Within the range, the intraperitoneal injection is carried out for impacting immunity of 25 mu g/500 mu L;

(3) cell fusion, namely selecting mouse spleen cells, carrying out in-vitro fusion with myeloma cells by using a PEG1500 reagent, using FtMT-6His as a coating antigen, screening hybridoma cell strains secreting anti-FtMT by using an indirect non-competitive E L ISA method, simultaneously using recombinant IGF protein containing His-tag as an unrelated antigen for cross screening, eliminating false positive aiming at His-tag epitopes, and carrying out subclass identification according to the operation instruction of an antibody subclass identification kit;

(4) preparing and identifying mAb ascites: preparing ascites by using liquid paraffin as a sensitizer and adopting an in vivo induction method, and purifying the ascites by using a method combining hydrophobic charge chromatography HCIC and Protein A affinity chromatography; the purified sample is identified and analyzed by 12 percent SDS-PAGE and Western blot;

(5) e L ISA-resolution screening, using FtMT-6His protein as screening antigen, adopting 'E L ISA-resolution' method to screen anti-FtMT PR-mAb, using 96-hole enzyme label plate, pH9.6 carbonate buffer solution to coat FtMT-6His, 10 mu g/ml, 100 mu l/hole, 4 ℃ to preserveStoring overnight, washing the plate by PBSB, and adding gelatin for sealing; adding 100 μ l/well of antibody sample, incubating at 37 deg.C for 1.5h, washing the plate with PBSB, adding polyal eluate or control eluate, and incubating at room temperature for 20 min; after PBST washing and drying, adding goat anti-mouse IgG-HRP diluted by 1: 5000 as a secondary antibody, using o-phenylenediamine OPD solution as a reaction substrate, and 2M H₂SO₄After the reaction is stopped at 30 mul/hole, measuring by an enzyme-labeling instrument at 492 nm; after the elution of the polyol eluent, the signal is reduced by more than 50 percent, thus obtaining the target anti-FtMTPR-mAb.

The polyol eluent in the step (5) is as follows: 40% propylene glycol + 0.75M (NH4)₂SO₄50mM Tris-HCl, pH 7.9; the control eluent was 50mM Tris-HCl, pH 7.9.

An application of a monoclonal antibody against tartary buckwheat metallothionein FtMT and FtMT PR-mAb in FtMT immunoaffinity chromatography comprises the following steps:

(1) preparation of FtMT immunoaffinity column: exchanging the obtained anti-FtMT PR-mAb into a coupling buffer solution through ultrafiltration, washing the anti-FtMT PR-mAb for multiple times by HCl with the concentration of 1mM and the pH value of 4.0, and then suspending the anti-FtMT PR-mAb in the coupling buffer solution; according to 15 mg of FtMT monoclonal antibody: mixing the activated matrix of 1g dry weight, sealing, shaking the table to incubate the coupling buffer solution to wash the surplus uncoupled mAb, and finally incubating with the sealing buffer solution I to seal the uncoupled activated sites; sequentially and alternately washing the matrix by using a blocking buffer solution II and an acetic acid buffer solution; resuspending in 0.01M PBS, pH7.4 buffer, adding 0.05% NaN₃Storing at 4 deg.C for use;

(2) non-fusion FtMT expression: expressing a non-fusion FtMT protein by referring to a FtMT-6His preparation method; wherein, PCR primers FtMT-F are used: 5' -G CCATGG CCTCTTGCTGCGGTGGAA-3' and FtMT-R: 5' - -A GGATCC CTAGCAGTTGCAGGGATT-3' after amplification, was subcloned into the phoA expression plasmid; compared with the preparation method of FtMT-6His, the stop codon is introduced in advance but the C section 6His label in the plasmid is not introduced, and the recombinant non-fusion FtMT is expressed;

(3) immunoaffinity purification: fully balancing the balance buffer solution, washing the sample to a baseline by using 50mM Tris-HCl after the sample is loaded on a column, eluting the hybrid protein by using an elution buffer solution I, finally eluting a target protein peak by using an elution buffer solution II, and immediately adjusting the pH value of the recombinant protein to 7.9; ultrafiltering the eluted components with 3000 MWCO ultrafiltering tube and concentrating to obtain stock solution; the recombinant FtMT exists in the bacterial supernatant in a soluble state, and is purified by a nickel column to obtain a recombinant protein with higher purity, the molecular weight of the recombinant protein is about 8 KDa, and the molecular weight is basically consistent with a theoretical value 8280 Da.

In the step (1): the coupling buffer was: 0.1M NaHCO₃0.5M NaCl, pH 8.3; the blocking buffer I was: 0.1M Tris-HCl, pH 8.2; blocking buffer II was: 0.1M Tris-HCl, 0.5M NaCl, pH 8.2; the acetic acid buffer solution is: 0.1M sodium acetate, 0.5M NaCl, pH 4.0.

In the step (3): the equilibrium buffer was: 50mM Tris-HCl, pH 7.9; elution buffer I was: 50mM Tris-HCl, 0.25M (NH)₄)₂SO₄pH 7.9; elution buffer II was: 40% propylene glycol + 0.75M (NH)₄)₂SO₄50mM Tris-HCl, pH 7.9; the storage liquid is as follows: 100mM Tris-HCl, 0.2mM PMSF, 10mM DTT, pH 7.9.

The specific expression method of the recombinant non-fusion FtMT in the step (2) comprises the following steps: synthesizing FtMT by an in-vitro chemical synthesis method according to the gene Access, KY643823.1, of the coding region of the tartary buckwheat metallothionein FtMT; FtMT is taken as a template, and a specific primer: phoA-FtMT-F: 5' -G CCATGG CCTCTTGCTGCGGTGGAA-3' containsNcoIA restriction enzyme site; phoA-FtMT-R: 5' -A GGATCC CTAGCAGTTGCAGGGATT-3' containsBamH ICarrying out PCR amplification on the enzyme digestion sites; the amplified product and phoA plasmid were digested simultaneously with NcoI and BamH I, ligated with T4 DNA ligase, and transformedE.coliDH5 α competent cell, choose the positive clone to shake the fungus, carry on colony PCR and plasmid enzyme digestion appraisal, DNA sequencing;

through sequencing identification, a correct recombinant expression vector phoA-FtMT/B L21 is converted into competent peptide Escherichia coli B L21 (DE 3) to construct a recombinant engineering strain phoA-FtMT/B L21 (DE 3), the engineering strain is inoculated into a L B culture medium, and shaking culture is carried out at 37 ℃ and 200r/min for overnight culture for 12 hours to prepare seeds;

the next day, inoculating the seeds into optimized and improved Neidhardt low-phosphorus culture medium according to the inoculation amount of 2.5% v/v for induction, and adding 0.5-1 mM ZnSO₄Increase protein stability, 30Performing induction culture at the temperature of 165 r/min for 20-24 h.

The low-phosphorus culture medium comprises 2.5 g/L tryptone, 0.25 g/L yeast extract, 3 g/L (NH4)₂SO₄,，5g/LNacl,，1g/LMgSO₄4 g/L glucose, phosphorus content ≦ 0.05 mmol/L.

The antigen is obtained by subcloning an FtMT coding region gene (Access: KY 643823.1) to a phoA expression plasmid by utilizing the technical scheme of a patent Z L201310430331.4.

Compared with the prior art, the invention has the advantages that: the invention provides an anti-tartary buckwheat metallothionein (FtMT) monoclonal antibody, namely an anti-FtMT PR-mAb, which can be eluted by polyhydroxy compounds and non-chaotropic sequence salt under the mild conditions of non-strong acidity and non-high salt in the presence of polyhydroxy compounds such as ethylene glycol, propylene glycol and butanediol and non-chaotropic sequence salt such as ammonium sulfate, sodium chloride and the like to realize dissociation with an antigen FtMT.

The antibody is used as a good immunoaffinity ligand to be applied to FtMT immunoaffinity chromatography, so that the characteristics and the activity of target protein can be ensured, the damage to a monoclonal antibody can be reduced, the service life of an immunoaffinity column can be prolonged, more importantly, the existence form of an MT/metal ion compound of FtMT in the purification process is ensured, and the high-efficiency separation and purification of non-fused MT with a natural structure and functions are realized.

Drawings

FIG. 1 shows Western blot identification of anti-FtMT mAbs. 1 is recombinant FtMT-6His protein, 2 is irrelevant protein IGF-6 His;

FIG. 2 is a drawing of E L ISA-resolution screening of anti-FtMT PR-mAb antibodies;

FIG. 3 is the reaction characteristic analysis of anti-FtMT PR-mAb antibody polyol salt;

FIG. 4 is an SDS-PAGE electrophoretic analysis of recombinant non-fused FtMT immunoaffinity chromatography purification.

Detailed Description

Example 1: a monoclonal antibody against tartary buckwheat metallothionein (FtMT) is prepared.

1. The antigen preparation comprises the steps of subcloning an FtMT coding region gene (Access: KY 643823.1) to phoA expression plasmid by using the technical scheme of a patent Z L201310430331.4, secreting the gene to periplasm under the induction of a low-phosphorus culture medium by using a phoA promoter and a secretion signal sequence of the plasmid, expressing soluble FtMT-6His fusion protein, and finally purifying by using nickel column affinity chromatography to obtain the FtMT-6His fusion protein.

2. Animal immunization, namely, after recombinant FtMT-6His is subjected to ultrafiltration concentration to storage solution (100 mmol/L Tris-HCl, 0.2 mmol/L PMSF, 10 mmol/L DTT and pH 7.8), bovine serum albumin BSA is used as standard protein, the concentration of the recombinant protein is determined by a Bradford method, the recombinant FtMT-6His is used as immune protein, the immune protein is quickly mixed with Quick Antibody-Mouse5W water-soluble immune adjuvant 1:1, 25 mu g/100 mu L is injected into calf muscles of mice according to immune dose, the mice BA L B/c with age of 5-6 weeks are immunized, tail vein blood is taken as negative control before immunization, the immunization is strengthened for 1 needle in the same way after 3 weeks, after 5 weeks, trace tail blood is taken for indirect E L ISA detection, and when the blood serum titer reaches 1:10⁴~1:10⁷In range, i.p. injection of ballistic immunization 25 μ g/500 μ L before fusion, mice were enucleated for blood removal and serum was isolated as a positive control.

3. And (2) cell fusion, namely selecting the splenocytes of the mice with the highest serum titer, mixing the splenocytes suspension of the immunized mice and myeloma cells Sp2/0 cells screened by 8-azaguanine according to the ratio of 1: 5-1: 10, fusing under the action of PEG1500, and carrying out rapid DMEM to stop reaction, screening and culturing by using a 96-well culture plate containing HAT culture medium and HT culture medium (containing 50m L/L DMEM/F12 culture medium, 10m L/L NCTC-109 culture medium, 20m L/L fetal calf serum and 1m L/L ITS mixture), and culturing after 14 days by using a common complete culture medium.

FtMT-6His protein is used as a screening antigen, and indirect E L ISA is adopted to screen hybridoma cell strains secreting anti-FtMT, meanwhile recombinant IGF protein containing His-tag is used as an unrelated antigen to carry out cross screening, and false positive aiming at His-tag epitope is eliminated.

The subcloning was repeated 3 times by limiting dilution method until 100% of hybridoma cell supernatants were positive, which was the standard for establishing strains. And (3) replacing the hybridoma cells which are in logarithmic growth phase and grow vigorously to culture in a DMEM/F12 culture medium without fetal calf serum, continuously culturing for 2-3 d, collecting cell culture supernatant, and performing subclass identification according to the operation instruction of the antibody subclass identification kit.

The results show that 3 hybridoma cell strains capable of stably secreting anti-FtMT antibody are obtained by screening after 3 times of subclone culture, and the hybridoma cell strains are respectively named as FtMT-mAb1 and FtMT-mAb 2.2 hybridoma cell strain serum-free culture supernatant and identified as IgG1 subtype by a sandwich E L ISA subclass.

4. Preparing and identifying mAb ascites by using liquid paraffin as sensitizer and in vivo induction method, selecting 22-week-old mouse with BA L B/c, injecting liquid paraffin 0.5 m L/mouse, expanding and culturing hybridoma cell strain obtained by screening, and culturing with 1 × 10⁵/mL～1×10⁷And inoculating the hybridoma cell strain at the inoculation density of/m L into the abdominal cavity of the mouse to produce ascites, collecting the ascites after 10-12 days, centrifuging at 3000 r/min for 15 min, and harvesting the supernatant.

The method comprises the steps of coating FtMT-6His Protein, measuring ascites titer by an indirect E L ISA method, collecting ascites supernatant, purifying ascites by a method combining hydrophobic charge chromatography HCIC and Protein A affinity chromatography, and carrying out operation steps according to the literature (Zhakumei, zilianhong, Yongji, and the like), the research of a method for quickly purifying an anti-rhTF 243 monoclonal antibody by HCIC, the method of Chinese immunology journal, 2010, 26 (6): 548 and 551), and the purified sample is identified and analyzed by 12% SDS-PAGE and a Westernblot method.

The results show that the allo-anti-FtMT antibody is a primary antibody, and the AP-labeled goat anti-mouse IgG is a secondary antibody for Westernblot identification. mAbs secreted from 2 hybridomas all recognized specifically the recombinant FtMT-6His protein, but no positive signal was observed for the recombinant IGF protein (FIG. 1).

5. E L ISA-resolution antibody screening, using FtMT-6His protein as screening antigen, screening anti-FtMT PR-mAb by adopting an E L ISA-resolution method, selecting a 96-hole enzyme label plate, coating FtMT-6His (10 mu g/ml and 100 mu l/hole) with a pH9.6 carbonate buffer solution, storing at 4 ℃ overnight, washing the plate by PBSB, and adding gelatin for sealing.

Adding antibody sample (100 μ l/well), incubating at 37 deg.C for 1.5h, washing the plate with PBSB, and adding poly (ol) eluent ((100 μ l/well))40% propylene glycol + 0.75M (NH4)₂SO₄50mM Tris-HCl, pH 7.9) or control eluent (50 mM Tris-HCl, pH 7.9) was incubated at room temperature for 20 min.

After PBST washing and drying, adding goat anti-mouse IgG-HRP diluted by 1: 5000 as a secondary antibody, using o-phenylenediamine (OPD) solution as a reaction substrate, and 2M H₂SO₄After the reaction was terminated (30. mu.l/well), the reaction was measured by a microplate reader at 492 nm. Compared with the control, after the target anti-FtMT PR-mAb is eluted by polyol eluent, the signal is reduced by more than 50 percent, and the target anti-FtMT PR-mAb is obtained.

The results showed that the signal of FtMT-mAb2 was reduced by about 63% in the presence of 40% propylene glycol and 0.75m ammonium sulfate (FIG. 2), which was the anti-FtMT PR-mAb of interest. On the basis, the reaction characteristics of the anti-FtMT PR-mAb to the polyalcohols with different concentrations are shown in an in-depth analysis (figure 3), and a foundation is laid for the selection of the immunoaffinity chromatography eluent.

PR-mAbs were first proposed in 1992 by the research group of professor Nancy E.Thompson, university of wisconsin-madison, USA (Thompson, N.E., Hager, D.A., Burgess, R.R.et al. Isolationand characterization of a polyol-responsive monoclonal antibody useful forgentle purification ofEscherichia coliRNA polymerase, Biochemistry, 1992,31, 7003-7008), they found that about 5-10% PR-MAbs existed in monoclonal antibodies prepared by hybridoma technology, and that such antibodies could dissociate antigen and antibody under mild conditions of non-strong acid and non-high salt in combination of polyhydroxy compounds (such as ethylene glycol, propylene glycol and butylene glycol) and non-chaotropic salts such as ammonium sulfate, sodium chloride, etc. PR-Mabs monoclonal antibody is a good immunoaffinity ligand, and can ensure the characteristics and activity of target protein in immunoaffinity chromatography, and can reduce the destruction of monoclonal antibody and prolong the life of immunoaffinity column (Espsch A. M., Thomon N.E., &lTtT translation = L &/TtTtam J.A., ber J.,et al. Production and characterization ofmonoclonal antibodies to estrogen-related receptor alpha (ERRα) and use inimmuno-affinity chromatography. Protein Expr Purif, 2012, 84: 47-58）。

example 2: application of anti-FtMT PR-mAb in FtMT immunoaffinity chromatography

1. Preparation of FtMT immunoaffinity column: the anti-FtMT PR-mAb obtained from the screening was exchanged by ultrafiltration into coupling buffer (0.1M NaHCO) according to the CNBr-activated Sepharose 4B protocol₃0.5M NaCl, pH 8.3) and washed several times with 1mM HCl (pH 4.0) and resuspended in coupling buffer.

According to 15 mg of FtMT monoclonal antibody: 1g dry weight of the activated matrix was mixed and blocked, excess unconjugated mAb was washed with shaking incubator buffer and finally incubated with blocking buffer (0.1M Tris-HCl, pH 8.2) to block the unconjugated activation sites. Finally, the matrix was washed alternately with blocking buffer (0.1M Tris-HCl, 0.5M NaCl, pH 8.2) and acetate buffer (0.1M sodium acetate, 0.5M NaCl, pH 4.0) in sequence. Resuspended in 0.01M PBS (pH 7.4) buffer and 0.05% NaN added₃Storing at 4 ℃ for later use.

2. The non-fusion FtMT is expressed by utilizing the technical scheme of a patent Z L201310430331.4 and referring to the preparation method of FtMT-6His in the invention to express the non-fusion FtMT protein compared with the preparation method of FtMT-6His, the non-fusion FtMT is recombined and expressed by introducing a stop codon in advance without introducing a C section 6His label in a plasmid, and the FtMT is synthesized by an in vitro chemical synthesis method according to an access: KY643823.1 gene of a fagopyrum tataricum metallothionein coding region.

FtMT is taken as a template, and a specific primer: phoA-FtMT-F: 5' -G CCATGG CCTCTTGCTGCGGTGGAA-3' (includingNcoIEnzyme cleavage site); phoA-FtMT-R: 5' -A GGATCC CTAGCAGTTGCAGGGATT-3' (includingBamH IEnzyme cutting site) PCR amplification; the amplified product and phoA plasmid were digested simultaneously with NcoI and BamH I, ligated with T4 DNA ligase, and transformedE.coliDH5 α competent cells, positive clone shake bacteria, colony PCR and plasmid restriction enzyme identification, DNA sequencing.

The correct recombinant expression vector phoA-FtMT/B L21 is identified by sequencing to transform competent peptide Escherichia coli B L21 (DE 3), a recombinant engineering strain phoA-FtMT/B L21 (DE 3) is constructed, the engineering strain is inoculated into a L B culture medium, shaking culture is carried out at 37 ℃ and 200r/min overnight culture is carried out for 12 hours to prepare seeds, and the next day, the inoculation amount is divided according to 2.5% (v/v)Respectively inoculating to optimized modified Neidhardt low-phosphorus culture medium (2.5 g/L tryptone, 0.25 g/L yeast extract, 3 g/L (NH4)₂SO₄,，5g/LNacl,，1g/LMgSO₄4 g/L glucose, phosphorus content ≦ 0.05 mmol/L), adding 0.5-1 mM ZnSO₄Increasing the stability of the protein, and carrying out induction culture at 30 ℃ and 165 r/min for 20-24 h.

3. Immunoaffinity purification: the thallus is treated through crushing, heating, centrifuging and other conventional steps, and the supernatant is taken for immunoaffinity purification. The immunoaffinity purification steps are as follows: the equilibration buffer (50 mM Tris-HCl, pH 7.9) was well equilibrated with the equilibration buffer, and the sample was loaded onto the column, washed to baseline with 50mM Tris-HCl, and then eluted with elution buffer (50 mM Tris-HCl, 0.25M (NH)₄)₂SO₄pH 7.9) and finally elution buffer (40% propylene glycol + 0.75M (NH)₄)₂SO₄50mM Tris-HCl, pH 7.9) eluted the desired protein peak and immediately adjusted the recombinant protein pH to 7.9. The eluted peak fractions were concentrated by ultrafiltration through a 3000 MWCO ultrafiltration tube to stock solution (100 mM Tris-HCl, 0.2mM PMSF, 10mM DTT, pH 7.9). SDS-PAGE results show (see figure 4), recombinant FtMT exists in the bacterial supernatant mainly in a soluble state, and the recombinant protein with higher purity can be obtained by nickel column purification, the molecular weight of the recombinant protein is about 8 KDa, and is basically consistent with the theoretical value (8280 Da).

4. The binding characteristics of recombinant FtMT metal under two elution conditions are compared, the purification under the traditional pH gradient elution condition is carried out by fully balancing immunoaffinity column with 0.01M PBS (pH 7.4) buffer solution, loading the filtered supernatant onto the column, similarly washing with PBS to baseline, eluting with elution buffer solution (10 mM Tris-HCl, 1M NaCl, pH 8.0) to obtain hybrid protein, eluting with elution buffer solution (100 mM sodium acetate, 0.5M NaCl, pH 3.9) to obtain target protein peak, immediately adjusting pH of recombinant protein to 7.9, ultrafiltering and concentrating the eluted peak component with 3000 MWCO ultrafilter tube to obtain solution (100 mM Tris-HCl, 0.2mM MSF, 10mM DTT, pH 7.9), using bovine serum albumin as standard protein, using Bradford protein concentration determination kit to determine recombinant FtMT protein concentration, taking 1M L recombinant protein purified sample, placing in nitrifying bottle, adding nitric acid, perchloric acid (1: 3) on MT plate, completely placing on the same, using ion coupling ion spectrometer to determine the concentration of recombinant FtMT protein, using infrared spectrometer (OEI-I) to obtain the sample, and using ion coupling, dissolving the protein in OES 84 under the same condition, and measuring CAP (USA) under the electric heating and measuring).

ESI-TOF MS analysis of the FtMT/Zn binding profiles under both elution conditions gave the results shown in Table 1.

Table 1: in vivo and in vitro MT-3/metal complex binding profiles

The results show that anti-FtMT PR-mAb was used under mild elution conditions (40% propylene glycol + 0.75M (NH)₄)₂SO₄50mM Tris-HCl, pH 7.9), the Zn/FtMT compound keeps the existing form in vivo, and the high-efficiency separation and purification of the non-fused MT with natural structure and function are realized. While the conventional gradient pH elution condition is used for immunoaffinity purification, the Zn/FtMT compound completely presents a single saturated Zn_7/Form FtMT.

Claims

1. A preparation method of an anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMT PR-mAb is characterized in that: the method comprises the following steps:

(2) animal immunization, namely, rapidly mixing recombinant FtMT-6His as an immune protein with Quick Antibody-Mouse5W water-soluble immunologic adjuvant 1:1, injecting the mixture into the calf muscle of a Mouse according to the immunization dose of 25 mu g/100 mu L, immunizing a female BA L B/c Mouse with the age of 5-6 weeks, enhancing the immunization for 1 needle in the same way after 3 weeks, taking tail blood for indirect E L ISA detection after 5 weeks, and when the serum titer reaches 1:10⁴~1:10⁷In range, intraperitoneal injection of ballistic immunization25 μg/500 μL；

(5) e L ISA-resolution screening, using FtMT-6His protein as screening antigen, adopting 'E L ISA-resolution' method to screen anti-FtMT PR-mAb, using 96-hole enzyme label plate, using carbonate buffer solution with pH9.6 to coat FtMT-6His, 10 mu g/ml, 100 mu l/hole, storing at 4 ℃ overnight, adding gelatin to seal after PBSB washing plate, adding antibody sample 100 mu l/hole, incubating at 37 ℃ for 1.5h, adding polyal eluent or contrast eluent to PBSB washing plate, incubating at room temperature for 20min, drying PBST washing plate, adding goat anti-mouse IgG-HRP diluted by 1: 5000 as secondary antibody, using o-phenylenediamine OPD solution as reaction substrate, 2M H₂SO₄After the reaction is stopped at 30 mul/hole, measuring by an enzyme-labeling instrument at 492 nm; after the elution of the polyol eluent, the signal is reduced by more than 50 percent, thus obtaining the target anti-FtMTPR-mAb.

2. The preparation method of the anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMT PR-mAb of claim 1, which is characterized in that: the polyol eluent in the step (5) is as follows: 40% propylene glycol + 0.75M (NH4)₂SO₄50mM Tris-HCl, pH 7.9; the control eluent was 50mM Tris-HCl, pH 7.9.

3. An application of a monoclonal antibody against tartary buckwheat metallothionein FtMT and FtMT PR-mAb in FtMT immunoaffinity chromatography is characterized in that: the method comprises the following steps:

4. The use of the anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMT PR-mAb in FtMT immunoaffinity chromatography according to claim 3, characterized in that: in the step (1): the coupling buffer was: 0.1M NaHCO₃0.5M NaCl, pH 8.3; the blocking buffer I was: 0.1M Tris-HCl, pH 8.2; blocking buffer II was: 0.1M Tris-HCl, 0.5M NaCl, pH 8.2; the acetic acid buffer solution is: 0.1M sodium acetate, 0.5M NaCl, pH 4.0.

5. According toThe use of the anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMT PR-mAb in FtMT immunoaffinity chromatography according to claim 3, characterized in that: in the step (3): the equilibrium buffer was: 50mM Tris-HCl, pH 7.9; elution buffer I was: 50mM Tris-HCl, 0.25M (NH)₄)₂SO₄pH 7.9; elution buffer II was: 40% propylene glycol + 0.75M (NH)₄)₂SO₄50mM Tris-HCl, pH 7.9; the storage liquid is as follows: 100mM Tris-HCl, 0.2mM PMSF, 10mM DTT, pH 7.9.

6. The use of the anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMT PR-mAb in FtMT immunoaffinity chromatography according to claim 3, characterized in that: the specific expression method of the recombinant non-fusion FtMT in the step (2) comprises the following steps: synthesizing FtMT by an in-vitro chemical synthesis method according to the gene Access, KY643823.1, of the coding region of the tartary buckwheat metallothionein FtMT; FtMT is taken as a template, and a specific primer: phoA-FtMT-F: 5' -G CCATGG CCTCTTGCTGCGGTGGAA-3' containsNcoIA restriction enzyme site; phoA-FtMT-R: 5' -A GGATCC CTAGCAGTTGCAGGGATT-3' containsBamH ICarrying out PCR amplification on the enzyme digestion sites; the amplified product and phoA plasmid were digested simultaneously with NcoI and BamH I, ligated with T4 DNA ligase, and transformedE.coliDH5 α competent cell, choose the positive clone to shake the fungus, carry on colony PCR and plasmid enzyme digestion appraisal, DNA sequencing;

the next day, inoculating the seeds into optimized and improved Neidhardt low-phosphorus culture medium according to the inoculation amount of 2.5% v/v for induction, and adding 0.5-1 mM ZnSO₄Increasing the stability of the protein, and carrying out induction culture at 30 ℃ and 165 r/min for 20-24 h.

7. The use of the anti-tartary buckwheat metallothionein FtMT monoclonal antibody anti-FtMT PR-mAb in FtMT immunoaffinity chromatography according to claim 4, characterized in thatThe low-phosphorus culture medium is 2.5 g/L tryptone, 0.25 g/L yeast extract, 3 g/L (NH4)₂SO₄,，5g/LNacl,，1g/LMgSO₄4 g/L glucose, phosphorus content ≦ 0.05 mmol/L.