CN114081941A - Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of medicine for resisting excessive immune response or resisting cytokine storm - Google Patents
Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of medicine for resisting excessive immune response or resisting cytokine storm Download PDFInfo
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
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- C12N15/09—Recombinant DNA-technology
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- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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Abstract
The invention provides a preparation method of B7-H4 protein and application thereof in preparing a medicine for resisting excessive immune reaction or resisting cytokine storm, belonging to the technical field of biological engineering. The preparation method of the B7-H4 protein comprises the following steps: s1, identifying the expression of B7-H4 protein; s2, protein expression and purification of B7-H4; b3. B7-H4 protein verifies that the B7-H4 protein prepared by the invention has good inhibition effect on excessive immune response and cytokine storm, and experiments prove that the B7-H4 protein inhibits mouse and human CD8+ T cells from secreting cytokines.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a preparation method of B7-H4 protein and application thereof in preparing medicines for resisting excessive immune response or resisting cytokine storm.
Background
Many serious infections, such as new corona virus, SARS, HIV, etc., cause the appearance of cytokine storms (cytokines storms), which lead to worsening of the disease and become critically ill patients. This is caused by an excessive immune response, which may lead to complications such as hypotension, blood coagulation disorders, multiple organ failure, etc., and ultimately death of the patient. Similarly, the composition is used for preventing acute graft-versus-host disease (GvHD) after bone marrow transplantation of leukemia patients, and can also cause the condition of excessive immune response, including organ transplantation, such as liver transplantation, heart and lung transplantation and the like. In addition, the use of Immune Checkpoint Inhibitors (ICIs), including inhibitors of programmed death receptor protein-1 (PD-1), inhibitors of programmed death receptor ligand-1 (PD-L1), and inhibitors of cytotoxic T lymphocyte-associated antigen 4(CTLA-4), has become one of the standard approaches in the treatment of advanced tumors due to their good antitumor activity. ICIs activate and strengthen the immune system (mainly T cell system) of the body and exert the anti-tumor capacity by blocking two main immune checkpoint pathways of PD-1/PD-L1 and CTLA-4 and releasing the 'immune brake' action of the two major immune checkpoint pathways. However, an overactivated immune system can cause damage to target organs, tissues and, thus, immune-related adverse events (irAEs). This also occurs in the case of hyperimmunization. Autoimmune diseases, metabolic syndrome can also lead to cytokine storm.
Glucocorticoid is often used to suppress this excessive immune response of cytokine storm. The sequelae (femoral head necrosis) induced by high dose hormone shock therapy is a very troublesome problem. Therefore, there is an urgent need to find new methods for safely suppressing such excessive immune responses.
Exosomes (exosomes) are micro-membrane vesicles secreted by cells and widely distributed in various body fluids, and can carry information such as various proteins, lipids, DNA and RNA to form a cell-cell signal transmission. B7-H4 (also known as B7x, B7-S1, and VTCN1) are immune modulatory molecules with homology to other B7 family members, including PD-L1. It is a type I transmembrane protein composed of IgV and IgC extracellular domains. B7-H4 was administered in exosomes, allowing direct access to the center of the cytokine storm.
Disclosure of Invention
The invention aims to provide a preparation method of B7-H4 protein and application thereof in preparing a medicine for resisting excessive immune reaction or cytokine storm, and experiments prove that the B7-H4 protein can inhibit mouse and human CD8+ T cells from secreting cytokines and inhibiting functions of the cytokines.
The technical scheme of the invention is realized as follows:
the invention provides an application of B7-H4 protein in preparing a medicine for resisting excessive immune response or cytokine storm.
The invention further protects a medicine containing the B7-H4 protein, and the administration route of the medicine is skin/mucous membrane external use, intravenous injection, intramuscular injection, oral administration, aerosol inhalation and inhalation by exosome containing the protein.
The invention further provides a preparation method of the B7-H4 protein, which comprises the following steps:
s1, identifying the expression of B7-H4 protein;
s2, protein expression and purification of B7-H4;
and S3.B7-H4 protein verification.
As a further improvement of the invention, the identification of the B7-H4 protein expression in the step S1 comprises the following steps:
(1) obtaining a B7-H4 gene cDNA fragment: B7-H4 gene cDNA fragments are obtained through the design of specific amplification primers, PCR amplification and cDNA fragment purification and recovery;
(2) double enzyme digestion: performing double enzyme digestion on the cDNA fragment and the plasmid of the B7-H4 gene by using endonuclease, performing agarose gel electrophoresis, and purifying and recovering the cDNA fragment;
(3) connection and recombination: the B7-H4 gene cDNA fragment and the linearized plasmid PET-30a (+) are subjected to recombinant connection by using ligase;
(4) and (3) clone bacterium transformation: mixing E.coli DH5 alpha competent cells with the ligation product, and culturing;
(5) and (5) cloning and identifying.
As a further improvement of the invention, the endonuclease is NdeI and HindIII restriction endonuclease.
As a further improvement of the invention, the ligase is T4 ligase.
As a further improvement of the invention, the plasmid is PET-30a (+).
As a further improvement of the invention, the B7-H4 protein expression purification in the step S2 comprises the following steps:
(1) B7-H4 recombinant protein expression: mixing E.coliBL21(DE3) competent cells with recombinant plasmids, culturing, randomly selecting a single clone on an LB agar plate to a Kan + LB liquid culture medium, adding IPTG (isopropyl-beta-thiogalactoside) to perform protein induction expression after culturing, and then performing protein identification;
(2) separating and purifying the B7-H4 recombinant protein; collecting thalli, carrying out ultrasonic disruption and centrifugation, taking supernatant, enriching target protein by using a His label carried by a PET-30a (+) prokaryotic expression vector and adopting a His gel affinity chromatography method, identifying a restriction enzyme sequence and a cutting site by 3C protease specificity, separating the target protein from the His label, thus obtaining the target protein with higher purity and better specificity, concentrating after identification, purifying by using an AKTA protein purification system, and collecting purified protein with the purity of more than 95%.
As a further improvement of the invention, the molecular sieve prepacked column in the AKTA protein purification system is Hiload superdex 200.
As a further improvement of the invention, the method for verifying the B7-H4 protein in the step S3 adopts Western Blot for verification.
The invention has the following beneficial effects: the B7-H4 protein prepared by the invention has good inhibition effect on excessive immunoreaction and cytokine storm, the B7-H4 protein can reach the center of the cytokine storm, namely lung, in an exosome form by nasal inhalation administration, and tests prove that the B7-H4 protein can reduce the number of CD8+ T cells and inhibit the CD8+ T cells from secreting the cytokine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art 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 for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Coli codon usage;
FIG. 2 is a SDS-PAGE and Western blot validation chart;
FIG. 3 is a map of the B7-H4 protein antibody;
FIG. 4 is an enlarged view of CD8+ T cells in the spleen of mouse C57 BL;
FIG. 5 is a CD8+ T cell loss cytometer identification chart;
FIG. 6 is a graph depicting the interaction between B7-H4 protein and the number of CD8+ T cells;
FIG. 7 is a determination of the proportion of CD8+ cells in human tumor infiltrating T cells (TILs);
FIG. 8 shows the function of B7-H4 protein at various concentrations to inhibit secretion of INF-gamma by TIL (Elispot method);
FIG. 9 is a graph showing that human T function is significantly inhibited with increasing concentrations of B7-H4.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of B7-H4 protein
S1.B7-H4 protein expression identification:
1. construction of B7-H4 Gene and plasmid construction
The B7-H4 gene recombinant plasmid PET-30a (+) -B7-H4 (Table 1) was reconstructed by using PET-30a (+) as a prokaryotic expression vector (the plasmid was verified to be correct by full-length sequencing and has a his tag).
TABLE 1
2. Obtaining a B7-H4 gene cDNA fragment
(1) Designing a specific amplification primer: the puc57-B7-H4 gene recombinant plasmid is used as an amplification template, enzyme cutting sites of NdeI and HindIII restriction enzymes are selected as the cutting sites of an amplification fragment which is connected into a PET-30a (+) vector, the sequence of the enzyme cutting sites, a protected base sequence, the coding direction of the plasmid, the GC content and the like are considered when a primer is designed, and the codon usage preference of E.coli is also considered (figure 1, pictures are from http:// www.geneinfinity.org/sp/sp _ codon _ coding. htmL). Finally, primers synthesized by the designed specific amplification primers are prepared into 50 mu M working concentration, and the working concentration is preserved at 4 ℃.
(2) And (3) PCR amplification:
the PCR reaction mixture was prepared according to Table 2, mixed by vortex shaker and centrifuged rapidly.
TABLE 2B 7-H4 Gene cDNA fragment amplification System (50. mu.L)
| Components | Volume of 1 part (μ L) |
| B7-H4-NdeI-F(50μM) | 0.5 |
| B7-H4-HindIII-R(50μM) | 0.5 |
| PET-30a(+) | 1 |
| |
1 |
| 5× |
10 |
| 2.5mM dNTPs | 5 |
| Nuclease-Free water | Make up volume to 50 |
Reaction conditions are shown in Table 3.
TABLE 3
(3) 1% agarose gel electrophoresis: PCR amplification products were identified and all amplification products were collected by running gel prepared with a thin and wide comb.
(4) B7-H4 gene cDNA fragment purification and recovery:
the DNA fragment was recovered from the agarose gel using a general-purpose DNA purification recovery kit from Tiangen Biotechnology (Beijing) Ltd.
Firstly, adding absolute ethyl alcohol with a corresponding volume according to a label on a rinsing liquid PW bottle before the rinsing liquid PW bottle is used for the first time.
Column balancing step: to adsorption column CB2 (adsorption column was put in the collection tube), 500. mu.L of the equilibrium solution BL was added, and centrifuged at 12000rpm (about 13400g) for 1 minute, the collection tube was discarded, and adsorption column CB2 was replaced in the collection tube again (for the day of treatment).
Cut the agarose gel (pad with clean plastic film, use the new blade without DNA pollution, in order to prevent the exogenous DNA pollution, cut the surplus part as far as possible) containing the single mesh strip under the ultraviolet lamp, put into the clean centrifuge tube and weigh.
Adding solution PC (calculated according to the volume of 0.1g of gel and the volume of the gel is regarded as 100 mu L) with equal volume into the gel block, placing the gel block in a water bath at 50 ℃ for 10 minutes, and turning the centrifugal tube up and down continuously and gently during the placing process to ensure that the gel block is fully dissolved and the solution is yellow. If the size of the rubber block is too large, the rubber block can be cut into pieces firstly. After the gel block is completely dissolved, the column is loaded after the temperature of the gel block is reduced to room temperature, and the adsorption column has stronger DNA binding capacity at room temperature. For recovery of small fragments <150bp the volume of solution PC can be increased 3-fold to increase the recovery efficiency.
Fifthly, adding the solution obtained in the previous step into an adsorption column CB2 (placing the adsorption column into a collection tube), centrifuging at 12000rpm for 1 minute, pouring the waste liquid in the collection tube, and placing an adsorption column CB2 into the collection tube.
Sixthly, 600 microliter of rinsing liquid PW is added into the adsorption column CB2, centrifugation is carried out for 1 minute at 12000rpm, waste liquid in the collecting pipe is poured out, and the adsorption column CB2 is placed into the collecting pipe.
And seventhly, repeating the previous step.
Eighthly, centrifuging at 12000rpm for 2 minutes to remove the rinsing liquid as much as possible. The adsorption column CB2 was left at room temperature for several minutes and was thoroughly dried to prevent the residual rinse from affecting the subsequent experiments.
Ninthly, putting the adsorption column CB2 into a clean centrifugal tube, hanging and dropping 50 mu L of ddH2O (which can be preheated in a water bath at 65-70 ℃) to the middle position of the adsorption film, placing the adsorption film at room temperature for 2 minutes, centrifuging the adsorption film at 12000rpm for 2 minutes, and collecting DNA solution. In order to increase the recovery amount of DNA, the solution obtained by centrifugation may be re-applied to a centrifugal adsorption column, left at room temperature for 2 minutes, centrifuged at 12000rpm for 2 minutes, and the DNA solution collected. Store at-20 ℃.
3. Double enzyme digestion
(1) The B7-H4 gene cDNA fragment and the plasmid PET-30a (+) were double digested simultaneously with NdeI and HindIII restriction enzymes as shown in Table 4 and incubated at 37 ℃ for 2 hours.
TABLE 4 double enzyme digestion System (80. mu.L)
| Components | Volume of 1 part (μ L) |
| |
1 |
| |
1 |
| B7-H4 cDNA or PET-30a (+) | 50 (about 1.5. mu.g) |
| 10×NEBuffer | 8 |
| Nuclease-Free water | Make up volume to 80 |
(2) 1% agarose gel electrophoresis: the gel was prepared using a thin and wide comb and run to collect all amplification products.
(3) And (3) purification and recovery: the procedure was as described for "purification and recovery of cDNA fragment from B7-H4", and finally eluted with 40. mu.L of ddH 2O.
4. Ligation recombination
The B7-H4 gene cDNA fragment and the linearized plasmid PET-30a (+) were recombinantly ligated with T4 ligase as in Table 5, and either incubated at room temperature for 3 hours, immediately transformed or stored at-20 ℃.
TABLE 5 recombinant ligation System (20. mu.L)
Note: the molar ratio of the target fragment to the plasmid in the ligation system is about 3:1, and the calculation formula is as follows:
5. transformation (clone bacteria)
Coli DH5 α competent cells were selected, the specific procedure was as follows:
(1) DH 5. alpha. competent cells were removed from-80 ℃ and placed on ice, 20. mu.L of ligation product (adjusted according to the preliminary experimental results) was added to 100. mu.L of freshly thawed DH 5. alpha. competent cell suspension, gently pipetted and mixed, and allowed to stand in ice bath for 30 minutes.
(2) And after heat shock is carried out for 90 seconds in a water bath at 42 ℃, the mixture is quickly transferred to ice and stands for 2-3 minutes.
(3) Adding 900 μ L LB liquid medium without antibiotic, mixing uniformly, placing in shaking table at 37 deg.C and 150 rpm, shaking and culturing for 45 minutes.
(4) And (3) absorbing 100 mu L of bacterial liquid (the amount of the plate-laying bacterial liquid needs to be adjusted according to the pre-experimental result, when the total amount of the converted DNA is more, 50 mu L or less of bacterial liquid can be absorbed for plate coating, on the contrary, 200-300 mu L of bacterial liquid can be absorbed for plate coating, when the amount of the clone is less, the bacterial liquid can be centrifuged at 4000rpm for 2 minutes, then part of culture liquid supernatant is discarded, and the plate is coated after heavy suspension) and added to an LB solid agar culture plate containing 100 mu g/mL Kan +, the bacterial liquid is evenly spread by using an alcohol lamp baked and cooled aseptic elbow glass rod or fine iron wire rod, the bacterial liquid is flatly laid for 10 minutes at room temperature and then placed in a 37 ℃ incubator for inverted culture for 12-16 hours.
6. Cloning and identification
(1) Selecting clone culture: randomly picked single colonies on LB agar plates were placed in 3mL LB liquid medium containing 100. mu.g/mLKan +, and shaken at 37 ℃ with 170rpm shaking table for 12 hours.
(2) Small-scale extraction of plasmids: the method comprises the following steps of extracting recombinant plasmids in a small quantity by using a rapid plasmid small extraction kit (centrifugal column type) of Tiangen Biochemical technology (Beijing) Co., Ltd:
firstly, RNase A and TIANRED are evenly mixed in a solution P1 before being used for the first time, and the mixture is stored at 4 ℃; and adding corresponding volume of absolute ethyl alcohol according to the label on the rinsing liquid PWT bottle.
And secondly, adding 3mL of bacteria liquid after 12-hour bacteria shaking into 1.5mL of centrifuge tubes (collecting for 2-3 times), centrifuging for 1 minute at 12000rpm (about 13400g), and discarding the supernatant as much as possible.
③ adding 150 μ L of the solution P1 into the centrifuge tube with the bacterial sediment, using a pipette to blow or swirl the bacterial sediment thoroughly, and mixing uniformly to obtain turbid red solution.
And fourthly, adding 150 mu L of solution P2 into the centrifuge tube, and gently turning the centrifuge tube up and down for 6-8 times to fully crack the thalli, wherein the fully cracked solution is clear purple.
And fifthly, adding 350 mu L of solution P5 into the centrifuge tube, immediately and quickly turning upside down for 12-20 times, fully and uniformly mixing until the solution generates flocculent precipitate, and centrifuging at 12000rpm for 2 minutes until the supernatant is clear yellow.
Sixthly, transferring the supernatant collected in the last step into an adsorption column CP3 (placing the adsorption column into a collecting pipe) by using a liquid transfer device, sucking out the precipitate as little as possible, centrifuging at 12000rpm for 30 seconds, pouring the waste liquid in the collecting pipe, and replacing the adsorption column CP3 in the collecting pipe again.
Adding 300 microliter rinsing liquid PWT into the adsorption column CP3, centrifuging at 12000rpm for 30 seconds, pouring the waste liquid in the collection tube, and replacing the adsorption column CP3 in the collection tube.
Eighthly, centrifuging at 12000rpm for 1 minute, uncovering the adsorption column CP3, and standing at room temperature for a plurality of minutes to remove residual rinsing liquid in the adsorption column.
Ninthly, placing the adsorption column CP3 in a clean 1.5mL centrifuge tube, dropwise adding 50 muL ddH2O (preheated in a water bath at 65-70 ℃) into the middle of the adsorption membrane, standing for 2 minutes at room temperature, centrifuging at 12000rpm for 30 seconds, collecting the plasmid eluent into the centrifuge tube, and adding the obtained solution into the adsorption column again for centrifugation again for collection. The concentration and purity of the plasmid are detected by the Nanodrop 2000c (the ratio of OD260/OD280 of the purified plasmid is usually 1.8-2.0), and the plasmid is stored at the temperature of-20 ℃ to be identified.
(3) And (3) identification: double enzyme digestion or PCR amplification can be selected, and then the preliminary identification and positive plasmid sequencing verification are carried out through agarose gel electrophoresis.
The double enzyme digestion identification system is shown in a table 6, the double enzyme digestion identification system is incubated in water bath at 37 ℃ for 30 minutes, electrophoresis is carried out on 1% agarose gel, and if bands are found in the sizes of both the original plasmid and the target fragment, the double enzyme digestion identification system can be further sequenced and verified.
TABLE 6 double restriction enzyme identification System (10. mu.L)
Secondly, PCR amplification of the recombinant plasmid is carried out by using universal primers pGEX5 'and pGEX3' (Table 7), the amplification system and conditions are the same as those of ordinary PCR (see the first part), 1.5% agarose gel electrophoresis is carried out, and if a band is seen in the size of the target fragment, the band is regarded as positive, and further sequencing verification can be carried out.
TABLE 7 Universal primers for PET-30a (+) plasmid
| Primer name | Primer sequence (5 '-3') |
| pGEX5' | GGGCTGGCAAGCCACGTTTGGTG |
| pGEX3' | CCGGGAGCTGCATGTGTCAGAGG |
And thirdly, taking a proper amount of the recombinant plasmid, taking universal primers pGEX5 'and pGEX3' as sequencing primers, performing bidirectional sequencing on the sequence between the plasmid pGEX5 'and the plasmid pGEX3', sequencing, connecting and splicing, and successfully constructing pGEX-WT if the sequencing result is completely correct.
S2, protein expression and purification of B7-H4:
1.B7-H4 recombinant protein small-scale expression
The B7-H4 recombinant protein was expressed in small amounts to determine experimental feasibility and to explore protein expression conditions, the following were determined to be suitable for the B7-H4 recombinant protein expression in small amounts:
(1) transformation (expression bacteria):
coli BL21(DE3) competent cells were selected as follows:
taking BL21(DE3) competent cells out of the mixture at-80 ℃, placing the competent cells on ice, subpackaging 50 mu L of the just melted cell suspension into a sterile precooled centrifuge tube, adding 1 mu L of recombinant plasmid, slightly blowing and stirring the mixture evenly, and standing the mixture in ice bath for 30 minutes.
② after 60 seconds of heat shock in 42 ℃ water bath, quickly transferring to ice and standing for 2 minutes.
③ adding 600 mu L of LB liquid culture medium without antibiotic, mixing evenly, placing in a shaker at 37 ℃ and shaking at 220rpm for 45 minutes.
And fourthly, sucking 100 mu L of bacterial liquid (the amount of the plate-paving bacterial liquid needs to be adjusted according to the pre-experimental result) and adding the bacterial liquid to an LB solid agar culture plate containing 100 mu g/mL kanamycin, lightly and uniformly paving the bacterial liquid by using a cooled sterile elbow glass rod or a thin iron wire rod after roasting by using an alcohol lamp, putting the bacterial liquid in a 37 ℃ incubator for upright culture for 1 hour, and then carrying out inverted culture for about 13 hours.
(2) Selecting clone culture: randomly picking single clone on LB agar plate to 3mL LB liquid culture medium containing 100. mu.g/mL Kan +, placing in 37 ℃ shaking table 220rpm to shake for 4 hours, and determining that OD600 reaches about 0.5 by NanoDrop 2000 c.
(3) Protein expression: adding 1mL of bacterial solution into 1mM IPTG, and continuously shaking for 3 hours at 37 ℃ by a shaking table at 220 rpm; the remaining bacteria solution was used as an uninduced control and shaken synchronously. Meanwhile, a plurality of groups of expression test experiments are carried out, and the influence of different expression temperatures (16 ℃, 22 ℃, 37 ℃) and IPTG concentrations (0.1-1 mM) on protein expression is groped.
(4) Protein identification: 200 mu.L of each of the induced bacterial liquid and the non-induced bacterial liquid (the rest of the non-induced bacterial liquid is frozen) is taken, centrifuged at 13000rpm for 1 minute, after the supernatant is removed, 50 mu.L of 5 xSDS-PAGE Protein loading buffer solution is added into the deposited bacteria, after the mixture is mixed evenly, the mixture is denatured in 100 ℃ boiling water for 10 minutes, after the centrifugation at 13000rpm for 10 minutes, 3 mu.L of loading solution is taken for SDS-PAGE, 5 mu.L of Prestained Protein Ladder is taken as a reference, and the gel is stained by Coomassie brilliant blue to identify Protein bands.
Preparing a gel (a common system is shown in table 8) by using an SDS-PAGE gel preparation kit, carrying out constant voltage electrophoresis at 80V until bromophenol blue enters the laminated gel, setting the voltage to 125V, and stopping electrophoresis when the bromophenol blue reaches the position near the bottom end of the gel.
TABLE 8 SDS-PAGE gel systems
Secondly, taking down all the gel, washing or rinsing the gel by milliQ water, placing the gel in protein fixing liquid for fixing, and incubating for 30 minutes to overnight by low-speed shaking on a horizontal shaking table; removing the stationary liquid, replacing the protein electrophoresis gel staining solution (0.25% Coomassie brilliant blue R250 solution), and incubating for 2-4 hours on a horizontal shaking table by shaking at a low speed until the gel is dyed into uniform blue and can not be seen by naked eyes in the staining solution, and finishing dyeing; putting the gel into protein electrophoresis gel destaining solution, incubating for 4 hours by low-speed shaking on a horizontal shaking table, and replacing destaining solution once per hour
(iii) observing the protein band of the gel in the ChemiDoc gel imaging system, the molecular weight under reducing condition should be about 26kDa (see FIG. 2) where M1Is protein Marker, GenScript, Cat. No. M00516; m2Protein Marker, GenScript, cat.no. m 00521; 1 is BSA (2.00. mu.g); 2 is B7-H4 protein (Reducing condition, 2.00. mu.g); 3 is B7-H4 protein (Reducing condition); original antibody: mouse anti-His mAb (GenScript, Cat. No. A00186).
2.B7-H4 recombinant protein mass expression
The previous experiment searches out the suitable conditions for the large-scale expression of the B7-H4 recombinant protein in E.coli cells, mainly comprising the expression temperature (16 ℃, 22 ℃, 37 ℃) and IPTG concentration (0.1-1 mM) and the like, and finally the conditions for the large-scale expression of the B7-H4 recombinant protein are as follows:
(1) 100 μ L of uninduced bacteria liquid which is identified to be successfully induced and expressed is added into 10mL LB liquid culture medium and placed in a 37 ℃ shaking table to shake at 220rpm overnight.
(2) Adding 5mL of overnight-cultured bacterial liquid into 1L of LB liquid culture medium, adding Kan + until the working concentration is 100 mu g/mL, placing the mixture in a shaker at 37 ℃ and shaking at 220rpm for 4 hours, detecting that OD600 reaches 0.6 by Nanodrop 2000c, reducing the shaker temperature to 16 ℃, maintaining the shaker at 100rpm for 1 hour, adding IPTG until the final concentration is 0.1mM, and shaking in a shaker at 220rpm at 16 ℃ overnight (14 hours).
3.B7-H4 recombinant protein separation and purification
Care was taken to maintain the protein sample in a 4 ℃ or ice bath environment throughout the experiment:
(1) and (3) collecting thalli: after centrifugation at 3500rpm at 4 ℃ for 10 minutes, 50mL of lysine buffer (50mM Tris pH8.0, 400mM NaCl and 10% glycerol) was added to the resulting suspension in 2L of the suspension, and the suspension was shaken to resuspend the cells, and PMSF was added to a final concentration of 1mM and DTT to a final concentration of 5 mM.
(2) Collecting the recombinant protein supernatant: ultrasonically breaking the bacterial cells until the bacterial liquid is clear (700W power, working for 5 seconds, interval of 10 seconds, whole process about 15 minutes, ice bath), reserving a little bacterial liquid for SDS-PAGE identification, centrifuging at 18000rpm at 4 ℃ for 50 minutes, collecting all supernatant, subpackaging into 50mL centrifuge tubes,
(3) affinity chromatography: as the PET-30a (+) prokaryotic expression vector carries a His label, the B7-H4 recombinant protein is highly enriched with the target protein by a His gel affinity chromatography method,
first, His gel (generally 5-8 times the volume of the gel) is equilibrated with lysine buffer.
② adding 1mL His gel into the collected supernatant according to every 50mL, and carrying out rotary combination for 2 hours under the condition of 4 ℃.
Thirdly, collecting the gel by using an affinity chromatography hollow column tube, and enabling the flow-through liquid to flow into a clean centrifugal tube.
And fourthly, after the flow-through liquid is dried, washing the His gel by using lysine buffer with the volume 50 times that of the gel to remove the foreign protein.
(4) And (3) recombinant protein enzyme digestion: the enzyme cutting sequence specifically recognized by 3C protease is arranged between the His label and the target protein in the PET-30a (+) prokaryotic expression vector used in the experiment (the amino acid sequence is LEVLFQ □ GP, □ represents a 3C protease cutting site). Therefore, after the target protein is efficiently enriched, the target protein and the His tag are separated by specifically recognizing the enzyme cutting sequence and the cutting site of the 3C protease, so that the target protein with higher purity and better specificity is obtained.
Firstly, blocking an affinity chromatography column tube, adding 15mL lysine buffer into every 2mL gel for heavy suspension, transferring the gel into a clean 15mL centrifuge tube, adding 150 mu L3C protease, uniformly mixing, and performing rotary incubation at 4 ℃ for 3 hours.
Secondly, transferring the enzyme digestion reaction solution to an affinity chromatography hollow column tube, and collecting the flow-through solution at the moment, namely the purified high-purity B7-H4 protein sample.
(5) SDS-PAGE identification: in the purification process, 10 mu L of samples are reserved in each step, the samples mainly comprise induced bacteria liquid (which is subjected to ultrasonic crushing), supernatant after bacteria breaking, flow-through liquid after first His gel combination, resuspension liquid before 3C protease is added, samples before column loading after enzyme digestion and B7-H4 sample flow-through liquid, the non-induced bacteria liquid is used as a control, the experimental method is approximately the protein identification of 2.1B7-H4 recombinant protein small-quantity expression, and the expression quantity, solubility, the combination condition with an affinity chromatography column, the concentration of the obtained target protein and the like are observed.
(6) Concentration: transferring the soluble target protein obtained by affinity chromatography to a concentration tube (the molecular size of the retention of the concentration tube is 10kD), centrifuging at 4000rpm and 4 ℃, gently blowing and uniformly mixing the protein solution by using a liquid transfer machine every 10 minutes of centrifugation so as to prevent the local concentration from being too high, and concentrating the finally obtained protein solution sample to about 8 mL.
(7) Purifying a molecular sieve: the protein is purified by an AKTA protein purification system, and a Hiload superdex200 is selected as a molecular sieve prepacked column. After equilibration of the Hiload superdex200 column with Elution buffer (25mM Tris pH8.0 and 150mM NaCl), the concentrated protein solution was added and samples were collected using an automated collector at a flow rate of 1mL/min for 1 mL/tube. Collecting sample tubes corresponding to the target protein peaks according to the peak shape diagram generated by a protein purification instrument in real time, carrying out SDS-PAGE detection, collecting purified protein with the purity of more than 95%, concentrating to about 500 mu L again, adding equal volume of glycerol and a final concentration of 2mM DTT solution, and preserving at-80 ℃.
And S3, protein verification of B7-H4:
western Blot was performed on the purified B7-H4 protein, and 3 distinct bands were visible after incubation of 3 home-made B7-H4 antibodies: about 27kD, 54kD (dimer) and 81kD (trimer) (see FIG. 3). FIG. 3 shows the detection of B7-H4 protein antibody, the sample loading of 4 lanes is the same, 3B 7-H4 bands are visible on antibody No. 1-3, the antibody No. 4 is an ab209242 batch of abcam, GR3283705-5, the background is not clear, and the Marker is a standard protein and can indicate the protein size of the position of the band.
Example 2B 7-H4 protein inhibition of T cells of mouse CD8+
(1) Extraction of mouse spleen T cells: taking 5 female SPF (specific pathogen free) grade C57BL/6 mice with age of 6-8 weeks, taking spleens after neck removal and death, grinding, filtering the cell suspension through a 70-micron filter screen, transferring the cell suspension to a centrifuge tube, centrifuging at the temperature of 4 ℃ for 5 minutes at 300g, and discarding the supernatant; adding 5ml of erythrocyte lysate, fully and uniformly mixing, incubating for 5 minutes at room temperature, adding 20ml of PBS, centrifuging for 5 minutes at the temperature of 4 ℃ at 300g, and removing supernatant; adding 20ml PBS, filtering with a 40um filter screen, centrifuging for 5 minutes at 300g at 4 ℃ after heavy suspension, and removing supernatant; resuspending the cells in 10ml PBS and counting, centrifuging at 300g at 4 ℃ for 5 minutes, and discarding the supernatant;
(2) separation, culture and identification of mouse spleen CD8+ T cells: at 107Each cell is a unit, every 107Adding 90ul MACS buffer, 10ul CD8+ Microbead to each cell, and incubating for 15 minutes at 2-8 ℃; transferring the cell suspension to a sorting column pre-rinsed in a magnetic field, passing through the column, and taking unlabeled cells, namely non-CD 8+ T cells, wherein the labeled cells are CD8+ T cells (see figure 4, which is a 100-fold magnified graph); the CD8+ T cells are incubated with the mouse CD8+ antibody for 15 minutes, the proportion of the antibody is detected by flow, the result is shown in figure 5, and the rate of CD8+ is 98.59%; the mouse CD8+ T cells are resuspended in a 24-well plate by using RPMI 1640 medium (+ 10% FBS + IL-2100U/ml + double antibiotics), the proportion is adjusted to 2 x 10^ 6/2 ml/well, T cell activating reagents CD3 and CD28 antibody magnetic beads 20ul are added into each well, and the cells are cultured for 1-2 days at 37 ℃ and 5% CO 2;
(3) B7-H4 experiments affecting IFN- γ secretion from mouse spleen T cells: the CD8+ T cells were centrifuged in PBS and resuspended, grouped as follows: group A: CD8+ T cells (NC: negative control); group B: CD8+ T cells +40ug/ml B7-H4 protein. Resuspending the cells in an incomplete T cell culture medium without IL-2, adjusting the cell concentration, planting the cells in an Elispot 96 pore plate coated with INF-gamma monoclonal antibodies according to groups, adding corresponding enzyme-labeled detection antibodies and substrate developing solutions after culturing for 48 hours, reading the plate after INF-gamma spots appear in the pores, observing and counting the number of INF-gamma + TILs, and finding that the B7-H4 protein can inhibit CD8+ T cells from secreting cytokines, as shown in figure 6.
Example 3 inhibition of human solid tumor infiltrating T cells by B7-H4 protein
(1) Solid tumor infiltrating T cells (TILs) isolation, culture and enrichment: removing impurities such as necrosis and blood clot from solid tumor tissue under sterile environment, and cutting into pieces of 1-8mm3Placing the culture medium in a 24-hole culture plate, adding a culture medium containing a large dose of IL-2 cytokines, incubating for 2-4 weeks, collecting corresponding TILs, amplifying, and freezing; meanwhile, the cell suspension is placed in a T25 culture flask for conventional culture, and after the cell suspension is amplified to a certain number, the cell suspension is sorted by using human CD8+ magnetic beads (MACS).
(2) Identification of CD8+ T cells within TILs: and (3) centrifuging the sorted T cells by PBS, closing to incubate the human CD8 flow antibody for 15 minutes, centrifuging and cleaning for 2 times by PBS, and detecting the CD8+ grouping in the TILs on a machine. See fig. 7.
(3) Determination of the inhibitory Effect of B7-H4 protein on human TILs: TILs are grouped as follows: negative control group: tils (nc); positive control group: TILs + CD3 antibody + CD28 antibody (PC); different concentration inhibition groups: TILs + CD3 antibody + CD28 antibody + varying concentrations of B7-H4 protein. Resuspending the cells in an incomplete T cell culture medium without IL-2, adjusting the cell concentration, planting the cells in an Elispot 96 pore plate coated with INF-gamma monoclonal antibodies according to groups, adding corresponding enzyme-labeled detection antibodies and substrate developing solutions after culturing for 48 hours, reading the plate after INF-gamma spots appear in the pores, and observing and counting the number of INF-gamma + TILs. See fig. 8: INF-gamma secreted by TIL of upstream Case1 was inhibited by different concentrations of B7-H4 protein; descending: TIL secretion of Case3 INF-. gamma.was inhibited by varying concentrations of B7-H4 protein. T cell function was significantly inhibited with increasing concentrations of B7-H4, as shown in FIG. 9.
Compared with the prior art, the B7-H4 protein prepared by the invention has good inhibition effect on over-immune reaction and cytokine storm, the B7-H4 protein can reach the center of the cytokine storm, namely the lung, in an exosome form by nasal inhalation administration, and experiments prove that the B7-H4 protein can reduce the number of CD8+ T cells and inhibit the CD8+ T cells from secreting the cytokine.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
- The application of B7-H4 protein in preparing medicine for resisting excessive immune reaction or cytokine storm.
- 2. The drug containing the B7-H4 protein is characterized in that the administration route of the drug is skin/mucous membrane external application, intravenous injection, intramuscular injection, oral administration, aerosol inhalation and inhalation by exosomes containing the protein.
- A method for preparing B7-H4 protein, which is characterized by comprising the following steps:s1, identifying the expression of B7-H4 protein;s2, protein expression and purification of B7-H4;and S3.B7-H4 protein verification.
- 4. The method of claim 2, wherein the identification of the B7-H4 protein expression in step S1 comprises:(1) obtaining a B7-H4 gene cDNA fragment: B7-H4 gene cDNA fragments are obtained through specific amplification primer design, PCR amplification and cDNA fragment purification and recovery;(2) double enzyme digestion: performing double enzyme digestion on the cDNA fragment and the plasmid of the B7-H4 gene by using endonuclease, performing agarose gel electrophoresis, and purifying and recovering the cDNA fragment;(3) connection and recombination: the B7-H4 gene cDNA fragment and the linearized plasmid PET-30a (+) are subjected to recombinant connection by using ligase;(4) cloning bacterium transformation: mixing E.coli DH5 alpha competent cells with the ligation product, and culturing;(5) and (5) cloning and identifying.
- 5. The process according to claim 4, wherein the endonuclease is NdeI or HindIII restriction endonuclease.
- 6. The method according to claim 4, wherein the ligase is T4 ligase.
- 7. The method according to claim 4, wherein the plasmid is PET-30a (+).
- 8. The method of claim 7, wherein the B7-H4 protein expression purification in the step S2 comprises:(1) B7-H4 recombinant protein expression: mixing E.coliBL21(DE3) competent cells with recombinant plasmids, culturing, randomly selecting a single clone on an LB agar plate to a Kan + LB liquid culture medium, adding IPTG (isopropyl-beta-thiogalactoside) to perform protein induction expression after culturing, and then performing protein identification;(2) separating and purifying the B7-H4 recombinant protein; collecting thalli, carrying out ultrasonic crushing, centrifuging, taking supernatant, enriching target protein by a His tag through a PET-30a (+) prokaryotic expression vector and adopting a His gel affinity chromatography method, specifically identifying an enzyme digestion sequence and a cutting site through 3C protease, separating the target protein from the His tag, thus obtaining the target protein with higher purity and better specificity, concentrating after identification, purifying by adopting an AKTA protein purification system, and collecting the purified protein with the purity of more than 95%.
- 9. The method of claim 8, wherein the molecular sieve pre-packed column of the AKTA protein purification system is Hiload superdex 200.
- 10. The method according to claim 2, wherein the B7-H4 protein verification method in step S3 is performed by Western Blot.
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