CN114057854B - Helicobacter pylori CD4+T cell tolerance polypeptide fusion antigen and application thereof - Google Patents
Helicobacter pylori CD4+T cell tolerance polypeptide fusion antigen and application thereof Download PDFInfo
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- CN114057854B CN114057854B CN202111164229.5A CN202111164229A CN114057854B CN 114057854 B CN114057854 B CN 114057854B CN 202111164229 A CN202111164229 A CN 202111164229A CN 114057854 B CN114057854 B CN 114057854B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
Abstract
The invention discloses helicobacter pylori CD4+The preparation process of the T cell tolerance polypeptide fusion antigen and the polyclonal antibody cow milk prepared by the antigen can be used for treating an infected person with helicobacter pylori natural immune tolerance, and have the advantages of no drug resistance, good patient compliance, easy large-scale production and the like.
Description
Technical Field
The invention relates to helicobacter pylori CD4+T cell tolerance polypeptide fusion antigen and polyclonal antibody cow milk prepared by the same.
Background
Helicobacter pylori (H.pylori: (Helicobacter pylori，Hp) Is a gram-negative bacterium that accompanies human evolution. At present forHpThe treatment is mainly a combination therapy of a proton pump in combination with two or three antibiotics. The increase of the antibacterial drug resistance, recurrent infection and high treatment cost limitHpThe treatment of (1). Prevention and cureHpThere is still a lack of ideal countermeasures for infection.
Helicobacter pylori infection can cause systemic and local immune reactions of the gastric mucosa. Usually, neutrophils are first recruited to enter the helicobacter pylori infected site of the gastric mucosa, if the neutrophils can not eliminate the helicobacter pylori in time, dendritic cells in the gastric mucosa will be activated, and then the neutrophils are recruitedB cells to the site of H.pylori infection of the gastric mucosa will recruit CD4 if the B cell response is not sufficient to eliminate H.pylori+T and CD8+T cells to the site of infection of gastric tissue. Activated CD4+Secretion of IFN-. gamma.and IL-2 by T will activate macrophages and CD8, respectively+T cells, activated macrophages and CD8+T cells can effectively remove micro-colonies, biofilms under mucosa and helicobacter pylori globiformis which hides in the cell space and bacteria in the cells; simultaneous activation of CD4+T can also enhance and amplify the immune response of B cells to eliminate H.pylori.
It is found in the research that,Hpthe reason why the drug can be parasitic in the stomach for a long time and is not easily cleared by the immune system of the body is that humans exhibit "innate tolerance" to it. That is to sayHpThe infection has vertical transmission of mother and fetus and central immune tolerance, i.e. the mother is pregnant and combinedHpThe infection is caused by the infection of the patient,Hpthe antigen substance is transmitted to embryo via blood-placenta, resulting in the formation of offspring in developmentHpCentral immune tolerance of the human body, thereby leading to continuous new generations in the human bodyHpIs a susceptible individual of (a). The reasons are as follows:
（1）Hpcan release Outer Membrane Vesicles (OMVs) containing bioactive substances, has a size of 20-400nm, can be remotely transmitted through blood circulation, and can be transmitted across the blood-placenta barrier.
(2) Pregnancy complicated with infectionHpThe number of the pregnant women is 25-62%, and the average is 46%.
(3) Infection during pregnancyHpHas injury effect on placenta and maternal infectionHpCan cause the fetal hypoevolutism, malformation and even the abortion of dead fetus. Andrea Kyburz et al report perinatal exposureHpCan influence the development of progeny regulatory T cells and can reduce the incidence rate of progeny respiratory allergic diseases.
(4) We start fromHpExtraction from culture solutionHpThe OMV is injected into a pregnant mouse after being marked by a fluorescent probe, and the marked OMV is observed and imaged in a fetal mouse body by using the animal integral imaging technology, so that the demonstration is thatHpThe ability of OMV to cross the placental barrier indicatesIn pregnant mother bodyHpThe OMVs produced, pass through the blood and can be transmitted across the placental barrier into the embryo.
Thus, OMV content can mediate progeny CD4+T cells undergo selective apoptosis and fail to effectively stimulate B cells and CD8+T cells and macrophages, which in turn cause progeny to exhibit innate tolerance to H.pylori and not be easily cleared.
Disclosure of Invention
The invention aims to provide the helicobacter pylori CD4 which can effectively break the immune tolerance and treat the helicobacter pylori infection+T cell tolerance polypeptide fusion antigen and polyclonal antibody cow milk prepared by the same.
The invention adopts the following technical scheme:
helicobacter pylori CD4+A T cell-tolerant polypeptide fusion antigen comprising an amino acid sequence as set forth in SEQ ID No. 1.
The method specifically comprises the following steps: DPRVPSSYVTQCGGNANGQKSTSSTTIFNNEPGYRGPGPGEHMDMLMVCHHLDKSIKEDVQFADSRGPGPGNLVVNTGKVKQTFNMGMRFLKKLYFPSYAQFLNLAPHFVIKKKRAYRPFQWGGPGPGINVSSQTFLKGMSNASSRTVLDSAAQKK is added.
A method for coding the helicobacter pylori CD4+A nucleic acid molecule comprising a T cell tolerance polypeptide fused to an antigen.
A vector comprising the nucleic acid molecule described above.
A method for preparing helicobacter pylori CD4+Polyclonal antibodies prepared from the T cell tolerance polypeptide fusion antigen.
The polyclonal antibody is used for treating a person infected with natural tolerance of helicobacter pylori.
A preparation method of helicobacter pylori CD4+T cell tolerance polypeptide fusion antigen.
The vaccine is used for preventing helicobacter pylori infection of people with natural immune tolerance of helicobacter pylori.
The helicobacter pylori CD4+T cell tolerant polypeptide fusion antigens or vaccines for stimulating an immune response in a host cow.
As described aboveHelicobacter pylori CD4+T cell tolerant polypeptide fusion antigens or vaccines for the production of polyclonal antibody bovine milk.
The invention has the beneficial effects that: the invention prepares the helicobacter pylori CD4+The T cell tolerance polypeptide fusion antigen can improve the eliminating capacity of the organism to the helicobacter pylori through a developed passive immune product, and is used for preventing and treating the helicobacter pylori infection.
Drawings
FIG. 1 is a diagram showing the mass spectrometric identification of polypeptides eluted from DC cells.
FIG. 2 shows the expression of WB to identify fusion polypeptides.
FIG. 3 is a mass spectrometric identification of the fusion polypeptides.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
(one)HpCulture of (2)
Will SS1HpInoculating into brain infusion broth (5 mg/L trimethoprim, 5mg/L polymixin-B, 5mg/L amphotericin-B, 10mg/L vancomycin) containing dextrin, and placing into a three-gas incubator (10% CO)2、85% N2、5% O2) Shaking culture is carried out for 3-10 days at the temperature of 37 ℃ and at the speed of 120 r/min.
(II) crude extractionHpOMV (OMV)
Will be cultured to logarithmic growth phaseHpCentrifuging the bacterial liquid at 10000 Xg for 10min, collecting the supernatant, filtering with a 0.22 μm bacteria filter, concentrating to 1/6 with the original volume by using an ultrafiltration centrifugal tube with the relative molecular mass cut-off of about 100KD, collecting the liquid, centrifuging at 39000 Xg for 60min, discarding the supernatant, and suspending the precipitate in a HEPES buffer solution (4-hydroxyethyl piperazine ethanesulfonic acid, N- (2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid, pH 6.8) of 180 μ L and 50mmol/L to obtain the crude extracted OMV.
(III) damping column purification OMV
3.1 column Balancing
(1) The Exosupur column was removed from the 4 ℃ freezer and mounted vertically on a mounting and separation assembly.
(2) Place waste liquid collecting tube (common centrifuge tube or beaker) under exclusion column, remove column top cover and bottom cover (note: remove top cover and then remove bottom cover), discard column sealing liquid (can pour directly or suck with pipettor) on column, and add PBS of not less than 20ml from top to wash column. During the washing process, the top sieve plate must be kept wet to avoid drying the column, and after the washing is completed, the bottom cover is covered and 1ml of PBS is added for standby.
3.2 sample Loading
(1) Taking off the bottom cover of the column, sucking PBS on the upper sieve plate by a pipettor, adding a crude OMV sample of not more than 1ml, and adding eluent (PBS) for elution after all the samples enter the sieve plate to prevent the samples from being diluted to influence the experimental result.
(2) After the sample completely enters the sieve plate and no liquid flows out from the bottom outlet, adding eluent (500 microliter of eluent is recommended to be added each time, and the next 500 microliter of eluent is added after the previous 500 microliter of eluent completely enters the exclusion column), collecting the effluent by a collecting tube (1.5 ml centrifuge tube), counting from the first drop of effluent, recording 1 fraction in every 500 microliter of effluent, and separating the first 3 fractions into void volumes without OMV components in the column of the type without collection.
3.3 OMV Collection
Void volume was then eluted by adding 2.5ml PBS directly and starting to collect OMVs into 1.5ml EP tubes and labeled OMV-4, OMV-5, OMV-6, OMV-7. 4 tubes were collected in total. After that, 1.5ml of PBS was added and collection of 1 tube was continued and labeled OMV-8.
3.4 Place the marked OMVs collected in the EP tubes in a freezer and store them in a-80 ℃ freezer.
(IV) Electron microscopy of OMV
And (3) respectively dripping purified OMV samples onto a carbon-coated copper net, dripping 2% uranyl acetate, drying at room temperature, and sending to an electron microscope room of Hebei medical university for detection.
(V) quantification and identification of OMV protein
The total protein concentration of OMVs was determined using the BCA method protein concentration quantification kit instructions. Protein spectrometers identify OMV content.
(VI) OMV-injected pregnant mouse
All animal experiments were conducted after review by the ethical committee on animal experiments at the university of medical science, north and river. Experimental mice (BALB/c) were provided by the animal experiments center of North Hei medical university. In each group, 2-3 female BALB/c mice and 1-2 male BALB/c mice were housed at about five pm in the first day, and female BALB/c mice were removed every other day at about nine am, and then pregnancy was confirmed when the formation of vaginal emboli was observed, and 0d was calculated, and they were marked and housed individually, and at 12d, the abdomen of the mice was observed to confirm pregnancy, and the non-pregnant mice were discarded. BALB/c pregnant mice were injected from tail vein or abdominal cavity at 6, 9, 12, 15 days of pregnancy, respectively, 50. mu.L/time/mouse, and PBS was used as a control group.
(VII) mouse DC culture
BALB/c mice (6-10 weeks old) were sacrificed, all femurs and tibiae were surgically removed, syringe needles were inserted into the medullary cavity from both ends of the bones, the bone marrow was washed out repeatedly into a petri dish, bone marrow suspension was collected, and small fragments and muscle tissues were filtered out with a 200 mesh nylon mesh; centrifuging the filtrate at 1200rpm for 5min, and discarding the supernatant; add 2ml of ammonium chloride erythrocyte lysate (1X), resuspend the cells, incubate at room temperature for 3-5 min. The obtained bone marrow cells were counted and then adjusted to a cell concentration of 2X 105Perml, 10ml of cells per 100mm cell culture dish in complete medium RPMI1640 containing 10% FBS, and recombinant small GM-CSF (200U/ml), 37 ℃, 5% CO2Culturing in an incubator; on day 3, 10ml of complete medium containing 20ng/ml recombinant mouse GM-CSF was added to the dish; half-volume changing the culture solution respectively at 6 th day and 8 th day, namely collecting the old culture solution, suspending the cell sediment again by using complete culture solution containing 20ng/ml recombinant mouse GM-CSF after centrifugation, and then putting the cell suspension back to the original dish; cells were collected at day 10, i.e., BMDCs.
(eight) elution of MHC-bound antigenic peptides by Weak acid elution
Mature BMDC (concentration 2X 10)5Piece/ml) with 10, 20, 40 and 60 mu g/ml OMV respectively, centrifuging, discarding the supernatant, adding 4ml of weak acid extract (0.131 mol/L citric acid, 0.066mol/L disodium hydrogen phosphate, adjusting pH to 3.3), shaking gently at room temperature for 5min, centrifuging, and absorbing the supernatant, and performing mass spectrometric identification. PBS was used as a control. A was measured according to the method indicated in the instructions of the Bradford protein concentration measuring kit595And calculating the polypeptide concentration according to a standard curve, and storing at-80 ℃ for later use.
(nine) elution polypeptide Mass Spectrometry identification
100uL of sample (the initial amount of the general treatment is about 100-200ug protein), add 200. mu.l of UA, centrifuge for 12,000g for 15 min; then adding 200 μ l of UA, centrifuging for 12,000g for 15 min; removing liquid in the centrifugal tube; adding 100 μ l IAA, mixing at 600 rpm for 1 min; incubating in dark for 20 min; centrifuging at 12,000g for 10 min; adding 100 μ l of UA, and centrifuging for 15 min; repeating twice; adding 100 mul ABC; centrifuging at 12,000g for 10 min; repeating twice; adding 40 mu l of ABC with trypsin; mixing, at 37 deg.C for 4-18 h; transferring into new collecting tube, 12,000 Xg for 10 min; adding 40 mul ABC; centrifuging for 12,000 Xg for 10 min; adding formic acid for acidification (the volume ratio of ABC to formic acid is 100: 1); freeze drying; desalting can be considered if the salt residue in the collection tube is high (mainly for removing ABC. SPE small columns are generally used, but sample loss is caused at the same time, and Trap column desalting is generally adopted in mass spectrometry); freeze drying; redissolving with 0.1% FA before sample introduction, and loading concentration is about 1 μ g.
Mass spectrometer detection analysis
A chromatograph: ThermoFisher EASY-nLC1200 nanoliter liquid phase; and (3) chromatographic column: c1875 μm by 150 mm, 3 μm, 100A; flow rate: 300 nl/min; mobile phase A: 0.1% Formic acid in Water; mobile phase B: 0.1% Formic acid in Acetonitrile; elution gradient time: and (5) 60 min. Ion source parameters: the spraying voltage is 2.1 kV; capillary temperature =275 oC; s-lens 60%; resolution setting: primary 60,000@ m/z 200; parent ion scan range: m/z 350-1600; and AGC setting: primary 3e6, secondary1e 5; maximum ion implantation time: first-stage 20ms and second-stage 45 ms; parent ion separation window: 1.6 Da. NCE collision energy: 27% HCD; and (3) dynamic exclusion: and 40 s.
Database search criteria
Proteome discover 2.2; protease: (ii) trypsin; missed cutting sites: 2; mass deviation of parent ion: 20 ppm; mass deviation of fragment ions: 0.02 Da; dynamic modification: oxidation/15.995Da (M), Deamidited/0.984 Da (N, Q); fixing and modifying: carbammidomethyl/57.021 Da (C); peptide fragment & spectrum false positive rate: less than or equal to 1 percent. The results of the identification are shown in FIG. 1.
(ten) analyzing the peptide fragments identified by mass spectrometry by using netMHCII 2.3 analysis software, and artificially synthesizing the peptide fragments for downstream screening, and storing at-20 ℃ for later use (10 mg/strip).
(eleven) screening of tolerance peptides by T-DC mix reaction
Peripheral Blood Mononuclear Cells (PBMCs) of the submouse were isolated by density gradient centrifugation, and CD4 was isolated from PBMCs by negative Magnetic Activated Cell Sorting (MACS) using a T cell isolation kit (Myltenil Biotec)+T cells.
In the mixed leukocyte reaction assay, 1, 2, 4, 8, 16. mu.g/ml artificially synthesized polypeptide-stimulated DCs and untreated DCs were cultured in complete RPMI1640 medium containing 10% FCS, l-glutamine, 2-mercaptoethanol (50. mu.M) and 1% diabody (penicillin, streptomycin). The DCs after stimulation with different polypeptides at different concentrations (0.5X 10)4、0.25×104、0.125×104) And CD4+T cells (96-well plate 1X 10)5/well) for 5 days. The blank control group was CD4 cultured alone+T cells. ELISA kit is used for detecting IL-2 and IFN-gamma of cell culture well supernatant, and the well without statistical significance with the blank control group is rat CD4+T cell tolerance peptide (P)>0.05); the wells statistically significant to the blank control group were the submouse CD4+T cell non-tolerant peptide fragment (P)<0.05）。
Expression of the (twelve) fusion polypeptide
12.1 construction of fusion polypeptide expression vectors
(1) According to the amino acid sequences (shown as SEQ ID No. 2-6) of the 5 screened tolerance peptides, a fusion gene segment is artificially synthesized, namely a nucleic acid sequence (with NdeI-XhoI enzyme cutting sites at two ends) shown as SEQ ID No.7, and the corresponding amino acid sequence is shown as SEQ ID No. 1.
(2) Enzyme cutting empty skeleton vector pET-22b (+) -T7 promoter-Gene-6XHis, agarose gel electrophoresis is carried out on the vector enzyme cutting product, and a target band is recovered.
Enzyme digestion system:
10x buffer 2μl
NdeI 1μl
XhoI 1μl
Plasmid 2-3μl
Add ddH2O to 20μl。
(3) ligation of synthetic genes to expression vectors
After obtaining a framework vector recovery product, cloning a synthetic gene into a pET-22b + vector by utilizing a seamless splicing kit according to the principle of homologous recombination, wherein the cloning site is as follows: NdeI (CATATG) -XhoI (CTAGAG).
(4) Transformation coating plate and bacterial plaque identification
And (3) converting 5-10 mu L of the seamlessly spliced product into 100 mu L of DH5a competent cells, carrying out heat shock for 1min by using a metal bath at 42 ℃, rapidly precooling for 2min on ice, adding 600 mu L of nonreactive medium into a super clean bench, carrying out shake culture for 1h by using a shaking table at 37 ℃, coating a proper amount of bacterial liquid on a flat plate containing Kan resistance, and carrying out inverted culture for 12-16 h in a constant temperature incubator. Positive clones were identified after colony PCR.
(5) Positive clone shake bacteria and plasmid extraction
3-4 single colony shake bacteria are selected, culture medium shake bacteria containing Kan is added overnight (8 ml LB liquid culture medium), and then plasmid extraction is carried out according to a plasmid extraction kit.
Preparation of polypeptide expression engineering bacteria
(1) Preparing BL21 competence;
(2) adding the extracted plasmid into BL21 to be competent, carrying out water bath heat shock at 42 ℃ for 90 seconds, quickly putting back on ice and standing for 5 minutes;
(3) adding 500 mu L of antibiotic-free sterile culture medium into a centrifuge tube, uniformly mixing, and recovering for 60 minutes at 37 ℃ at 200 rpm;
(4) carrying out instantaneous centrifugation at 3000rpm to collect bacteria, reserving about 100 mu L of supernatant, gently blowing and beating a heavy suspension bacteria block, and coating the heavy suspension bacteria block on an LB culture medium containing Kan;
(5) after the plate is placed upright for 30min, the plate is placed upside down in an incubator at 37 ℃ for overnight culture.
(6) And (5) obtaining positive bacterial colonies, shaking the bacteria, and preparing glycerol bacteria for freezing and storing for later use.
Enrichment of engineering bacteria
(1) Taking 10ml of LB liquid culture medium and putting into a 15ml centrifuge tube;
(2) inoculating BL21 engineering bacteria to LB liquid culture medium containing Kan, fixing with adhesive tape, and placing on a shaker at 37 deg.C for 16-18h, wherein the cover is not too tight;
(3) the next day, pouring the mixture into 200ml of LB liquid culture medium, and shaking the mixture overnight;
(4) taking out on the third day, subpackaging into 50ml centrifuge tubes, centrifuging (5000 r, 10-15 min), washing once with PBS, and storing at-80 ℃.
Cracking BL21 engineering bacteria
(1) 10mg/ml lysozyme (dissolved in PBS, diluted to 1mg/ml with RIPA lysate);
(2) adding 1ml of lysozyme into 9ml of RIPA lysate, adding protease inhibitor, performing lysis for 30 minutes, uniformly mixing by using a gun head every 10 minutes, performing ultrasonic lysis, centrifuging (12000 g,10 min), and storing supernatant.
Bacterial split protein quantification
Mu.l of PBS + 1. mu.l of sample, and then 200. mu.l of working solution (A: B = 1: 50) were added, incubated at 37 ℃ for 30 minutes, and the absorbance was measured at 570 nm.
Purification of fusion polypeptides by protein purification apparatus
(1) Filtering the prepared bacterial lysis protein by using a 0.22 micron filter, and then diluting the concentration of the bacterial lysis protein to 1 mg/ml;
(2) connecting a purification column, washing a pump by using ultrapure water, and performing column volume of 3-5;
(3) washing 3-5 column volumes with binding buffer;
(4) loading 10 column volumes, then washing 5-10 column volumes with binding buffer;
(5) washing with eluent, once a peak is seen, immediately pausing the elution for 2 minutes to allow the peak to fully react, and then starting collecting;
(6) after the collection is completed, the column is disassembled and washed with binding buffer solution for 5-10 column volumes, and then washed with 20% ethanol;
(7) the collected fusion polypeptide is stored in a refrigerator at the temperature of minus 80 ℃ for standby.
Capture His tag
(1) Freeze-drying the purified fusion polypeptide, then performing SDS-PAGE, and dividing the gel sample into two parts (one part is used for exposing and capturing the His tag, and the other part is used for mass spectrum identification);
(2) a half of film rotation;
(3) putting the PVDF membrane after the membrane conversion into a sealing liquid, and sealing on a shaking table for 1 hour;
(4) washing off blocking solution with PBST (3 times, 5-10min each time), incubating primary antibody (1: 3000), and incubating overnight at 4 deg.C with shaking;
(5) washing the incubated PVDF membrane with PBST for 3 times in the next day, 5-10min each time;
(6) incubating the secondary antibody (1: 1000), incubating for 1 hour in the dark (gentle shaking), washing for 3 times with PBST, and keeping out of the light;
(7) exposure was performed to see if His tag was present. The results are shown in FIG. 2.
Identification by mass spectrometry
(1) Baking and dyeing the other half of the glue, and dyeing for half an hour on a shaking table;
(2) decolorizing, changing 1 time of decolorizing solution every 1h, and then incubating overnight at 4 ℃;
(3) the next day, the objective band on the gel was cut off with a scalpel blade, cut into 1mm pieces, and placed in 1.5mL EP tubes;
(4) adding 300ul of color solution into each tube, decolorizing at room temperature, cleaning to be transparent, and removing supernatant; the replacement of the decoloring solution is recommended every 1 to 2 hours to accelerate the decoloring process; according to different conditions, the decolorization generally needs 4 to 8 hours;
(5) drying glue: adding 300ul 100% ACN thermo Mixer, shaking for 5min until the colloidal particles turn white, removing ACN by suction, and lyophilizing for 3 min;
(6) 300ul 10mM DTT/50mM NH was added to each tube4HCO3Oscillating and mixing evenly until the gel block is swollen and transparent, at 56 ℃ for 1h, and then discarding the supernatant;
(7) drying the glue: adding 300uL 100% ACN thermo Mixer, shaking for 5min until the colloidal particles turn white, removing ACN by suction, and lyophilizing for 3 min;
(8) 300ul of 60mM IAA/50mM NH was added to each tube4HCO3Oscillating and mixing uniformly in a dark place until the rubber block is swollen and transparent, and reacting for 30 minutes in a dark place;
(9) drying glue: adding 300uL 100% ACN ThermoMixer, shaking for 5min until the micelle turns white, removing ACN by suction, and freeze-drying for 3 min;
(10) adding 50-80ul of 50mM ammonium bicarbonate solution into each tube, adding 1-2ug pancreatin (or mixing the two and adding into the sample, submerging the adhesive tape), crushing the gel with a glass rod, and incubating at 37 deg.C for more than 6 hr;
(11) adding 200ul acetonitrile containing 0.1 percent of FA into each tube, shaking for 5 minutes, and sucking supernatant into a clean EP tube;
(12) adding 30ul of 0.1 percent FA into the gel, shaking for 5 minutes, adding 200ul of acetonitrile containing 0.1 percent FA, shaking for 5 minutes, sucking the supernatant, combining the two supernatants, and freeze-drying for more than 3 hours;
(13) desalination
Methanol activation: centrifuging 200 μ l methanol at room temperature 1200g for 5min, and discarding the effluent
Buffer B: 200ul, 2 times, 4000g,2min
Buffer A: 200 mul, 3 times, 6000g, 2min
The sample was diluted to 200. mu.l and added to a desalting column, 2000g, 5min
Buffer A: 200 mul, 3 times, 6000g, 2min
Buffer B: 180 mul, 2 times of centrifugation, 2000g, 2 min; 4000g,2 min.
The two eluates were placed in new EP tubes and lyophilized.
(14) Upper mass spectrum
The results are shown in FIG. 3.
(thirteen) preparation of polyclonal antibody cow milk
13.1 preparation of fusion polypeptide vaccines
13.1.1 enrichment of bacteria
(1) Taking 10ml of LB liquid culture medium and putting into a 15ml centrifuge tube;
(2) inoculating BL21 engineering bacteria to LB liquid culture medium containing Kan, covering the cover not too tight, fixing with adhesive tape, and placing on a shaking table at 37 deg.C for 16-18 h;
(3) the next day, pouring the mixture into 200ml of LB liquid culture medium, and shaking the mixture overnight;
(4) taking out on the third day, subpackaging into 50ml centrifuge tubes, centrifuging (5000 r, 10-15 min), washing once with PBS, and storing at-80 ℃.
13.1.2 lysis BL21 engineering bacteria
(1) 10mg/ml lysozyme (dissolved in PBS, diluted to 1mg/ml with RIPA lysate);
(2) adding 1ml of lysozyme into 9ml of RIPA lysate, adding protease inhibitor, performing lysis for 30 minutes, uniformly mixing by using a gun head every 10 minutes, performing ultrasonic lysis, centrifuging (12000 g,10 min), and storing supernatant.
13.1.3 quantification of bacterial lysis protein
Mu.l of PBS + 1. mu.l of sample, and then 200. mu.l of working solution (A: B = 1: 50) were added, incubated at 37 ℃ for 30 minutes, and the absorbance was measured at 570 nm.
13.1.4 purification of fusion polypeptide by protein purification instrument
(1) Filtering the prepared bacterial lysis protein by using a 0.22 micron filter, and then diluting the concentration of the bacterial lysis protein to 1 mg/ml;
(2) connecting a purification column, washing a pump by using ultrapure water, and performing column volume of 3-5;
(3) washing 3-5 column volumes with binding buffer;
(4) loading 10 column volumes, and then washing 5-10 column volumes with binding buffer;
(5) washing with eluent, once a peak is seen, immediately pausing the elution for 2 minutes to allow the peak to fully react, and then starting collecting;
(6) after the collection is completed, the column is disassembled and washed with binding buffer solution for 5-10 column volumes, and then washed with 20% ethanol;
(7) the collected fusion polypeptide vaccine is stored in a refrigerator at the temperature of minus 80 ℃ for standby.
Fused polypeptide immune milk cow
Selecting physically healthy pregnant cows of 3-4 years old, immunizing before delivery at 63d, 42 d, 14d and after delivery at 21d for 4 times; preparing 4ml of 100mg of the fusion polypeptide and an aluminum hydroxide adjuvant for the first time, and performing intramuscular injection on two sides of the neck of the cow; the latter three times were made up to 4ml with 50mg of the fusion polypeptide plus aluminium hydroxide adjuvant. And further performing boosting immunization according to the detection result of the antibody titer.
Detecting the antibody titer of cow milk
13.3.1 preparation of whey
Loading cow milk into a 50ml centrifuge tube at 3000r/min for 10 min; after degreasing, adjusting the pH value to 4.5-4.6 by using 1mol/L HCl, placing the mixture in a water bath box at 40 ℃ for 1h to completely separate out casein, centrifuging the mixture for 10min at 3000r/min again, pouring out supernatant, adjusting the pH value to 6.8-7.0 by using 1mol/L NaOH to obtain whey, and subpackaging the whey for later use.
13.3.2 Indirect ELISA for detecting antibody titer
Pretreating a 96-pore plate by using 150 mu l/pore of 2.5% glutaraldehyde solution for 1 hour, washing the 96-pore plate by using ultrapure water at 37 ℃ for 4 times, and wiping the plate dry each time; respectively coating the enzyme-labeled plate with 100 mul (0.05 mg/ml) of the synthesized polypeptide and the fusion polypeptide, drying at 37 ℃, washing the plate 3 times with a PBST (PBS buffer solution containing 0.5ml/L Tween 20) washing solution, 3min each time, and completely drying by throwing each time; blocking with 3% BSA in PBST for 1 hour, and washing as above; adding whey with different dilutions, 100 mu l/well and 3 multiple wells per sample, and incubating for 1 hour at 37 ℃; after washing 3 times, rabbit anti-bovine IgG-HRP (1: 8000), 100. mu.l/well was added and incubated for 1 hour at 37 ℃; adding 100ul of tetramethylbenzidine substrate working solution after washing for 3 times, and adding 50 mu l H in each hole after reacting for 15 minutes at 37 DEG C2SO4(2 mol/L) the reaction was stopped, and the absorbance at 450nm was measured immediately on a microplate reader as OD450The maximum dilution factor of 0.5 is the antibody titer.
Collecting and sanitizing milk
Collecting milk with the antibody titer reaching and stabilizing above 1: 1200; sterilizing cow milk at 62.8-65.6 deg.C for 30min or 71.7 deg.C for 15 s.
SEQUENCE LISTING
<110> Hospital of Hebei university of medical science, Hebei university of medical science
<120> helicobacter pylori CD4+ T cell tolerance polypeptide fusion antigen and application thereof
<130> 2021
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 156
<212> PRT
<213> Artificial Synthesis
<400> 1
Asp Pro Arg Val Pro Ser Ser Tyr Val Thr Gln Cys Gly Gly Asn Ala
1 5 10 15
Asn Gly Gln Lys Ser Thr Ser Ser Thr Thr Ile Phe Asn Asn Glu Pro
20 25 30
Gly Tyr Arg Gly Pro Gly Pro Gly Glu His Met Asp Met Leu Met Val
35 40 45
Cys His His Leu Asp Lys Ser Ile Lys Glu Asp Val Gln Phe Ala Asp
50 55 60
Ser Arg Gly Pro Gly Pro Gly Asn Leu Val Val Asn Thr Gly Lys Val
65 70 75 80
Lys Gln Thr Phe Asn Met Gly Met Arg Phe Leu Lys Lys Leu Tyr Phe
85 90 95
Pro Ser Tyr Ala Gln Phe Leu Asn Leu Ala Pro His Phe Val Ile Lys
100 105 110
Lys Lys Arg Ala Tyr Arg Pro Phe Gln Trp Gly Gly Pro Gly Pro Gly
115 120 125
Ile Asn Val Ser Ser Gln Thr Phe Leu Lys Gly Met Ser Asn Ala Ser
130 135 140
Ser Arg Thr Val Leu Asp Ser Ala Ala Gln Lys Lys
145 150 155
<210> 2
<211> 28
<212> PRT
<213> Helicobacter pylori
<400> 2
Tyr Val Thr Gln Cys Gly Gly Asn Ala Asn Gly Gln Lys Ser Thr Ser
1 5 10 15
Ser Thr Thr Ile Phe Asn Asn Glu Pro Gly Tyr Arg
20 25
<210> 3
<211> 26
<212> PRT
<213> Helicobacter pylori
<400> 3
Glu His Met Asp Met Leu Met Val Cys His His Leu Asp Lys Ser Ile
1 5 10 15
Lys Glu Asp Val Gln Phe Ala Asp Ser Arg
20 25
<210> 4
<211> 20
<212> PRT
<213> Helicobacter pylori
<400> 4
Asn Leu Val Val Asn Thr Gly Lys Val Lys Gln Thr Phe Asn Met Gly
1 5 10 15
Met Arg Phe Leu
20
<210> 5
<211> 30
<212> PRT
<213> Helicobacter pylori
<400> 5
Leu Tyr Phe Pro Ser Tyr Ala Gln Phe Leu Asn Leu Ala Pro His Phe
1 5 10 15
Val Ile Lys Lys Lys Arg Ala Tyr Arg Pro Phe Gln Trp Gly
20 25 30
<210> 6
<211> 26
<212> PRT
<213> Helicobacter pylori
<400> 6
Ile Asn Val Ser Ser Gln Thr Phe Leu Lys Gly Met Ser Asn Ala Ser
1 5 10 15
Ser Arg Thr Val Leu Asp Ser Ala Ala Gln
20 25
<210> 7
<211> 480
<212> DNA
<213> Artificial Synthesis
<400> 7
catatggatc cgcgtgttcc gagcagctat gtgacgcaat gtgggggtaa tgcaaatggt 60
cagaaaagca ccagcagcac caccattttt aacaatgaac cgggttatcg tggtcctggt 120
ccaggtgagc acatggacat gcttatggtg tgccaccact tggataaaag cattaaagaa 180
gatgtccagt tcgctgattc aaggggtcct ggtccaggta acctggttgt taataccggt 240
aaagttaaac agaccttcaa tatgggtatg cgcttcctga agaaattata tttccccagt 300
tacgcgcaat ttttaaactt agcacctcac tttgtcatta aaaaaaagcg cgcttataga 360
ccctttcaat gggggggtcc tggtccaggt atcaatgtgt cttctcaaac ttttttaaaa 420
ggaatgagca acgcttcttc acgaacagtg cttgattcag ccgctcagaa gaaactagag 480
Claims (8)
1. Helicobacter pylori CD4+The T cell tolerance polypeptide fusion antigen is characterized in that the amino acid sequence of the T cell tolerance polypeptide fusion antigen is shown as SEQ ID No. 1.
2. A gene encoding the helicobacter pylori CD4 of claim 1+A nucleic acid molecule comprising a T cell tolerance polypeptide fused to an antigen.
3. A vector comprising the nucleic acid molecule of claim 2.
4. Use of the helicobacter pylori CD4 of claim 1+Polyclonal antibodies prepared from the T cell tolerance polypeptide fusion antigen.
5. Use of a polyclonal antibody as defined in claim 4 in the manufacture of a medicament for the treatment of an infected person who is naturally immune-tolerant to H.pylori.
6. Use of helicobacter pylori CD4 according to claim 1+T cell tolerance polypeptide fusion antigen.
7. Helicobacter pylori CD4 according to claim 1+The T cell tolerance polypeptide fusion antigen is applied to the preparation of cow vaccines for stimulating the immune response reaction of cows.
8. Use of the helicobacter pylori CD4 of claim 1+Immune cow milk prepared by the T cell tolerance polypeptide fusion antigen.
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| CN1246799A (en) * | 1996-12-05 | 2000-03-08 | 阿斯特拉公司 | Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof |
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| 表达结核杆菌融合抗原的重组酿酒酵母免疫效果研究;江佳稀;《中国优秀硕士学位论文全文数据库》;20101231(第03期);全文 * |
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