CN107550748B - Polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine and preparation and application thereof - Google Patents
Polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine and preparation and application thereof Download PDFInfo
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- CN107550748B CN107550748B CN201710770237.1A CN201710770237A CN107550748B CN 107550748 B CN107550748 B CN 107550748B CN 201710770237 A CN201710770237 A CN 201710770237A CN 107550748 B CN107550748 B CN 107550748B
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- protein
- staphylococcus aureus
- enterotoxin
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- procyanidine
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- CWEZAWNPTYBADX-UHFFFAOYSA-N Procyanidin Natural products OC1C(OC2C(O)C(Oc3c2c(O)cc(O)c3C4C(O)C(Oc5cc(O)cc(O)c45)c6ccc(O)c(O)c6)c7ccc(O)c(O)c7)c8c(O)cc(O)cc8OC1c9ccc(O)c(O)c9 CWEZAWNPTYBADX-UHFFFAOYSA-N 0.000 claims abstract description 13
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of daily chemicals, and particularly relates to a polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine, and preparation and application thereof. The polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine comprises the following components: PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, sodium dodecyl sulfate, saccharin sodium, spice, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate, staphylococcal enterotoxin-B protein, procyanidins and water. Through reasonable proportion, the invention ensures that the superantigen staphylococcus aureus type-B enterotoxin protein can stably and effectively exist in the polishing paste, and the staphylococcus aureus type-B enterotoxin protein and procyanidine have synergistic interaction, thereby effectively preventing and treating periodontitis.
Description
Technical Field
The invention belongs to the field of daily chemicals, and particularly relates to a polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine, and preparation and application thereof.
Background
Among staphylococcus (staphylococcus) s (s. Aureus) is most pathogenic, often causing food poisoning. Staphylococcus aureus is gram-positive coccus with a diameter of 0.8-1.0 μm and is in a grape shape. No bud holding and no flagella. Aerobic and facultative anaerobes, the growth temperature is between 6.5 and 46 ℃ and the optimal temperature is between 30 and 37 ℃. Can grow in the pH range of 4.0-9.8, and optimally grows to pH7.4. Can grow in 15% NaCl and 40% bile. On the common broth solid culture medium, smooth, low-protruding, flashing and clean-edged colonies can be formed, and the colony pigment is unstable, but most of the colonies are golden yellow.
The strain can produce enterotoxin causing food poisoning at 20-37 ℃. Among them, staphylococcus aureus enterotoxin B is a bacterial superantigen secreted by staphylococcus aureus, and has various biological activities. The superantigen activates T cells in a manner different from the usual antigen, and can be directly combined with MHC class II molecules and the V region of TCR-beta chain without the internalization and degradation process of Antigen Presenting Cells (APCs), so that a large number of T cells can be selectively amplified and activated, and cells expressing MHC class II molecules can be combined with the superantigen activating T cells, and the activated T cells can generate cytotoxicity on target cells expressing MHC class II molecules, which is called superantigen-dependent cell mediated cytotoxicity (SDCC). Meanwhile, the production of tumor sensitive cytokines such as IFN-gamma, TNF-alpha and IL-2 can be induced, thereby achieving the effects of indirectly killing tumor cells and inhibiting tumor growth. Compared with common antigen, the superantigen SEB does not need APC treatment, is not limited by MHC class II molecules, and can react in T cell library about 5% -20% of cells aiming at a specific superantigen due to the polymorphism of V beta gene. Based on this, superantigen SEB is increasingly applied to tumor treatment, and becomes an important direction of immunological research.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine.
The invention also aims to provide a preparation method of the polishing paste containing the staphylococcus aureus type B enterotoxin protein and the procyanidine.
It is still another object of the present invention to provide the use of the polishing paste containing staphylococcal enterotoxin type B protein and procyanidins described above.
The aim of the invention is achieved by the following technical scheme:
a polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine comprises the following components in percentage by mass:
the polishing paste containing the staphylococcus aureus type-B enterotoxin protein and the procyanidine preferably comprises the following components in percentage by mass:
the amino acid sequence of the staphylococcus aureus enterotoxin B protein is shown as SEQ ID NO. 1;
the nucleotide sequence of the gene for encoding the staphylococcus aureus type-B enterotoxin protein is as follows:
GAGAACCTGTACTTCCAGGGCGAAAGCCAGCCGGACCCGAAACCGGATGAACTGCACAAAAGCAGCAAATTCACCGGCCTGATGGAAAACATGAAAGTGCTGTACGACGACAACCACGTCTCCGCCATCAACGTGAAAAGCATCGATCAGTTCCTGTACTTCGACCTGATCTACTCCATCAAAGACACCAAACTGGGTAACTACGACAACGTCCGCGTCGAGTTCAAAAACAAAGACCTGGCGGACAAGTACAAAGACAAATACGTGGACGTTTTCGGCGCGAACTACTACTACCAGTGCTACTTCTCCAAAAAGACCAACGACATCAACTCCCACCAGACCGATAAACGCAAGACCTGCATGTACGGCGGCGTGACCGAACACAACGGCAACCAGCTGGACAAATACCGCAGCATCACCGTGCGCGTGTTCGAGGACGGCAAAAACCTGCTGAGCTTCGATGTGCAGACCAACAAGAAAAAAGTGACCGCGCAGGAACTGGACTACCTGACCCGTCACTACCTGGTGAAAAACAAAAAACTGTACGAGTTCAACAACTCCCCGTACGAAACCGGCTACATCAAGTTCATCGAAAACGAAAACAGCTTCTGGTACGACATGATGCCGGCGCCGGGCGACAAGTTCGACCAGTCCAAGTACCTGATGATGTACAACGACAACAAAATGGTTGACAGCAAAGACGTGAAAATCGAAGTGTACCTGACCACCAAAAAGAAATAATGA
the preparation method of the staphylococcus aureus type-B enterotoxin protein preferably comprises the following steps:
(1) The gene of the recombinant staphylococcus aureus type B enterotoxin protein shown as SEQ ID NO.1 is obtained through total gene synthesis;
(2) Carrying out PCR amplification on the gene obtained in the step (1) by using a primer X5523-1 and a primer X5523-34 to obtain a target fragment, and recovering and purifying;
(3) Connecting the target product recovered and purified in the step (2) with a carrier to obtain a recombinant plasmid;
(4) Transferring the recombinant plasmid prepared in the step (3) into a host cell to obtain a strain for expressing recombinant staphylococcus aureus type-B enterotoxin protein;
(5) Taking the strain expressing the recombinant staphylococcus aureus type-B enterotoxin protein prepared in the step (4) as an expression strain, and carrying out induced expression based on an escherichia coli expression system; centrifugally collecting thalli, washing and suspending, then ultrasonically crushing, centrifuging, collecting supernatant, and purifying to obtain staphylococcus aureus type-B enterotoxin protein;
the nucleotide sequences of the primer X5523-1 and the primer X5523-34 described in step (2) are as follows:
primer X5523-1: GACACGGTACCGAGAACCTGTACTTCCAG;
primer X5523-34: GTGTCCTCGAGTCATTATTTCTTTTTGGTGGTCAGG;
the vector in the step (3) is preferably pET-32a;
the host cell described in step (4) is preferably E.coli BL21 (DE 3);
the specific procedure for inducing expression described in step (5) is preferably: performing liquid amplification culture on a strain expressing recombinant staphylococcus aureus type-B enterotoxin protein until the OD value reaches 0.5-0.6, adding IPTG with the final concentration of 0.5mM, and inducing at 220rpm and 37 ℃ for 4 hours;
the liquid expansion culture comprises the following steps:
selecting single colony of strain expressing recombinant staphylococcus aureus enterotoxin B protein, culturing in LB liquid medium containing 50 mug/mL ampicillin at 37 deg.C and 220rpm overnight to obtain seed liquid; the cultured seed liquid is prepared according to the following ratio of 1:100 volume ratio is inoculated in LB liquid medium containing 50 mug/mL ampicillin, and cultured at 37 ℃ and 220 rpm;
the wash suspended reagent in step (5) is preferably a disruption Buffer of 1 XPBS Buffer containing 0.1% w/v Triton X-114 at pH 7.4;
the conditions of the ultrasonic disruption described in step (5) are preferably: under ice bath, the ultrasonic power is 400-450W, the ultrasonic time is 5-25 min, wherein the ultrasonic is carried out for 2s and the suspension is carried out for 6s in a cycle;
the purification described in step (5) comprises the steps of:
first Nickel Sepharose affinity chromatography
(1) Loading Ni-IDA (nickel-iminodiacetic acid, affinity chromatography medium) into a column, and cleaning the balance column with a Binding buffer1 with a volume of 3 times of the column bed, wherein the flow rate is 5mL/min;
(2) ultrasonically crushing, centrifuging, and collecting the supernatant, and loading the supernatant onto a column, wherein the flow rate is 2mL/min;
(3) 5 times of the column bed volume of Binding buffer1 to clean the column, and the flow rate is 3mL/min;
(4) wash Buffer washing impurities at a flow rate of 3mL/min, and collecting penetrating fluid;
(II) enzymatic cleavage of protein tag
Dialyzing the penetrating fluid collected in the step (I), then adding TEV enzyme, and cutting off the tag by enzyme to obtain the penetrating fluid after enzyme cutting;
(III) second Nickel agarose affinity chromatography
(1) Filling the Ni-IDA column, and cleaning the balance column with Binding buffer2 with the volume of 3 times of the bed volume, wherein the flow rate is 5mL/min;
(2) loading the cut penetrating fluid obtained in the step (II) to a column, wherein the flow rate is 2mL/min, and collecting the penetrating fluid;
(IV) filtration
Filtering the penetrating fluid collected in the step (III) through a 0.22 mu m CA (cellulose acetate) filter membrane to obtain staphylococcus aureus type B enterotoxin protein;
the Binding Buffer1 described in step (I) is preferably a 1 XPBS Buffer with 0.1% w/v Triton X-114, pH 7.4;
the Wash buffer in step (I) is preferably a 1 XPBS buffer containing 20mM Imidazole, and has a pH of 7.4;
the dialysis in step (II) is preferably a dialysis with 1 XPBS buffer to a pH of 7.4 for 2 hours;
the conditions for the cleavage in step (II) are preferably cleavage overnight at 25 ℃;
the Binding Buffer2 in the step (III) is preferably 1 XPBS Buffer with pH value of 7.4;
the preparation method of the polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine comprises the following steps:
(1) Mixing components PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, saccharin sodium, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate and water of a polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine to obtain paste 1;
(2) Adding procyanidine and staphylococcus aureus type-B enterotoxin protein into the paste 1 prepared in the step (1), and uniformly mixing to obtain a paste 2;
(3) Adding the rest components (sodium dodecyl sulfate and perfume) into the paste 2 prepared in the step (2), and uniformly mixing to obtain a paste 3;
(4) Vacuumizing the paste 3 to obtain polishing paste containing staphylococcus aureus enterotoxin B protein and procyanidine;
the polishing paste containing the staphylococcus aureus type-B enterotoxin protein and the procyanidine can be further packaged, for example: filling;
the polishing paste containing the staphylococcus aureus B-type enterotoxin protein and the procyanidine is applied to the fields of medicine and daily chemicals;
compared with the prior art, the invention has the following advantages and effects:
(1) The invention adds the superantigen staphylococcus aureus type B enterotoxin protein into the tooth polishing paste, and can effectively prevent and treat periodontitis.
(2) Through reasonable proportion, the invention ensures that the superantigen staphylococcus aureus type B enterotoxin protein can stably and effectively exist in the polishing paste.
(3) The procyanidine is added into the polishing paste, can produce a synergistic effect with SEB protein, has the effects of eliminating or relieving periodontitis reddening and swelling, gingival bleeding and the like, and also has an anti-sensitivity effect.
(4) The superantigen staphylococcus aureus type-B enterotoxin protein provided by the invention has the advantages of high purity, strong activity, simple preparation method, short time consumption and low cost.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis detection of a gene encoding a recombinant S.aureus type B enterotoxin protein obtained by the second round of amplification in example 1; wherein M: a DNA marker;1: a gene encoding a recombinant staphylococcal enterotoxin type B protein.
FIG. 2 is a diagram showing agarose gel electrophoresis of vector pET-32a after cleavage in example 1; wherein M: a DNA marker;1 and 2: vector pET-32a after KpnI and XhoI cleavage.
FIG. 3 is an agarose gel electrophoresis detection diagram of the recombinant vector pET32a-SEB constructed in example 1 after cleavage; wherein M: a marker;1: the recombinant plasmid pET32a-SEB after enzyme digestion.
FIG. 4 is a SDS-PAGE analysis of the best induction conditions selected for the pilot culture of example 3; wherein M: protein Marker;1: a negative control; 2: supernatant at 20 ℃;3: precipitating at 20 ℃;4: supernatant at 37 ℃;5: precipitation was carried out at 37 ℃.
FIG. 5 is a diagram of SDS-PAGE analysis of the first nickel agarose affinity chromatography purification of example 3; wherein M: a marker;1: bacterial disruption supernatant; 2: outflow (penetrating fluid a); 3:20mM Imidazole eluting component (permeate B); 4:50mM Imidazole eluting component (penetration C); 5:500mM Imidazole eluting component (eluent D), loading containing reducing agent.
FIG. 6 is a diagram showing SDS-PAGE analysis of the second nickel agarose affinity chromatography purification in example 3; wherein M: protein marker;1: penetrating fluid B before enzyme cutting; 2: the penetration liquid B after enzyme cutting; 3, outflow (penetrating fluid E); 4 to 5:20mM Imidazole eluting component (penetration F); 6:500mM Imidazole eluting component (eluent G), loading containing reducing agent.
FIG. 7 is a SDS-PAGE analysis of the purified and filtered protein of interest of example 3.
FIG. 8 is a Western Blot analysis of purified fusion proteins and unlabeled proteins of interest; wherein M: prestained Marker;1: a fusion protein; 2: the target protein is subjected to label removal.
FIG. 9 is a standard graph of BSA content of the standard protein constructed in example 3.
FIG. 10 is a diagram showing the morphology of a polishing paste containing Staphylococcus aureus type B enterotoxin protein and procyanidins obtained in example 1.
FIG. 11 is a graph showing the therapeutic effect of polishing paste containing Staphylococcus aureus type B enterotoxin protein and procyanidins prepared in example 1 after three days, wherein A: before treatment; b: after three days of use.
FIG. 12 is a graph showing the therapeutic effect of the polishing paste containing Staphylococcus aureus type B enterotoxin protein and procyanidins prepared in example 1 after one week; wherein A: before use; b: after one week of use.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
EXAMPLE 1 construction of recombinant plasmid pET32a-SEB
(1) Amplifying gene encoding recombinant staphylococcus aureus enterotoxin B protein
According to the published staphylococcus aureus type B enterotoxin protein sequence (NCBI Reference Sequence:WP_ 072497559.1), the staphylococcus aureus type B enterotoxin protein sequence is redesigned, 34 synthetic primers are designed according to the optimized gene sequence, and a sufficient amount of target product DNA is amplified through a first round of PCR, wherein a reaction system (50 mu L) is as follows:
wherein, the primer mix is the primers X5523-1 to X5523-34, 34 total, 0.4 muL×34=13.6 muL; the primer sequences are shown below:
X5523-1：GACACGGTACCGAGAACCTGTACTTCCAG；
X5523-2：CGGGTCCGGCTGGCTTTCGCCCTGGAAGTACAGGTTCTCG；
X5523-3：GCCAGCCGGACCCGAAACCGGATGAACTGCACAAAAGCAG；
X5523-4：TCCATCAGGCCGGTGAATTTGCTGCTTTTGTGCAGTTCAT；
X5523-5：CACCGGCCTGATGGAAAACATGAAAGTGCTGTACGACGAC；
X5523-6：ACGTTGATGGCGGAGACGTGGTTGTCGTCGTACAGCACTT；
X5523-7：TCTCCGCCATCAACGTGAAAAGCATCGATCAGTTCCTGTA；
X5523-8：ATGGAGTAGATCAGGTCGAAGTACAGGAACTGATCGATGC；
X5523-9：TCGACCTGATCTACTCCATCAAAGACACCAAACTGGGTAA
X5523-10：TCGACGCGGACGTTGTCGTAGTTACCCAGTTTGGTGTCTT；
X5523-11：CAACGTCCGCGTCGAGTTCAAAAACAAAGACCTGGCGGAC；
X5523-12：GTCCACGTATTTGTCTTTGTACTTGTCCGCCAGGTCTTTG；
X5523-13：TACAAAGACAAATACGTGGACGTTTTCGGCGCGAACTACT；
X5523-14：TTTTGGAGAAGTAGCACTGGTAGTAGTAGTTCGCGCCGAA；
X5523-15：CCAGTGCTACTTCTCCAAAAAGACCAACGACATCAACTCC；
X5523-16：TCTTGCGTTTATCGGTCTGGTGGGAGTTGATGTCGTTGGT；
X5523-17：AGACCGATAAACGCAAGACCTGCATGTACGGCGGCGTGAC；
X5523-18：TTGTCCAGCTGGTTGCCGTTGTGTTCGGTCACGCCGCCGT；
X5523-19：GCAACCAGCTGGACAAATACCGCAGCATCACCGTGCGCGT；
X5523-20：TCAGCAGGTTTTTGCCGTCCTCGAACACGCGCACGGTGAT；
X5523-21：CGGCAAAAACCTGCTGAGCTTCGATGTGCAGACCAACAAG；
X5523-22：GTTCCTGCGCGGTCACTTTTTTCTTGTTGGTCTGCACATC；
X5523-23：TGACCGCGCAGGAACTGGACTACCTGACCCGTCACTACCT；
X5523-24：CGTACAGTTTTTTGTTTTTCACCAGGTAGTGACGGGTCAG；
X5523-25：GTGAAAAACAAAAAACTGTACGAGTTCAACAACTCCCCGT；
X5523-26：GAACTTGATGTAGCCGGTTTCGTACGGGGAGTTGTTGAAC；
X5523-27：ACCGGCTACATCAAGTTCATCGAAAACGAAAACAGCTTCT；
X5523-28：CGCCGGCATCATGTCGTACCAGAAGCTGTTTTCGTTTTCG；
X5523-29：GACATGATGCCGGCGCCGGGCGACAAGTTCGACCAGTCCA；
X5523-30：TCGTTGTACATCATCAGGTACTTGGACTGGTCGAACTTGT；
X5523-31：TACCTGATGATGTACAACGACAACAAAATGGTTGACAGCA；
X5523-32：ACACTTCGATTTTCACGTCTTTGCTGTCAACCATTTTGTT；
X5523-33：GACGTGAAAATCGAAGTGTACCTGACCACCAAAAAGAAAT；
X5523-34：GTGTCCTCGAGTCATTATTTCTTTTTGGTGGTCAGG；
the first round of PCR procedure was: 3min at 95 ℃;95℃22sec,55℃20sec,72℃30sec,20cyc;72 ℃ for 5min;
(2) And taking the PCR product of the first round of PCR as a template, and performing a second round of PCR to obtain a gene sequence for encoding the recombinant staphylococcus aureus type B enterotoxin protein (or directly synthesizing the gene sequence by a synthesis company to obtain an optimized sequence), wherein a reaction system (50 mu L) is as follows:
second round PCR procedure: 3min at 95 ℃;95℃22sec,58℃20sec,72℃30sec,24cyc;72 ℃ for 5min;
(3) Vector pET-32a was subjected to double cleavage with the following cleavage system (50. Mu.L): pET-32a 1 μg;10 XFD Buffer 5. Mu.L; kpnI 1. Mu.L (10U/. Mu.L); xhoI 1. Mu.L (10U/. Mu.L); ddH 2 O makes up 50. Mu.L; reacting for 2h in a water bath kettle with constant temperature of 37 ℃;
(4) Recovering and purifying the PCR product (shown in figure 1) obtained by amplifying in the step (2), and then connecting the PCR product with the carrier (shown in figure 2) obtained by enzyme digestion in the step (3); wherein the connection system (20. Mu.L) is: seamless cloning of enzyme mix 8.5. Mu.L; the target fragment: 5. Mu.L; 2 mu L of enzyme cutting carrier; ddH2O was supplemented to 20. Mu.L. The reaction was carried out at 50℃for 1 hour using a PCR instrument.
(5) Converting the ligation product obtained in the step (4) into top10 competence, screening out positive clones by enzyme digestion detection (figure 3), extracting plasmid pET32a-SEB and sequencing, wherein the sequencing result is shown as follows (transverse line + italic are KpnI and XhoI enzyme digestion sites):
GAGAACCTGTACTTCCAGGGCGAAAGCCAGCCGGACCCGAAACCGGATGAACTGCACAAAAGCAGCAAATTCACCGGCCTGATGGAAAACATGAAAGTGCTGTACGACGACAACCACGTCTCCGCCATCAACGTGAAAAGCATCGATCAGTTCCTGTACTTCGACCTGATCTACTCCATCAAAGACACCAAACTGGGTAACTACGACAACGTCCGCGTCGAGTTCAAAAACAAAGACCTGGCGGACAAGTACAAAGACAAATACGTGGACGTTTTCGGCGCGAACTACTACTACCAGTGCTACTTCTCCAAAAAGACCAACGACATCAACTCCCACCAGACCGATAAACGCAAGACCTGCATGTACGGCGGCGTGACCGAACACAACGGCAACCAGCTGGACAAATACCGCAGCATCACCGTGCGCGTGTTCGAGGACGGCAAAAACCTGCTGAGCTTCGATGTGCAGACCAACAAGAAAAAAGTGACCGCGCAGGAACTGGACTACCTGACCCGTCACTACCTGGTGAAAAACAAAAAACTGTACGAGTTCAACAACTCCCCGTACGAAACCGGCTACATCAAGTTCATCGAAAACGAAAACAGCTTCTGGTACGACATGATGCCGGCGCCGGGCGACAAGTTCGACCAGTCCAAGTACCTGATGATGTACAACGACAACAAAATGGTTGACAGCAAAGACGTGAAAATCGAAGTGTACCTGACCACCAAAAAGAAATAATGA
the amino acid sequence of the recombinant S.aureus type B enterotoxin protein with his protein tag is as follows:
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTENLYFQGESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFLYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANYYYQCYFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK
recombinant staphylococcus aureus type-B enterotoxin protein with his protein removed label has the amino acid sequence as shown below:
ENLYFQGESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFLYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANYYYQCYFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK
EXAMPLE 2 construction of strains expressing recombinant Staphylococcus aureus type B enterotoxin protein
Taking 1 mu L of recombinant plasmid pET32a-SEB prepared in the step (5) of the example 1 to transform BL21 (DE 3), performing heat shock at 42 ℃ for 90s, and standing on ice for 2min; then coating the plasmid on an LB solid plate containing ampicillin with a final concentration of 50 mug/mL, culturing the plasmid at 37 ℃ overnight, picking up a monoclonal extracted plasmid, and carrying out sequencing verification to obtain the strain for expressing the recombinant staphylococcus aureus type B enterotoxin protein.
EXAMPLE 3 preparation and purification of recombinant Staphylococcus aureus enterotoxin B protein
1. The best induction conditions were selected for the pilot culture
(1) Single colonies of the recombinant S.aureus type B enterotoxin protein-expressing strain in example 2 were picked, 3mL of LB liquid medium containing 50. Mu.g/mL ampicillin was added to the tube, and cultured overnight at 37℃and 220 rpm;
(2) Inoculating the bacterial liquid cultured overnight into 4mL of LB liquid medium containing 50 mug/mL ampicillin at a volume ratio of 1:100, and culturing at 37 ℃ and 220 rpm;
(3) When the OD reached 0.6, IPTG was added at a final concentration of 0.5mM, at 220rpm, and the following treatments were performed: induced overnight at 20 ℃ or induced for 4 hours at 37 ℃ without IPTG inducer as negative control.
(4) Centrifuging at 4000rpm for 10min, collecting thallus, discarding supernatant, washing thallus with 500 μl PBS (pH 7.4) buffer solution, suspending, and ultrasonic crushing thallus in ice bath for 6min with power of 400W for more than 0.5s and stopping for 1.5s; supernatant and pellet were collected by centrifugation, and pellet was dissolved in 500. Mu.L inclusion body lysate (8M Urea,50mM Tris-HCl,300mM NaCl,pH8.0), and 40. Mu.L sample and 10. Mu.L 5X protein loading buffer were mixed, respectively, and subjected to boiling water bath for 10min, SDS-PAGE detection.
The detection results are shown in FIG. 4, and the induction condition at 37℃for 4 hours is optimal.
2. Bulk culture and protein purification
(1) culturing the bacterial liquid as shown in the small test culture steps (2) and (3), and then inoculating the cultured bacterial liquid into 4L of LB liquid medium containing 50 mug/mL ampicillin with a final concentration according to a volume ratio of 1:100, and culturing at 37 ℃ and 220 rpm; when the OD value reaches 0.6, adding IPTG with the final concentration of 0.5mM, inducing at 37 ℃ at 220rpm for 4 hours, and centrifugally collecting cell thalli;
(2) Ultrasonic breaking thalli (control endotoxin)
(1) The collected bacterial cells were suspended by washing with a disruption Buffer (1 XPBS Buffer containing 0.1% w/v Triton X-114, pH 7.4), sonicating the cells in an ice bath, power 400W,20min (ultrasound 2S, pause 6S is one cycle);
(2) centrifuging at 15000rpm and 4 ℃ for 20min after the completion of the ultrasonic treatment, and collecting the supernatant for further purification;
(3) First nickel sepharose affinity chromatography (control endotoxin)
(1) 10mL Ni-IDA column packed, 3 times bed volume Binding buffer1 (containing 0.1% w/v Triton X-114 in 1X PBS buffer, pH 7.4) to wash the equilibrated column, flow rate 5mL/min;
(2) bacteria are crushed, the supernatant is put on a column, the flow rate is 2mL/min, and penetrating fluid A is collected;
(3) 5 times of the column bed volume of Binding buffer1 to clean the column, and the flow rate is 3mL/min;
(4) wash Buffer1 (1 XPBS Buffer solution containing 20mM Imidazole, pH 7.4) is used for washing impurities, the flow rate is 3mL/min, and the penetrating fluid B is collected to obtain purified fusion protein (the protein size is 45.7 KD);
(5) wash Buffer2 (1 XPBS Buffer containing 50mM Imidazole, pH 7.4) was used for washing impurities at a flow rate of 3mL/min, and the penetration solution C was collected;
(6) the Elution Buffer (1 XPBS Buffer, pH7.4, containing 500mM Imidazole) was eluted at a flow rate of 2mL/min, and eluent D was collected; FIG. 5 shows the results of SDS-PAGE electrophoresis of bacterial disrupted supernatants, permeate A, permeate B, permeate C and eluent D.
(4) Enzymatic cleavage of protein tags
Dialyzing the permeate B collected in (3) in 1 XPBS buffer (pH 7.4) for 2 hours, then adding TEV enzyme into the dialysis bag, and performing enzyme digestion at 25 ℃ overnight to remove the tag;
(5) Second Nickel agarose affinity chromatography
(1) Taking 5mL of Ni-IDA to fill a column, washing the balance column with 3 times of Binding buffer2 (1 XPBS buffer, pH 7.4) with the flow rate of 5mL/min;
(2) loading the cut penetrating fluid B obtained in the step (4) to a column, wherein the flow rate is 2mL/min, and collecting penetrating fluid E;
(3) 2 times of the column bed volume of Binding buffer2 to clean the column, and the flow rate is 3mL/min;
(4) wash Buffer1 (1 XPBS Buffer containing 20mM Imidazole, pH 7.4) was washed with impurities at a flow rate of 3ml/min, and a penetration solution F was collected;
(5) the Elution Buffer (1 XPBS Buffer, pH7.4, containing 500mM Imidazole) was eluted at a flow rate of 2mL/min, and the eluate G was collected; FIG. 6 shows the results of SDS-PAGE electrophoresis of the penetrating fluid B before the enzyme digestion, the penetrating fluid B after the enzyme digestion, the penetrating fluid E, the penetrating fluid F and the eluting fluid G.
(6) Filtering the penetrating fluid E collected in the step (5) through a 0.22 mu m CA filter membrane to obtain staphylococcus aureus type B enterotoxin protein (the label-removed target protein has the protein size of 28.4 KD), wherein FIG. 7 is a diagram of the SDS-PAGE electrophoresis detection result of the protein.
Western Blot analysis is carried out on the purified fusion protein (penetrating fluid B) obtained in the step (3) and the unlabeled target protein obtained in the step (6), wherein the primary antibody is a rabbit anti-his tag (Sangon Biotech, code: D110002), and the secondary antibody is a sheep anti-rabbit (Sangon Biotech, code: D110058); the results are shown in FIG. 8, where FIG. 8, lane 2, has no band to demonstrate that the protein tag is removed cleanly.
The concentration of the target protein after label removal (volume of the sample to be measured was 10. Mu.L) was measured using SK3071 non-interfering protein quantification kit, and the target protein concentration was 2.01mg/mL as understood from the protein absorption values measured in FIG. 9 and Table 1.
TABLE 1 protein amounts and absorbance at 480nm wavelength corresponding to the measurements
The plasmid construction of the invention takes 10 working days; expression detection takes 5 working days; expression purification and detection took 10 working days. Short time consumption, simple operation and low cost, and the prepared staphylococcus aureus type-B enterotoxin protein has high purity and good activity. Further packaging 500 μl/tube, storing at-80deg.C, or lyophilizing; the lyophilized staphylococcal enterotoxin type B protein was used in examples 4-6 to prepare polishing pastes.
Example 4
A polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine comprises the following components in percentage by mass:
the preparation method of the polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine comprises the following steps:
(1) Mixing components PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, saccharin sodium, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate and water of a polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine to obtain paste 1;
(2) Adding staphylococcus aureus type B enterotoxin protein (prepared in example 3) and procyanidine into the paste 1 prepared in the step (1), and uniformly mixing to obtain a paste 2;
(3) Adding the rest components (sodium dodecyl sulfate and perfume) into the paste 2 prepared in the step (2), and uniformly mixing to obtain a paste 3;
(4) And vacuumizing the paste 3 to obtain the polishing paste containing the staphylococcus aureus enterotoxin B protein and the procyanidine.
Example 5
A polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine comprises the following components in percentage by mass:
the preparation method of the polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine comprises the following steps:
(1) Mixing components PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, saccharin sodium, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate and water of a polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine to obtain paste 1;
(2) Adding staphylococcus aureus type B enterotoxin protein (prepared in example 3) and procyanidine into the paste 1 prepared in the step (1), and uniformly mixing to obtain a paste 2;
(3) Adding the rest components (sodium dodecyl sulfate and perfume) into the paste 2 prepared in the step (2), and uniformly mixing to obtain a paste 3;
(4) And vacuumizing the paste 3 to obtain the polishing paste containing the staphylococcus aureus enterotoxin B protein and the procyanidine.
Example 6
A polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine comprises the following components in percentage by mass:
the preparation method of the polishing paste containing staphylococcus aureus type-B enterotoxin protein and procyanidine comprises the following steps:
(1) Mixing components PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, saccharin sodium, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate and water of a polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine to obtain paste 1;
(2) Adding staphylococcus aureus type B enterotoxin protein (prepared in example 3) and procyanidine into the paste 1 prepared in the step (1), and uniformly mixing to obtain a paste 2;
(3) Adding the rest components (sodium dodecyl sulfate and perfume) into the paste 2 prepared in the step (2), and uniformly mixing to obtain a paste 3;
(4) And vacuumizing the paste 3 to obtain the polishing paste containing the staphylococcus aureus enterotoxin B protein and the procyanidine.
Comparative example (without Staphylococcus aureus type B enterotoxin protein and procyanidins)
A polishing paste which does not contain staphylococcus aureus type-B enterotoxin protein and procyanidine comprises the following components in percentage by mass:
the preparation method of the polishing paste which does not contain staphylococcus aureus type B enterotoxin protein and procyanidine comprises the following steps:
(1) Mixing components of a polishing paste which does not contain staphylococcus aureus enterotoxin protein B and procyanidine, namely PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, saccharin sodium, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate and water to obtain paste 1;
(2) Adding the rest components (sodium dodecyl sulfate and perfume) into the paste 1 prepared in the step (1), and uniformly mixing to obtain a paste 2;
(4) And vacuumizing the paste 2 to obtain the polishing paste which does not contain the staphylococcus aureus enterotoxin B protein and the procyanidine.
Effect examples
(1) Polishing paste stability test
The polishing pastes prepared in examples 4 to 6 were left at 50℃for one week, then transferred to-3℃for one week, and the operation was repeated 7 times, whereby the polishing pastes were stabilized (FIG. 10).
(2) Evaluation of treatment Effect of polishing paste
Patients with periodontitis were randomly divided into treatment groups (examples 4 to 6) and control groups (comparative examples). The two groups of patients have no significant difference (P < 0.05) in terms of age, sex, disease severity and the like through statistical analysis, and have comparability.
The treatment method comprises the following steps: both groups of the preparation are used for oral cavity conventional full-mouth cleaning before administration, caries cavities are filled, gingival flap forming operation and periodontal splint ligation fixation can be performed if necessary, and the treatment group uses the polishing paste prepared in the examples 4-6 to polish the tooth surface (once every week); the control group polished the tooth face (once a week) using the polishing paste prepared in the comparative example. Both groups take half a month as 1 course of treatment, 3 courses of treatment are continuously observed, and the clinical curative effects of the two groups are subjected to comparative analysis.
Curative effect judgment criteria: the effect is shown: the symptoms are obviously improved, the gum appearance is normal, the periodontal congestion, the edema and the bleeding are resolved or are relieved before, the periodontal pocket is disappeared or partially disappeared, and the teeth are slightly loosened; the method is effective: symptom relief, basically normal appearance, relief of periodontal congestion, edema and bleeding, and loosening of teeth; invalidation: no change occurs before and after treatment. Total effective rate= (significant effect + effective)/n×100%.
The results are shown in Table 2, and the polishing paste containing Staphylococcus aureus type B enterotoxin protein and procyanidins has a remarkable therapeutic effect on periodontitis as compared with the comparative example. FIGS. 11 and 12 are graphs showing the effect of three days and one week after the use of the polishing paste containing Staphylococcus aureus type B enterotoxin protein and procyanidins prepared in example 1, and it can be seen that the symptoms were significantly improved, the gum appearance was normal, and periodontal congestion, edema, bleeding was resolved or earlier relieved.
Table 2 evaluation of periodontitis treatment effect of polishing pastes prepared in examples 4 to 6
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> Zeng Qingming
<120> polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine, and preparation and application thereof
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 246
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Glu Asn Leu Tyr Phe Gln Gly Glu Ser Gln Pro Asp Pro Lys Pro Asp
1 5 10 15
Glu Leu His Lys Ser Ser Lys Phe Thr Gly Leu Met Glu Asn Met Lys
20 25 30
Val Leu Tyr Asp Asp Asn His Val Ser Ala Ile Asn Val Lys Ser Ile
35 40 45
Asp Gln Phe Leu Tyr Phe Asp Leu Ile Tyr Ser Ile Lys Asp Thr Lys
50 55 60
Leu Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys Asn Lys Asp Leu
65 70 75 80
Ala Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly Ala Asn Tyr
85 90 95
Tyr Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn Asp Ile Asn Ser His
100 105 110
Gln Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly Val Thr Glu His
115 120 125
Asn Gly Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr Val Arg Val Phe
130 135 140
Glu Asp Gly Lys Asn Leu Leu Ser Phe Asp Val Gln Thr Asn Lys Lys
145 150 155 160
Lys Val Thr Ala Gln Glu Leu Asp Tyr Leu Thr Arg His Tyr Leu Val
165 170 175
Lys Asn Lys Lys Leu Tyr Glu Phe Asn Asn Ser Pro Tyr Glu Thr Gly
180 185 190
Tyr Ile Lys Phe Ile Glu Asn Glu Asn Ser Phe Trp Tyr Asp Met Met
195 200 205
Pro Ala Pro Gly Asp Lys Phe Asp Gln Ser Lys Tyr Leu Met Met Tyr
210 215 220
Asn Asp Asn Lys Met Val Asp Ser Lys Asp Val Lys Ile Glu Val Tyr
225 230 235 240
Leu Thr Thr Lys Lys Lys
245
<210> 2
<211> 744
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gagaacctgt acttccaggg cgaaagccag ccggacccga aaccggatga actgcacaaa 60
agcagcaaat tcaccggcct gatggaaaac atgaaagtgc tgtacgacga caaccacgtc 120
tccgccatca acgtgaaaag catcgatcag ttcctgtact tcgacctgat ctactccatc 180
aaagacacca aactgggtaa ctacgacaac gtccgcgtcg agttcaaaaa caaagacctg 240
gcggacaagt acaaagacaa atacgtggac gttttcggcg cgaactacta ctaccagtgc 300
tacttctcca aaaagaccaa cgacatcaac tcccaccaga ccgataaacg caagacctgc 360
atgtacggcg gcgtgaccga acacaacggc aaccagctgg acaaataccg cagcatcacc 420
gtgcgcgtgt tcgaggacgg caaaaacctg ctgagcttcg atgtgcagac caacaagaaa 480
aaagtgaccg cgcaggaact ggactacctg acccgtcact acctggtgaa aaacaaaaaa 540
ctgtacgagt tcaacaactc cccgtacgaa accggctaca tcaagttcat cgaaaacgaa 600
aacagcttct ggtacgacat gatgccggcg ccgggcgaca agttcgacca gtccaagtac 660
ctgatgatgt acaacgacaa caaaatggtt gacagcaaag acgtgaaaat cgaagtgtac 720
ctgaccacca aaaagaaata atga 744
<210> 3
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gacacggtac cgagaacctg tacttccag 29
<210> 4
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cgggtccggc tggctttcgc cctggaagta caggttctcg 40
<210> 5
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gccagccgga cccgaaaccg gatgaactgc acaaaagcag 40
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tccatcaggc cggtgaattt gctgcttttg tgcagttcat 40
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
caccggcctg atggaaaaca tgaaagtgct gtacgacgac 40
<210> 8
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
acgttgatgg cggagacgtg gttgtcgtcg tacagcactt 40
<210> 9
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
tctccgccat caacgtgaaa agcatcgatc agttcctgta 40
<210> 10
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atggagtaga tcaggtcgaa gtacaggaac tgatcgatgc 40
<210> 11
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tcgacctgat ctactccatc aaagacacca aactgggtaa 40
<210> 12
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
tcgacgcgga cgttgtcgta gttacccagt ttggtgtctt 40
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
caacgtccgc gtcgagttca aaaacaaaga cctggcggac 40
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gtccacgtat ttgtctttgt acttgtccgc caggtctttg 40
<210> 15
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
tacaaagaca aatacgtgga cgttttcggc gcgaactact 40
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ttttggagaa gtagcactgg tagtagtagt tcgcgccgaa 40
<210> 17
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ccagtgctac ttctccaaaa agaccaacga catcaactcc 40
<210> 18
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tcttgcgttt atcggtctgg tgggagttga tgtcgttggt 40
<210> 19
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
agaccgataa acgcaagacc tgcatgtacg gcggcgtgac 40
<210> 20
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
ttgtccagct ggttgccgtt gtgttcggtc acgccgccgt 40
<210> 21
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gcaaccagct ggacaaatac cgcagcatca ccgtgcgcgt 40
<210> 22
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
tcagcaggtt tttgccgtcc tcgaacacgc gcacggtgat 40
<210> 23
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
cggcaaaaac ctgctgagct tcgatgtgca gaccaacaag 40
<210> 24
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
gttcctgcgc ggtcactttt ttcttgttgg tctgcacatc 40
<210> 25
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
tgaccgcgca ggaactggac tacctgaccc gtcactacct 40
<210> 26
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
cgtacagttt tttgtttttc accaggtagt gacgggtcag 40
<210> 27
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gtgaaaaaca aaaaactgta cgagttcaac aactccccgt 40
<210> 28
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gaacttgatg tagccggttt cgtacgggga gttgttgaac 40
<210> 29
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
accggctaca tcaagttcat cgaaaacgaa aacagcttct 40
<210> 30
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cgccggcatc atgtcgtacc agaagctgtt ttcgttttcg 40
<210> 31
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gacatgatgc cggcgccggg cgacaagttc gaccagtcca 40
<210> 32
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
tcgttgtaca tcatcaggta cttggactgg tcgaacttgt 40
<210> 33
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tacctgatga tgtacaacga caacaaaatg gttgacagca 40
<210> 34
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
acacttcgat tttcacgtct ttgctgtcaa ccattttgtt 40
<210> 35
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gacgtgaaaa tcgaagtgta cctgaccacc aaaaagaaat 40
<210> 36
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gtgtcctcga gtcattattt ctttttggtg gtcagg 36
Claims (7)
1. The polishing paste containing the staphylococcus aureus B-type enterotoxin protein and the procyanidine is characterized by comprising the following components in percentage by mass:
the amino acid sequence of the staphylococcus aureus type-B enterotoxin protein is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for encoding the staphylococcus aureus type-B enterotoxin protein is shown as SEQ ID NO. 2.
2. The polishing paste containing staphylococcus aureus type B enterotoxin protein and procyanidine according to claim 1, which is characterized by comprising the following components in percentage by mass:
3. the polishing paste containing staphylococcus aureus type B enterotoxin protein and procyanidins according to claim 1, wherein:
the preparation method of the staphylococcus aureus type-B enterotoxin protein comprises the following steps:
(1) The gene of the recombinant staphylococcus aureus type B enterotoxin protein shown as SEQ ID NO.1 is obtained through total gene synthesis;
(2) Carrying out PCR amplification on the gene obtained in the step (1) by using a primer X5523-1 and a primer X5523-34 to obtain a target fragment, and recovering and purifying;
(3) Connecting the target product recovered and purified in the step (2) with a carrier to obtain a recombinant plasmid;
(4) Transferring the recombinant plasmid prepared in the step (3) into a host cell to obtain a strain for expressing recombinant staphylococcus aureus type-B enterotoxin protein;
(5) Taking the strain expressing the recombinant staphylococcus aureus type-B enterotoxin protein prepared in the step (4) as an expression strain, and carrying out induced expression based on an escherichia coli expression system; and centrifugally collecting thalli, washing and suspending, then ultrasonically crushing, centrifuging, collecting supernatant, and purifying to obtain staphylococcus aureus type-B enterotoxin protein.
4. A polishing paste containing staphylococcal enterotoxin type B protein and procyanidins according to claim 3 wherein:
the nucleotide sequences of the primer X5523-1 and the primer X5523-34 described in step (2) are as follows:
primer X5523-1: GACACGGTACCGAGAACCTGTACTTCCAG;
primer X5523-34: GTGTCCTCGAGTCATTATTTCTTTTTGGTGGTCAGG.
5. A polishing paste containing staphylococcal enterotoxin type B protein and procyanidins according to claim 3 wherein:
the vector in the step (3) is pET-32a;
the host cell in the step (4) is escherichia coli BL21 (DE 3).
6. A polishing paste containing staphylococcal enterotoxin type B protein and procyanidins according to claim 3 wherein:
the purification described in step (5) comprises the steps of:
first Nickel Sepharose affinity chromatography
(1) Filling the Ni-IDA column, and cleaning the balance column with a Binding buffer1 with a volume of 3 times of the bed volume, wherein the flow rate is 5mL/min;
(2) ultrasonically crushing, centrifuging, and collecting the supernatant, and loading the supernatant onto a column, wherein the flow rate is 2mL/min;
(3) 5 times of the column bed volume of Binding buffer1 to clean the column, and the flow rate is 3mL/min;
(4) wash Buffer washing impurities at a flow rate of 3mL/min, and collecting penetrating fluid;
(II) enzymatic cleavage of protein tag
Dialyzing the penetrating fluid collected in the step (I), then adding TEV enzyme, and cutting off the tag by enzyme to obtain the penetrating fluid after enzyme cutting;
(III) second Nickel agarose affinity chromatography
(1) Filling the Ni-IDA column, and cleaning the balance column with Binding buffer2 with the volume of 3 times of the bed volume, wherein the flow rate is 5mL/min;
(2) loading the cut penetrating fluid obtained in the step (II) to a column, wherein the flow rate is 2mL/min, and collecting the penetrating fluid;
(IV) filtration
And (3) filtering the penetrating fluid collected in the step (III) through a 0.22 mu m CA filter membrane to obtain the staphylococcus aureus type B enterotoxin protein.
7. The method for preparing a polishing paste containing a staphylococcal enterotoxin type B protein and procyanidins according to any one of claims 1 to 6, comprising the steps of:
(1) Mixing components PEG-400, sorbitol, glycerol, cloud stone powder, silicon dioxide, saccharin sodium, sodium phytate, calcium monofluorophosphate, xanthan gum, hydroxypropyl guar gum, disodium hydrogen phosphate, sodium pyrophosphate and water of a polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine to obtain paste 1;
(2) Adding procyanidine and staphylococcus aureus type-B enterotoxin protein into the paste 1 prepared in the step (1), and uniformly mixing to obtain a paste 2;
(3) Adding the rest components into the paste 2 prepared in the step (2), and uniformly mixing to obtain a paste 3;
(4) And vacuumizing the paste 3 to obtain the polishing paste containing the staphylococcus aureus enterotoxin B protein and the procyanidine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710770237.1A CN107550748B (en) | 2017-08-31 | 2017-08-31 | Polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine and preparation and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710770237.1A CN107550748B (en) | 2017-08-31 | 2017-08-31 | Polishing paste containing staphylococcus aureus B-type enterotoxin protein and procyanidine and preparation and application thereof |
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| Publication Number | Publication Date |
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| CN107550748B true CN107550748B (en) | 2024-01-30 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101692998A (en) * | 2009-10-22 | 2010-04-14 | 浙江大学 | Toothpaste containing grape pip procyanidin extract and application |
| WO2012085224A1 (en) * | 2010-12-22 | 2012-06-28 | Laboratoires Expanscience | Avocado flesh and/or skin extract rich in polyphenols and cosmetic, dermatological and nutraceutical compositions comprising same |
-
2017
- 2017-08-31 CN CN201710770237.1A patent/CN107550748B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101692998A (en) * | 2009-10-22 | 2010-04-14 | 浙江大学 | Toothpaste containing grape pip procyanidin extract and application |
| WO2012085224A1 (en) * | 2010-12-22 | 2012-06-28 | Laboratoires Expanscience | Avocado flesh and/or skin extract rich in polyphenols and cosmetic, dermatological and nutraceutical compositions comprising same |
Non-Patent Citations (3)
| Title |
|---|
| 《Chain A，Enterotoxin Type B》;Varshney,A.K.等;《GenBank Database》;20140401;DEFINITION、SOURCE、ORIGIN部分 * |
| 《金黄色葡萄球菌肠毒素B的原核表达及纯化》;张亮等;《中国生物制品学杂志》;20130630;第26卷(第6期);第805页材料与方法 * |
| 肖振忠.《SEB》.《突发灾害应急医学救援》.2007,第458-459页. * |
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| CN107550748A (en) | 2018-01-09 |
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