CN101322842A - Recombinant Staphylococcus aureus enterotoxin G oral preparation and use - Google Patents

Recombinant Staphylococcus aureus enterotoxin G oral preparation and use Download PDF

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CN101322842A
CN101322842A CNA2008100624758A CN200810062475A CN101322842A CN 101322842 A CN101322842 A CN 101322842A CN A2008100624758 A CNA2008100624758 A CN A2008100624758A CN 200810062475 A CN200810062475 A CN 200810062475A CN 101322842 A CN101322842 A CN 101322842A
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enterotoxin
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陈枢青
丁丁
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Zhejiang University ZJU
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Abstract

The invention provides a recombinant staphylococcus aureus enterotoxin G oral preparation which has an amino acid sequence of SEQ ID NO.1 and also comprises excipients in medicine or carriers allowed by the preparation. By the experiment on the Caco-2 monolayer cell trans-membrane transport, the oral preparation proves that the proteins can enter the general blood circulation from intestinal epithelial cells in the form of complete molecules and keep promoting the splenic lymphocyte proliferation and the activity of the superantigen preventing the tumor cells from growing, and can be applied to the preparation of drugs for curing the malignant tumor and other serious complications.

Description

Recombinant Staphylococcus aureus enterotoxin G oral preparation and application
Technical field
The invention belongs to biological engineering, relate to recombinant Staphylococcus aureus enterotoxin G (StaphylococcalEnterotoxin G, preparation SEG) and with the preparation of the oral formulations of the main effective ingredient of SEG and the application in oncotherapy.Specifically, utilize gene engineering method to prepare high-purity recombinant Staphylococcus aureus enterotoxin G albumen and associated protein goods thereof exactly, and it is prepared into oral formulations, be used for the treatment and the rehabilitation of tumor patient.
Background technology
Staphylococcus aureus enterotoxin (Staphylococcal Enterotoxin) is produced by staphylococcus aureus.Kind surplus the enterotoxin that has at present obtained to identify has 20, comprising the enterotoxin of classics such as SEA, SEB, SEC, SED, SEE etc., and the enterotoxin SEG～SEQ that finds recently.Variety classes enterotoxin molecular weight is that 25～33kD does not wait, all kinds of enterotoxin aminoacid sequences have certain homology (28%～98%), by protein crystal diffraction and Computerized three-dimensional structural modeing technique as can be known, the three dimensional structure of all kinds of enterotoxins is quite similar, all have two domains, most enterotoxins comprise one to keeping the crucial disulfide bond of three dimensional structure.
The gold staphylococcal enterotoxin is the bacillary superantigen of a quasi-representative, can cause a large amount of propagation and the activation of T cell in the host at short notice.Discover, the enterotoxin molecule can be incorporated into antigen presenting cell (Antigen presenting cells with complete molecule, APC) antigenic peptides of Biao Mian MHC-II quasi-molecule is in conjunction with the groove outside, and then by (T cell receptor, TCR) V β district specificity is in conjunction with activated T cell with TXi Baoshouti.Different with common antigen is, enterotoxin does not need simultaneously by each several part common identification such as the V α of TCR molecule, J α, J β, V β and D β, it is restricted also not have MHC-II, making only needs extremely low concentration by golden staphylococcal enterotoxin (0.01～10ng/mL) can activate 2～30% initial T lymphocyte, is 10 of common antigen activated T cells quantity 3～10 6Doubly.Simultaneously, enterotoxin also can promote to be subjected to its activated T cells secretion various kinds of cell factor, indirect activation B cell, NK cell, and therefore golden staphylococcal enterotoxin can stimulate efficiently with enhance immunity and replys.
Between this character, the enterotoxin superantigen is applied to the existing a large amount of reported in literature of research of oncotherapy both at home and abroad.People such as Frank are SEB (10 μ g/mL) intravesical perfusion lotus RT112 and RT4 cell mouse, six week back about 76% mice intravesical tumors disappear, the intravesical tumor cell of all the other mices then produces the obvious accent phenomenon of dying.Experiment shows that the SEA activated T cell makes it proliferation function and be dose-dependence in every mice 0.1-100 μ g scope in the body that people such as Hedlund carry out, and injects to occur ceiling effect in back 1 day, and multiplication effect was kept 4 days.The monoclonal antibody Fab fragment of human colon cancer related antigen C242 such as Litton and SEA prepare fusion rotein, prove that this fusion rotein can promote efficiently that in vivo T cell and mononuclear cell are assembled at the tumor tissues place, infiltration, and inducing T cell produces a large amount of cytokines, induce colon cancer cell to produce cytokines such as IL-4, IL-10, TNF-α simultaneously, and cause that cancerous cell IFN-γ receptor, Fas expression of receptor raise, thereby effectively the inducing tumor cell accent is died.Kodama etc. have prepared the mutant mSEAD227A of SEA, and this mutain is compared the binding ability that has reduced with the MHC-II molecule with natural SEA, but do not change the ability that stimulates proliferation to the T cell.Experimental results show that rabbit is 500 times of natural SEA to the tolerance of this mutant.Utilize this mutain and the antigenic monoclonal antibody MUSE11 of anti-MUC1 to prepare fusion rotein, prove this fusion rotein in vivo with the external specific cell toxic action that all can strengthen the T-LAK cell efficiently to the cholangiocarcinoma cell of expressing MUC1.
Abroad, be that the medicine of main effective ingredient has entered clinical research with golden staphylococcal enterotoxin and fusion rotein thereof.The I phase clinical experiment of the PNU-214565 that finishes as people such as Giantonio (fusion rotein of the Fab produced in fragments of reorganization SEA and C242 monoclonal antibody) shows, issuable cytotoxicity of patient and dosage are closely related, and dosage depends on anti-SEA antibody concentration in patient's body weight and the blood circulation.In the administration process, fervescence in various degree appears in 76% patient, and the blood pressure that 62% patient occurs in various degree reduces, and its principal element is to induce the IL-2 and the TNF of generation, and toxic and side effects is temporary, and is controlled easily.They find that the interior anti-SEA antibody of patient body has reduced toxicity to a certain extent before the treatment beginning, by taking all factors into consideration whose body weight and anti-SEA antibody concentration, can determine the dosage of a side effect minimum.People such as Jonathan have finished the I phase clinical experiment of the second filial generation product P NU-214936 of PNU-214565, purpose is at nonsmall-cell lung cancer (non-small-cell lung cancer, NSCLC) patient realizes individual administration on one's body, and use the Bayesian model to carry out dosage in conjunction with excessive dosage control method and amplify, determined the maximum drug resistance dosage (MTD) of this medicine.
In China, (SEC2) exceeded 10 years for the golden Portugal bacterium filtrate preparation of main effective ingredient is applied to clinical cancer therapy with Enteromycin C 2.Data shows, such preparation not only can significantly strengthen the body immunity of middle and advanced stage tumor patient, simultaneously can alleviate the toxic and side effects that causes because of chemicotherapy, the prolongation of malignant tumor patient life cycle and the improvement of life quality are had important clinic value and meaning.Studied after 56 routine epithelial ovarian cancer patient's chemotherapy and body cell immune state after the plain auxiliary treatment of high poly-golden Fructus Vitis viniferae as Zhu Genhai etc., after the plain combined chemotherapy in the high consor gold of therapeutic outcome display application Portugal, CD4 +/ CD8 +Ratio, the simple chemotherapy of NK cytoactive significantly improve, malignant tumor specificity growth factors such as CA125, TSGF are reduced to normal routine number more than simple chemotherapy group in 2 course of therapy bleeding from anus of the high simultaneously plain combined chemotherapy group in poly-golden Portugal, and the subnormal routine number of leukocyte or platelet count is less than simple chemotherapy group.Luo Feng etc. use the plain topical therapeutic in high consor gold Portugal to 35 routine shallow table metastatic tumo(u)rs, treats that total effective rate reaches 82.9% after one month, and confirm that the tumor cell average cell is transferred the rate increase (P＜0.01) of dying behind high consor gold Portugal extract for treating.Tang Wei etc. to early stage oral cancer adopt local golden Portugal extract for treating and to late period oral cancer use the plain combined chemotherapy in golden Portugal, observe the curative effect and pathological change, finding to use golden Portugal element can make big amount lymphocyte and fibrous tissue accumulate near the cancer nests, and can observe tumor cell and significantly reduce, even the some cases tumor cell disappears.Wu Jie etc. have observed high poly-golden Portugal element during esophageal carcinoma radical radiation therapy, short term effect, gastrointestinal reaction, leukocyte are changed and to the assosting effect of overall health of patients, result's demonstration is compared with matched group, observation group changes significant difference at gastrointestinal reaction, the quantity of leucocyte of radiation period, show obviously leukocyte increasing quantity of high poly-golden Portugal element, alleviate gastrointestinal reaction.Simultaneously,＞10 minutes patient of the Kps of observation group scoring increase accounts for 86.6%, and matched group only accounts for 36.6% (P＜0.05), illustrates that high poly-golden Portugal have the tangible effect that improves overall health of patients.But then, owing to contain a large amount of impurity albumen in the preparation, toxin protein comprising multiple golden Portugal Pseudomonas, as hemolysin, leukocyte killer factor, the abrasion factor etc., when the golden Portugal of application bacterium filtrate ejection preparation is used for the tumor patient treatment, find that toxic and side effects such as low grade fever, hypotension generally appear in tumor patient when using, also can cause simultaneously injection or symptoms such as perfusion position redness, pain, to the treatment and the life quality generation certain influence of tumor patient.
It is generally acknowledged, protein and polypeptides matter are after entering gastrointestinal tract, multiple enzymes such as a large amount of trypsin that exist, Chymotrypsin, polypeptidase have very large influence to the biologic activity of protein and polypeptide and the integrity of molecule in low pH environment of gastric and the gastrointestinal tract, thereby most of albumen and polypeptides matter can be decomposed into the aminoacid utilization that is absorbed by the body very soon.At present, it is comparatively extensive to carry out ground for the systemic research of gastrointestinal tract of oligopeptide molecules such as dipeptides, tripeptides, part dipeptides, three peptide transport proteins have also been searched out on the Epithelium of intestinal villus surface, above-mentioned result of study has also given new replenishing to metabolism approach such as the decomposition of albumen, polypeptide in traditional human body, absorptions, but, research for the gastrointestinal absorption of complete albumen and peptide molecule still is in the starting stage, and also having only only a few albumen to be inferred at present may be able to directly be absorbed by human body with complete form.
At present, the action principle and the research of mode in gastrointestinal tract is carried out ground not deeply for enterotoxin, and how golden staphylococcal enterotoxin is caused the alimentary toxicosis symptom and whether can and then produce the superantigen effect by oral absorption still failing to obtain common recognition.Can simulate symptom when producing alimentary toxicosis owing to give ectogenic IL-2, therefore the alimentary toxicosis symptom that has the scholar to think that enterotoxin causes may be relevant with its superantigen character, and conjecture intestinal epithelial cell surface existence can with the bonded receptor of enterotoxin molecule, they point out to exist in the intestinal MHC-II +The M cell, such cell can combine with the enterotoxin molecule.Enterotoxin produces the superantigen effect by such cell just.But also having research worker to think that the vomiting effect that causes of enterotoxin and superantigen effect there is no contacts directly, they utilize the method for amino acid mutation has found respectively material impact to these two kinds of activity of enterotoxin molecule amino acid residue, thereby conjecture gastrointestinal tract epithelial cell surface exists the receptor of other kinds or transport molecule and its to interact.Hamad etc. have confirmed MHC-II -The Caco-2 cell can realize two-way transhipment (Apical → Basolateral to SEB, Basolateral → Apical), and the integrity that in transport process, can keep the SEB molecule, Shupp etc. also utilize experiment in vitro to confirm that the epithelial T84 cell line of the little intestinal crypt of simulation can realize uniport to SEA, SEB, and prompting enterotoxin molecule might produce the superantigen effect after passing through the gastrointestinal tract epithelium to enter blood circulation.Ma Haiying etc. with golden Portugal bacterium filtrate preparation through gastric infusion, can make the cell immunocompetent and the humoral immune function of immunologic hypofunction model mice obtain good improving because of the injection caused by cyclophosphamide, wherein particularly evident with improving of cellular immune function.It is to be noted, enterotoxin may not only limit to play a role in certain single mode in intestinal, may there be more complicated mechanism, though find to activate the V β 8 that is present in mesentery lymph node and Pi Shi trifle place in a large number rapidly after the orally give low dosage enterotoxin as people such as Gerburg +The T cell, but the T lymphocyte in the systemic circulatory system be there is no significant stimulation.As seen change dosage that enterotoxin gives and time and enterotoxin in intestinal and the biological effect of whole body exist delicate relation.
Be that the theoretical research of oral anti-tumor agent of main component and treatment use that to carry out ground also comparatively slow with golden staphylococcal enterotoxin, and be object of study mainly with classical enterotoxin such as SEA, SEB, SEC etc.Disclose in a kind of golden Portugal fermented liquid as Chinese patent CN 1509762A and to have extracted metabolite and be used for the treatment of and prevent AIDS, wherein main component has comprised SEA, SEB, SEC1, SEC2 and SED, can improve anti-virus ability after patient is oral; Chinese patent CN 1509725A discloses to extract in a kind of golden Portugal fermented liquid and has obtained the oral formulations that SEA, SEB, SEC, SED and TSST-1 preparation are used for the human body immunity improving function, and patient can improve quantity of leucocyte after using, improve health status; Chinese patent CN 1283264C discloses and has extracted SEA, SEB, SEC1, SEC2, SEC3, SED and TSST-1 in a kind of golden Portugal bacterium metabolite and be used to prepare oral formulations, is applied to anti-curing oncoma, diabetes, hepatitis B, drug rehabilitation, antiviral, anti-AIDS, raising immunity.But it is certain not enough that the above-mentioned method of extracting enterotoxin from golden Portugal bacterium fermentating metabolism product exists, simple as preparation technology, lack the strict quality control standard, unstable between batch, introduce other albumen impurity etc. easily, thereby influence purity, activity and the quality of final products.
Summary of the invention
An object of the present invention is to provide recombinant Staphylococcus aureus enterotoxin G oral preparation, described recombinant Staphylococcus aureus enterotoxin G is the superantigen albumen that utilizes gene engineering method to prepare, aminoacid sequence with SEQ ID NO.1, the express recombinant Staphylococcus aureus enterotoxin G be recombiant plasmid pGEX-4T-1-SEG, this plasmid is formed through recombination to construct by the nucleotide sequence of pGEX-4T-1 and SEQ ID NO.2, described dosage form is to be the oral formulations of main effective ingredient with recombinant Staphylococcus aureus enterotoxin G and associated protein goods thereof, and this oral formulations also contains preparation allowable pharmaceutical excipients or carrier.
The present invention has confirmed that by the test of Caco-2 cell monolayer transmembrane transport this albumen can see through the superantigen activity that intestinal epithelial cell enters the systemic blood circulation and can keep its short spleen lymphocyte proliferation and suppress growth of tumour cell with the form of complete molecule, can be applied to the preparation of oral anti-tumor agent.
The oral formulations that the present invention describes can be called as " oral ", " oral administration ", " intestinal ", " enteral administration ", " parenteral is outer ", " the parenteral external administration " etc., to be indicated as the approach or the mode of individuality being carried out administration along digestive tract.This " oral " or " intestinal " route of administration include but not limited to for example swallow solid (pill, tablet, capsule) or liquid (syrup) chemical compound or compositions; The Sublingual; Nasal cavity intestinal tube or gastrostomy tube; Duodenal administration; Rectallies etc. simultaneously, can adopt one or more oral or enteral administration approach, wherein preferred oral administration.
" pharmaceutically acceptable " is meant not can be in biology, medical science, chemically or with any other mode and health chemistry and the inconsistent material of metabolism.
In the oral anti-tumor agent prepared in accordance with the present invention except that contain with recombinant Staphylococcus aureus enterotoxin G and associated protein goods thereof as the main effective ingredient, component that contains simultaneously and adjuvant comprise and prevent the degrade component of above-mentioned main effective ingredient of pepsin, prevent the degrade component of above-mentioned main effective ingredient of Chymotrypsin, prevent the component of the above-mentioned main effective ingredient of intestinal trypsin degradation, in absorption enhancer and other biological goods and/or the genetic engineering medicine in acceptable adjuvant and the component one or more, thus can use in the individual blood so that the above-mentioned main effective ingredient of effective dose arrives enteral and finally is absorbed into.
Recombinant Staphylococcus aureus enterotoxin G of the present invention obtains by following steps:
(1) design has the upstream and downstream primer that is used to clone Staphylococcus aureus enterotoxin G mature peptide sequence of BamH I and Xho I restriction enzyme site respectively:
SEQ ID NO.3:gta Caa ccc gat cct aaa tta gac (the line part is a BamH I restriction enzyme site),
SEQ ID NO.4:gga
Figure A20081006247500072
Tca gtg agt att aag aaa tac (the line part is an Xho I restriction enzyme site);
(2) be template with staphylococcus aureus (FRI S6) genome, adopt pcr amplification to obtain the genetic fragment of the golden staphylococcal enterotoxin G albumen mature peptide of coding, it is connected into multiple clone site on the pGEM-T plasmid vector, successfully make up the pGEM-T-SEG recombiant plasmid.The gene order (AY920260) of the coding same protein among the gene fragment order (seeing SEQ ID NO.2) that confirms the coding gold staphylococcal enterotoxin G albumen mature peptide that obtains by pcr amplification by order-checking and the GenBank data base is in full accord;
(3) be template with the pGEM-T-SEG plasmid, pcr amplification obtains the gene order that two ends have the coding gold staphylococcal enterotoxin G albumen mature peptide of restriction enzyme site, with the said gene fragment with restriction endonuclease BamH I with after Xho I carries out enzyme action with handle with identical restriction endonuclease after the pGEX-4T-1 plasmid be connected successful construction expression plasmid pGEX-4T-1-SEG;
(4) the pGEX-4T-1-SEG expression plasmid is converted into e. coli bl21 (DE3), the reorganization GST-SEG fusion rotein of abduction delivering band GST label.The centrifugal collection supernatant of results thalline and broken back adopts affinity chromatograph, ion chromatography, gel chromatography to obtain the reorganization gold staphylococcal enterotoxin G of high-purity (＞98%) successively.This purified product is promptly got the recombinant Staphylococcus aureus enterotoxin G lyophilized powder through desalination, lyophilization.
The golden staphylococcal enterotoxin of recombinating is striden the film experiment:
Set up monolayer Caco-2 cell transmembrane transhipment model, that detects the reorganization enterotoxin G strides the film effect.Confirmed that the golden staphylococcal enterotoxin G of reorganization can see through intestinal epithelial cell with complete molecular forms and enter the blood of human body circulation.
Stride active detection of superantigen of the reorganization enterotoxin molecule behind the film:
Adopt mtt assay, with the positive contrast of mitogen ConA, set up suitable external model and stride reorganization gold staphylococcal enterotoxin G behind the film to the proliferation function of mouse spleen lymphocyte and be subjected to the lethal effect of its mouse spleen lymphocyte that stimulates proliferation tumor cell with observation, and compare with staphylococcus aureus enterotoxin C 2 (SEC2), the reorganization gold staphylococcal enterotoxin G protein molecular that result's demonstration is striden behind the film still keeps typical superantigen activity, and is suitable with the effect of SEC2.
Of the present invention is that the preparation method of the oral formulations of main effective ingredient is with recombinate golden staphylococcal enterotoxin G and associated protein goods thereof:
With recombinate golden staphylococcal enterotoxin G and associated protein goods thereof is main effective ingredient, prepares oral anti-tumor agent by adding pharmaceutically acceptable component and adjuvant.Its preparation can be tablet, micro tablet, capsule, microencapsulation, granule, powder, effervescence solid, can chew solid tablet, softly coagulate in agent, capsule sheet, aqueous solution, suspensoid, emulsion, microemulsion, syrup or the elixir one or more, but is not limited to above-mentioned dosage form.
At present, yet there are no with non-classical golden staphylococcal enterotoxin and prepare the report that oral formulations is used for oncotherapy, United States Patent (USP) (PCT/US2005/02263) has been reported with deriving from enterotoxin genes bunch (egc, enterotoxingene cluster) golden staphylococcal enterotoxin (SEG, SEI, SEM, SEN, SEO) has good antitumor action, and be applied to malignant tumor and severe complication (hydrothorax, ascites etc.) patient's treatment and rehabilitation, obtained gratifying curative effect, but this patent application does not also propose above-mentioned enterotoxin is applied to oral anti-tumor agent.Existing Chinese patent (publication number CN1962692, CN1900119, CN101066993, CN101037478, CN101045924) relates to the golden staphylococcal enterotoxin SEE of reorganization, SEI, SEM, the preparation of SEN, SEO and the application in antitumor drug, does not also relate to the application of oral formulations.The present invention obtains the high-purity golden staphylococcal enterotoxin G that recombinates by engineered method, prove that simultaneously the golden staphylococcal enterotoxin G that obtains by method for preparing can be absorbed by the body by intestinal epithelial cell and continues to bring into play the superantigen effect with complete molecular forms, can stimulate mouse lymphocyte propagation and strengthen lymphocytic tumor-killing effect.Therefore, we have proved that the golden staphylococcal enterotoxin G of reorganization can absorb by human gastrointestinal tract, can enter blood circulation and produce the effective antitumour effect, have the application prospect that good preparation becomes oral anti-tumor agent.Simultaneously, the high-purity of acquisition reorganization enterotoxin G albumen does not contain golden Portugal Pseudomonas and other gram positive bacterias belong to all kinds of toxin proteins, can effectively reduce the probability that various toxic and side effects take place in the clinical use, therefore can be applied to the preparation of oral formulations.
Another purpose of the present invention provides the application of recombinant Staphylococcus aureus enterotoxin G in preparation treatment malignant tumor and other severe complication medicines.
The characteristics of the inventive method are: (1) provides the golden staphylococcal enterotoxin G of highly purified reorganization proteic preparation method, and confirm that this enterotoxin albumen can see through intestinal epithelial cell with the form of complete molecule, and can keep its superantigen activity, the preparation (2) that can be applied to oral anti-tumor agent provides a kind of with golden staphylococcal enterotoxin G and associated protein goods thereof the oral anti-tumor agent as effective ingredient of recombinating, the rehabilitation and the treatment (3) that can be applied to tumor patient provide the recombinant expression plasmid pGEX-4T-1-SEG that contains coding Staphylococcus aureus enterotoxin G mature peptide gene (seg), this plasmid is constructed by nucleotide shown in pGEX-4T-1 and the SEQ ID NO.2 and forms, can be transformed into suitable expressive host, contain strong promoter, under derivant is induced, can efficiently express the reorganization GST-SEG fusion rotein (4) that has the GST label and use anion-exchange chromatography, gel permeation chromatography has obtained highly purified reorganization SEG albumen (purity＞98%) to making with extra care purification by affinity purification and through the reorganization SEG albumen of enzyme action.
The inventive method utilizes engineered method to obtain highly purified recombinant Staphylococcus aureus enterotoxin G.The recombinate expression intensity height of golden staphylococcal enterotoxin G accounts for about the 10-20% of whole bacterial protein, and is solubility expression.Reorganization GST-SEG fusion rotein is behind the thrombin enzyme action, and resulting reorganization enterotoxin G is compared with corresponding natural intestine toxin protein G mature peptide, only has more 2 aminoacid (Gly-Ser) at the N end.The inventive method adopts the complete reorganization gold staphylococcal enterotoxin G molecule of Caco-2 cell monolayer model validation can see through intestinal epithelial cell and enters the systemic blood circulation, and the complete reorganization enterotoxin G molecule that utilizes the mtt assay proof to see through the Caco-2 cell monolayer can keep its superantigen characteristic.The present invention by specific activity, the superantigen of reorganization gold staphylococcal enterotoxin G that has confirmed to see through the Caco-2 cell monolayer is active with golden staphylococcal enterotoxin SEC2 suitable, because the main effective ingredient of the golden Portugal bacterium filtrate preparation of using in clinical acquisition is SEC2, thereby the golden staphylococcal enterotoxin G that recombinates can be applied to the preparation of oral anti-tumor agent.Simultaneously, component that contains in oral effective preparation that the inventive method provides and adjuvant comprise and prevent the degrade component of above-mentioned effective ingredient of pepsin, prevent the degrade component of above-mentioned effective ingredient of Chymotrypsin, prevent the component of the above-mentioned effective ingredient of intestinal trypsin degradation, in absorption enhancer and other biological goods and/or the genetic engineering medicine in acceptable adjuvant and the component one or more, thus can be so that the above-mentioned main effective ingredient of effective dose passes through oral, gastrointestinal tract, thereby parenteral route waits administering mode to arrive enteral outward and finally is absorbed in the blood that uses individuality.On the other hand, according to the identification experiment result, in the plain ejection preparations in existing three kinds of commercially available golden Portugals (high consor, think to win again, En Gefei) except containing micro-Enteromycin C 2 as main effective ingredient, also contain a large amount of different types of golden Portugal Pseudomonas albumen and polypeptides matter, wherein serine protease (serine protease), Enolase (enolase), dihydrolipoamide dehydrogenase three kinds of enzyme content such as (dihydrolipoamide dehydrogenase) have surpassed more than 95% of total protein content in the preparation.Simultaneously, also contain multiple golden Portugal Pseudomonas extracellular toxin and virulence factor in three kinds of preparations, comprising alpha hemolysin, γ-hemolysin, leukocidin, autolysin, fibronectin is conjugated protein and other different types of lipase, peptidase, ribozyme etc.Above-mentioned various protease structure and definite functions thereof, do not possess the superantigen activity of stimulation, enhancing human body immunity system, but probably be the immediate cause that causes in the clinical practice the various toxic and side effects (heating, hypotension, allergy, local pain etc.) that generally occur.The high-purity reorganization enterotoxin G that obtains by the present invention does not contain above-mentioned each eka-gold Portugal Pseudomonas toxin protein and virulence factor, in clinical use from now on, cause the probability of related side effects to reduce greatly, the present invention obtains the high-purity golden staphylococcal enterotoxin G that recombinates by engineered method, prove that simultaneously the golden staphylococcal enterotoxin G that obtains by method for preparing can be absorbed by the body by intestinal epithelial cell and continues to bring into play the superantigen effect with complete molecular forms, can stimulate mouse lymphocyte propagation and strengthen lymphocytic tumor-killing effect.Therefore, we have proved that the golden staphylococcal enterotoxin G of reorganization can absorb by human gastrointestinal tract, can enter blood circulation and produce the effective antitumour effect.Simultaneously, the high-purity of acquisition reorganization enterotoxin G albumen does not contain golden Portugal Pseudomonas and other gram positive bacterias belong to all kinds of toxin proteins, can effectively reduce the probability that various toxic and side effects take place in the clinical use, has good potential applicability in clinical practice.Given this, it is the preparation of the oral anti-tumor agent of main effective ingredient that the inventive method provides with recombinate golden staphylococcal enterotoxin G and protein product thereof, is used for the rehabilitation and the treatment of tumor patient.
Description of drawings
Fig. 1 contains the agarose gel electrophoresis figure of the coding SEG albumen mature peptide gene of restriction enzyme site for the pcr amplification gained.
Fig. 2 after by pcr amplification the gene order sequencing result figure of survey coding SEG mature peptide.
Fig. 3 is reorganization pGEX-4T-1-SEG plasmid double digestion agarose gel electrophoresis figure.
Fig. 4 is reorganization SEG expression plasmid pGEX-4T-1-SEG sketch map, and " SEG " of sign is the gene order on position of coding SEG albumen mature peptide.
Fig. 5 is reorganization SEG abduction delivering electrophoretogram in BL21 (DE3) escherichia coli.
Fig. 6 is reorganization SEG purification electrophoretogram.
Fig. 7 is the anion chromatography collection of illustrative plates of the reorganization SEG behind affinitive layer purification and the enzyme action, and solid line is a ultraviolet absorption value, and dotted line is an electric conductivity value, and wherein first eluting peak is reorganization SEG.
Fig. 8 is the gel chromatography collection of illustrative plates through the reorganization SEG behind the anion-exchange chromatography purification.
Fig. 9 for the Caco-2 cell monolayer transport experiment of reorganization SEG time-relationship between quality figure (A side to B side).
Figure 10 for the Caco-2 cell monolayer transport experiment of reorganization SEG time-relationship between quality figure (B side to A side).
Figure 11 is respectively the Western Blot testing result figure that (swimming lane 4) behind (swimming lane 3) behind (swimming lane 2) behind (swimming lane 1) behind the 6hr, the 12hr, the 18hr, the 24hr sees through the reorganization SEG of Caco-2 cell monolayer (the A side is to the B side).
Figure 12 is respectively behind the 6hr behind the 6hr behind (swimming lane 1), the 12hr behind (swimming lane 2), the 18hr WesternBlot testing result figure that (swimming lane 4) behind (swimming lane 3), the 24hr sees through the reorganization SEG of Caco-2 cell monolayer (the B side is to the A side).
Figure 13 sees through the reorganization SEG and the proliferation function (48h) of reorganization SEC2 standard substance to the ICR mouse spleen lymphocyte of Caco-2 cell monolayer for through behind the 24hr.
Figure 14 be respectively the ICR mouse spleen lymphocyte that stimulates of the reorganization SEG through seeing through the Caco-2 cell monolayer and reorganization SEC2 standard substance to the chronic marrow originality of adriamycin-resistant leukaemia's (K562-AD) inhibitory action (48h).
The specific embodiment
The present invention is further illustrated in conjunction with the accompanying drawings and embodiments in the present invention.Be pointed out that following each embodiment is not to be further qualification to content of the present invention, the equivalence that content technologies of the present invention has been done is replaced or corresponding the improvement, still belong within protection scope of the present invention.
In this context, unless specialize, otherwise any technical term of being quoted has those of ordinary skills' implication of common sense in the art, and not marked experimental technique is to carry out or carry out according to the operating instruction that supplier advised according to the normal experiment method.
Embodiment 1: the pcr amplification that contains the coding SEG albumen mature peptide gene order of restriction enzyme site: design following primer sequence:
SEQ ID NO.3:gta
Figure A20081006247500121
Caa ccc gat cct aaa tta gac (the line part is a BamH I restriction enzyme site)
SEQ ID NO.4:gga Tca gtg agt att aag aaa tac (the line part is Xho I restriction enzyme site) is a template with staphylococcus aureus (FRI S6) genome, and golden staphylococcal enterotoxin SEG mature peptide genetic fragment seg is used to increase;
The pcr amplification condition is as follows, and amplification obtains coding SEG mature peptide genetic fragment, and wherein each genetic fragment 5 ' end has BamH I restriction enzyme site, has Xho I restriction enzyme site after 3 ' the end terminator.
The PCR system:
H 2O： 60μL
Buffer(10×)： 10μL
Mg 2+(25mmol/L)： 8μL
BSA(5mg/mL)： 10μL
Primer-up(25μmol/L)： 4μL
Primer-down(25μmol/L)： 4μL
dNTP(20mmol/L)： 2μL
Template(Genome of S.aureus，10ng/μL)： 1μL
KOD Plus Polymerase(5U/μL)： 1μL
The PCR program:
1,95 ℃ of pre-degeneration 3min
2,95 ℃ of degeneration 30s, 55 ℃ of annealing 60s, 72 ℃ are extended 1min
3, according to step 2 circulation 34 times
4,72 ℃ are extended 10min
Electrophoresis detection PCR product, the result shows that PCR product purity and output are all higher, the genetic fragment molecular weight correct position that amplification obtains, PCR agar electrophoresis figure as a result sees accompanying drawing 1, wherein swimming lane 1: nucleic acid Marker, swimming lane 2: the PCR product that contains the coding SEG albumen mature peptide gene of restriction enzyme site.
Embodiment 2: the structure of recombiant plasmid pGEM-T-SEG that contains the coding enterotoxin SEG albumen mature peptide gene seg of restriction enzyme site:
The gene fragment clone of the coding SEG albumen mature peptide of the band restriction enzyme site of pcr amplification gained is gone into the pGEM-T plasmid, make up the pGEM-T-SEG recombiant plasmid, above-mentioned recombinant plasmid transformed is increased to bacillus coli DH 5 alpha.Extract above-mentioned reorganization pGEM-T-SEG plasmid, sample presentation order-checking after enzyme action is identified, sequencing result shows the gene order of coding SEG albumen mature peptide of band restriction enzyme site of above-mentioned acquisition and GenBank to be gone up corresponding gene order to remove restriction enzyme site of 5 ' increase all the other sites in full accord, the aminoacid sequence of the enterotoxin G mature peptide that it is corresponding is gone up corresponding protein mature peptide sequence with GenBank and is only compared how held two aminoacid (Gly-Ser), all the other amino acid sites (AAX11325) in full accord at N.The gene order sequencing result is seen accompanying drawing 2.
Embodiment 3: the structure that contains the pGEX-4T-1-SEG recombinant expression plasmid of the coding SEG mature peptide gene of being with restriction enzyme site:
With BamH I and Xho I respectively enzyme action by the pGEM-T-SEG recombiant plasmid and the pGEX-4T-1 plasmid of the genetic fragment of the coding SEG albumen mature peptide that contains restriction enzyme site that obtains among the embodiment 2.Reclaim the genetic fragment of the coding SEG mature peptide that contains restriction enzyme site behind the enzyme action and the big fragment of pGEX-4T-1 plasmid enzyme restriction product respectively, and connect, made up expression plasmid pGEX-4T-1-SEG.Above-mentioned recombinant expression plasmid is changed over to the bacillus coli DH 5 alpha amplification and extracts plasmid, identify through BamH I and Xho I enzyme action, the result shows that target gene fragment inserted vector plasmid, electrophoresis result is seen accompanying drawing 3, swimming lane 1 among the figure: nucleic acid Marker, swimming lane 2: do not pass through the reorganization pGEX-4T-1-SEG plasmid of enzyme action, swimming lane 3: reorganization pGEX-4T-1-SEG plasmid BamH I and Xho I double digestion product.
PGEX-4T-1-SEG recombinant expression plasmid sketch map is seen accompanying drawing 4.The detailed sequence of genes of interest on the pGEX-4T-1-SEG plasmid seen SEQ ID NO.2
Embodiment 4: the structure of reorganization GST-SEG expressing fusion protein bacterial strain
From the bacillus coli DH 5 alpha that contains the pGEX-4T-1-SEG recombinant expression plasmid, extract this plasmid, be converted in the e. coli bl21 (DE3), by the amicillin resistance screening positive clone, the engineering bacterial strain that acquisition can great expression GST-SEG fusion rotein.
Embodiment 5: reorganization GST-SEG Expression of Fusion Protein
It is dull and stereotyped that the engineering bacteria liquid that will contain the pGEX-4T-1-SEG recombinant expression plasmid is drawn the ampicillin screening, 37 ℃ leave standstill cultivate 16-20hr after picking list colony inoculation contain in the LB culture medium of ampicillin in 5mL, 6hr are cultivated in 37 ℃ of joltings, as seed liquor.The inoculum concentration of this seed liquor with 1-5% is inoculated in the 2 * YT culture medium that contains ampicillin, and 4hr is cultivated in 37 ℃ of joltings, and the volume ratio of pressing 0.01%-0.1% adds 0.1mol/L IPTG abduction delivering 5-8hr.
Embodiment 6: the affinity chromatograph sample pretreatment of reorganization GST-SEG fusion rotein
The pretreatment: get by among the embodiment 5 through the inductive bacterium liquid of IPTG in 4 ℃, 10000r.p.m is centrifugal, abandons collecting precipitation behind the supernatant.With the resuspended precipitation of PBS of original bacteria liquid 1/10 volume, suspension is put in the FRENCH cell breakage instrument, in the 700-900psi fragmentation, it is thick that suspension is.Ultrasonication 5-10min is continued with Ultrasonic Cell Disruptor in the back makes nucleolysis, and the cellular lysate fluid viscosity reduces.Ultrasonic back adds glycerol to final concentration in cellular lysate liquid be 2%, the vibration mixing, and ice bath leaves standstill 30min.After leaving standstill with cellular lysate liquid in 4 ℃, the centrifugal 30min of 12000r.p.m, it is stand-by to get the supernatant cryopreservation.Supernatant sampling carrying out SDS-PAGE detects, and at molecular weight 50kD place tangible band is arranged, and this is the reorganization GST-SEG fusion rotein of abduction delivering.Quantity One image analysis software shows that the fusion rotein band accounts for the 10-20% of full bacterium total protein content greatly.The swimming of cellular lysate liquid eggs white appliances the results are shown in accompanying drawing 5 after inducing, and swimming lane 1 among the figure: protein Marker, swimming lane 2: through the bacterial protein of abduction delivering.
Embodiment 7: the affinity chromatograph of reorganization GST-SEG fusion rotein:
Get by among the embodiment 6 through pretreated GST-SEG fusion rotein solution, behind 0.45 μ m membrane filtration, be added to the Glutathione Sepharose of pre-equilibration TMIn the 4 Fast Flow affinity columns, mixing gently is in conjunction with flowing to end liquid behind the 30min.Wash pillar 3-5 time with PBS, flow to end liquid.Xiang Zhuzhong adds the reduced glutathion eluent of certain volume, mixing gently.Fully collect elute soln behind the reaction 20min, survey protein concentration with ultraviolet spectrophotometer.The elution step that repeats to go on foot is lower than 0.1mg/mL until protein concentration and stops to collect.The elute soln component of collecting is checked purity and concentration with SDS-PAGE, the result shows the about 50kD of GST-SEG fusion protein molecule amount that preliminary purification obtains, electrophoresis result is seen accompanying drawing 6, swimming lane 1 among the figure: protein Marker, swimming lane 2: through the GST-SEG fusion rotein solution example behind the affinity purification, GST behind the swimming lane 3:GST-SEG fusion rotein process thrombin enzyme action and reorganization SEG albumen mixed protein solution example, swimming lane 4: through the reorganization SEG solution example that the anion chromatography purification obtains, swimming lane 5: the solution example that contains high-purity reorganization SEG that obtains through gel chromatography again behind the anion chromatography.
Embodiment 8: the enzyme action of reorganization GST-SEG fusion rotein
Above-mentioned collection component by the reorganization GST-SEG fusion rotein that obtains through affinity purification among the embodiment 10 is merged, and desalination is removed the glutathion in the solution and albumen place solution is replaced as and contains 0.05mol/LTris, 1.5mol/LNaCl, 2.5mmol/LCaCl 2(pH7.4) thrombin buffer solution.Every 1mL protein solution adds 20mg/mL thrombin 2 μ L, 25 ℃ of reaction 24h, and the sampling electrophoresis detects the endonuclease reaction degree.SDS-PAGE result shows that endonuclease reaction is complete substantially, and the GST label separates with reorganization SEG, and restriction enzyme digestion and electrophoresis the results are shown in Figure of description 6.
Embodiment 9: the refining purification of reorganization SEG
To be replaced as 0.05mol/L Tris (pH8.52) and carry out anion-exchange chromatography behind the buffer solution by mixed protein solution (comprising reorganization SEG, GST albumen label, thrombin and other small amount of impurities albumen) desalination that enzyme action among the embodiment 8 obtains, ion-exchange packing is selected Q Sepharose for use TMHP, after treating in the solution albumen and the anion chromatography post fully combining, adopt the linear gradient elution mode, improve salt ion intensity eluting gradually, eluent is 0.05mol/L Tris, 1mol/L NaCl (pH8.52) collects first eluting peak, is the reorganization SEG albumen (purity＞95%) of higher degree.To concentrate through the reorganization SEG solution component that ion chromatography obtains, utilize the Sephacryl-200 gel chromatography column that reorganization SEG albumen is made with extra care purification, flow velocity 0.75ml/min collects uv absorption summit corresponding solution component, is high-purity reorganization SEG (purity＞98%).The chromatography collection of illustrative plates is seen accompanying drawing 7,8, and solid line is a ultraviolet absorption value among Fig. 7, and dotted line is an electric conductivity value, and wherein first eluting peak is reorganization SEG; Solid line is a ultraviolet absorption value among Fig. 8, and this peak is highly purified reorganization SEG.Utilize SDS-PAGE to detect, show the single band of the golden staphylococcal enterotoxin G of reorganization for about 28kD position, purification result is seen accompanying drawing 6.
Embodiment 10: the preservation of high-purity reorganization SEG
After the high-purity reorganization enterotoxin G solution that obtains in the foregoing description 9 was replaced as ammonium formate solution, freezing draining promptly was prepared into reorganization enterotoxin G lyophilized powder ,-20 ℃ of preservations
Embodiment 11: the film of striding of reorganization SEG is measured
Set up monolayer Caco-2 cell transmembrane transhipment model, that detects the reorganization enterotoxin G strides the film effect.Confirmed that the golden staphylococcal enterotoxin G of reorganization can see through intestinal epithelial cell with complete molecular forms and enter the blood of human body circulation.
Concrete steps:
Get high-purity reorganization SEG that a certain amount of prepared in laboratory preserves and, be dissolved in HBSS, utilize BCA determination of protein concentration test kit to carry out accurately quantitatively, preparation reorganization SEG and HRP storing solution as HRP (horseradish peroxidase) powder of negative control.By a certain percentage with two kinds of storing solution dilutions and mix homogeneously, make reorganization SEG and HRP final concentration be respectively 7.5 μ g/mL and 10 μ g/mL with HBSS, use preceding 0.22 μ m membrane filtration degerming.
With the Caco-2 cell culture in Transwell TMIn the cell, cultivated 18-25 days, treat differentiation fully after, inhale and abandon original culture fluid, add the HBSS solution of 37 ℃ of preheatings, 37 ℃, 5%CO 2Leave standstill 20min under the condition, suction is abandoned, and adds to be preheated to 37 ℃ HBSS solution, 37 ℃, 5%CO again 2Leave standstill 20min under the condition, suction is abandoned.The mixed protein solution that will contain reorganization SEG (7.5 μ g/mL) and HRP (10 μ g/mL) adds Transwell respectively TMTop side (the Apical of cell, the A side) or bottom side (Basolateral, the B side), wherein the A side adds 500 μ L, and the B side adds 1.5mL, respective side adds HBSS solution, wherein the A side adds 500 μ L, and the B side adds 1.5mL, in 37 ℃ of shaking table low speed jolting (35-55r.p.m) hatch 6,12,18, behind the 24hr, add the implantation site according to mixed protein solution and collect opposite side solution, after utilize BA-ELISA and development process to measure respectively through the reorganization SEG albumen of Caco-2 cell monolayer and the content of HRP.
(1) the BA-ELISA method is measured SEG concentration
After 96 hole ELISA Plate are spent the night with mouse anti SEG monoclonal antibody bag, discard coating buffer, with 37 ℃ of sealings of the PBS that contains 10%FCS 2hr.Discard confining liquid, wash with PBS and add sample solution and reorganization SEG series concentration standard solution behind the plate, hatch 2hr for 37 ℃.Discard aforesaid liquid, wash with PBS and add anti-SEG rabbit polyclonal antibody behind the plate, hatch 2hr for 37 ℃.Discard polyclonal antibody solution, PBS adds the anti-rabbit igg of biotinylated goat after washing plate, hatches 0.5hr for 37 ℃.Discard the anti-rabbit igg solution of biotinylated goat, PBS adds HRP mark streptavidin after washing plate, hatches 0.5hr for 37 ℃.Discard HRP mark streptavidin solution, PBS washes to add behind the plate and now joins ELISA colour developing liquid, hatches to add now to join behind 1～5min for 37 ℃ to end the liquid stopped reaction.Read OD with microplate reader 450Value is according to reorganization SEG content, wherein each three multiple holes of each sample in the reorganization SEG series concentration standard curve calculation sample.
(2) determination of color HRP concentration
Sample solution and HRP series concentration standard solution are added in 96 well culture plates, add TMB, hatch to add behind 1～5min for 37 ℃ and end the liquid stopped reaction, read OD with microplate reader as chromogenic substrate 450Value, according to HRP concentration in the HRP series concentration standard curve calculation sample, wherein each three in each sample is answered holes.
Experimental result shows that HRP compares with negative control, and (A → B, B → A) efficient all is significantly higher than HRP, and the amount of transporting in 24hr changes in time to present and increases progressively trend in the Caco-2 cell bilateral transhipment of reorganization SEG.The Caco-2 cell monolayer transport experiment of reorganization SEG the results are shown in accompanying drawing 9,10.Solid line is reorganization SEG among Fig. 9, and the negative contrast of dotted line HRP, slogan banner are the time, vertical is designated as transhipment/through the quality of the reorganization SEG of Caco-2 cell monolayer.Solid line is reorganization SEG among Figure 10, and the negative contrast of dotted line HRP, slogan banner are the time, vertical is designated as transhipment/through the quality of the reorganization SEG of Caco-2 cell monolayer.
Embodiment 12: the integrity detection that sees through the reorganization gold staphylococcal enterotoxin G of Caco-2 cell monolayer
Utilize Western Blot method to detect through the proteic integrity of reorganization SEG behind the Caco-2 cell monolayer.Get each the time point sample solution among the embodiment 11, change film behind the conventional SDS-PAGE, film is spent the night with the PBS solution sealing that contains 10% alipoidic milk power.Discard confining liquid, PBS places the anti-SEG polyclonal antibody of rabbit solution with film after washing film, hatches 2hr in 37 ℃ of slow joltings.Discard an anti-solution, PBS places HRP enzyme mark goat anti-rabbit igg solution with film after washing film, hatches 2hr in 37 ℃ of slow joltings.PBS gets Lumi-Light Western Blotting Substrate solution 1,2 each 0.5mL mixing after washing film, drips on film, and 2min is placed in the dark place, preservative film envelope film is fixed in the magazine tabletting, after pressing 20sec, 1min and 5min respectively, automatic film developer is developed a film.
The result shows no matter the reorganization SEG that sees through the Caco-2 cell monolayer is all can keep its integrity from the A side to the B side or from the B side to the A side, the SEG albumen of promptly recombinating can see through the Caco-2 cell monolayer with complete molecular forms, confirms that reorganization SEG albumen can be entered blood circulation by the absorption of human small intestine's epithelial cell with complete molecular forms.Western Blot experimental result is seen Figure of description 11,12.
Embodiment 13:MTT method is measured the external antitumor activity of the reorganization enterotoxin SEG that sees through monolayer Caco-2 cell
Adopt mtt assay, with the positive contrast of mitogen ConA, observation is striden reorganization gold staphylococcal enterotoxin G behind the film to the proliferation function of mouse spleen lymphocyte and be subjected to the lethal effect of its mouse spleen lymphocyte that stimulates proliferation to tumor cell, and compares with staphylococcus aureus enterotoxin C 2 (SEC2) standard substance.
If zeroing hole (only containing culture medium), tumor cell control wells (culture medium+tumor cell), lymphocyte background release aperture [contains blank well (culture medium+ICR mouse spleen lymphocyte), negative control hole (culture medium+ICR mouse spleen lymphocyte+reorganization GST albumen), positive control hole (culture medium+ICR mouse spleen lymphocyte+Con A), SEC2 standard substance hole (culture medium+ICR mouse spleen lymphocyte+SEC2 standard substance) and experimental port (culture medium+ICR mouse spleen lymphocyte+see through the reorganization enterotoxin SEG of monolayer Caco-2 cell)] and the tumor-inhibiting action hole [contain blank well (culture medium+ICR mouse spleen lymphocyte+tumor cell), negative control hole (culture medium+ICR mouse spleen lymphocyte+tumor cell+reorganization GST albumen), positive control hole (culture medium+ICR mouse spleen lymphocyte+tumor cell+Con A), SEC2 standard substance hole (culture medium+ICR mouse spleen lymphocyte+tumor cell+SEC2 standard substance) and experimental port (culture medium+ICR mouse spleen lymphocyte+tumor cell+see through the reorganization enterotoxin SEG of monolayer Caco-2 cell)], establish three multiple holes for every kind.Wherein, Con A final concentration is 10 μ g/mL, and the GST final concentration is 10 μ g/mL, and SEC2 standard substance final concentration is 100ng/mL, and seeing through monolayer Caco-2 cell SEG final concentration is 90～150ng/mL (according to actual transit dose), in the background release aperture 5 * 10 6Individual/mL ICR mouse spleen lymphocyte adds with 100 μ L/ holes, in the function of tumor hole 5 * 10 6Individual/mL ICR mouse spleen lymphocyte and 2.5 * 10 5Individual/chronic marrow the originality of mL adriamycin-resistant leukaemia's (K562-AD) mixing suspension joins in the experimental port with 100 μ L/ holes, sees through monolayer Caco-2 cell SEG sample solution 50 μ L/ holes and adds each hole, and carefully blow and beat mixing.
44hr adds the MTT solution in 15 μ L/ holes behind the bed board in treating gaging hole, behind the careful piping and druming mixing, after continuing in the incubator to cultivate 4hr, the careful supernatant of absorbing, adding DMSO by 120 μ L/ holes, place 10min in 37 ℃ of incubators behind the piping and druming hydrotropy, microplate reader double wave regular way (570nm is for measuring wavelength, and 630nm is a reference wavelength) is measured each hole OD 570nm-OD 630nmValue is only to contain the zeroing hole zeroing of culture medium.Be calculated as follows tumour inhibiting rate: tumour inhibiting rate (%)=100-[(tumor-inhibiting action hole-lymphocyte background release aperture)/the tumor cell control wells] * 100, mapping analysis is subjected to see through the inhibitory action of the activated ICR mouse spleen lymphocyte of complete reorganization enterotoxin SEG molecule of Caco-2 cell monolayer to growth of tumour cell, and does statistical analysis with student t check.
Utilizing mtt assay to measure the external antitumor activity result of the complete reorganization enterotoxin SEG that sees through monolayer Caco-2 cell monolayer shows, with the ICR mouse spleen lymphocyte is function cells, with K562-AD is target cell, compare positive control Con A and see through the reorganization enterotoxin SEG of the complete molecular forms of Caco-2 cell monolayer (the mouse spleen lymphocyte tumour inhibiting rate all has significantly and increases behind 90～150ng/mL) the effect 48hr with pressing down the tumor negative control hole.Detect the lethal effect of the activated ICR mouse spleen lymphocyte of reorganization enterotoxin SEC2 standard substance by same experimental regime to the K562-AD cell, through relatively, suitable to the lethal effect of K562-AD cell with the activated ICR mouse spleen lymphocyte of reorganization enterotoxin SEC2 standard substance lethal effect by the activated ICR mouse spleen lymphocyte of complete reorganization enterotoxin SEG molecule that sees through the Caco-2 cell monolayer.Experimental result is seen Figure of description 13,14.Compare with the blank group among Figure 13,14: * * *: P＜0.001; *: P＜0.01.
Embodiment 14: commercially available golden Portugal bacterium ejection preparation component analysis
Carry out electrophoresis after the plain injection in commercially available three kinds of golden Portugals are utilized 40 times of ultrafiltration pipe ultrafiltration and concentration.Use the dyeing of Colloidal Coomassie Blue dyeing liquor after electrophoresis finishes, gel is divided into several portions, carry out in-gel digestion and peptide section respectively and extract according to different protein band shades and position in each sample on the gel.The peptide section that will extract obtain is with LC sample solution (2% acetonitrile, 0.5% formic acid) 20 μ L dissolving, the centrifugal 10min of 10000r.p.m after the vibration several seconds, and 30 ℃ of ultrasonic 10min of water-bath, the centrifugal 10min of 10000rpm gets supernatant and carries out LC/MS/MS and analyze.
According to mass spectrometry results, identify that altogether acquisition derives from different Pseudomonas protein for about 250 kinds in three kinds of golden Portugal bacterium ejection preparations (high consor, the multiple victory of think of, En Gefei), 100 kinds of golden Portugal Pseudomonas albumen are wherein arranged approximately, golden staphylococcal enterotoxin C2 and multiple golden Portugal Pseudomonas toxin protein and virulence factor have been comprised, as alpha hemolysin, γ-hemolysin, leukocidin, autolysin, fibronectin is conjugated protein and other different types of lipase, peptidase, ribozyme etc.Above-mentioned each albuminoid and golden Portugal verticillium toxin protein structure definite functions do not possess the superantigen activity of stimulation, enhancing human body immunity system, but the direct virulence factor of various clinical disease.Point out that as Dinges etc. alpha hemolysin can cause cutaneous necrosis, cause vasoconstriction, cause the ischemia necrosis, and finally may cause pulmonary edema, the pyogenic infection of respiratory distress syndrome and each organ; Can the cause inflammation generation of the factor of γ-hemolysin causes various inflammatory reactions (Martin M.Dinges et al.Exotoxins of Staphylococcus aureus.Clin Microbiol Rev.2000; Vol.13, No.1:16-34).Iwatsuki etc. point out, leukocidin can cause skin and necrosis of subcutaneous tissue, also can cause child and young necrotizing pneumonia (Keiji Iwatsuki et al.Staphylococcal cutaneousinfections:Invasion, evasion, and aggression.J Dermatol Sci.2006 Jun; Vol.42, No.3:203-214).Menzies points out that fibronectin albumen then is important factor (the Menzies BE.The role of fibronectin binding proteins in the pathogenesis ofStaphylococcus aureus infections.Curr Opin Infect Dis.2003 Jun that causes infective endocarditis; Vol.16, No.3:225-229).The existence of above-mentioned various golden Portugals bacterium virulence factor very likely is to cause existing three kinds of plain ejection preparations in commercially available golden Portugal to produce the immediate cause of multiple side effect (heating, hypotension, allergy, local pain etc.) in clinical practice, therefore must strict control and removal as far as possible in exploitation, development and the production process of similar medicine from now on.The golden Portugal important toxin protein of Pseudomonas that obtains in three kinds of injections (high consor, En Gefei, think of win again) to identify sees Table 1.First column contains GenBank protein sequence number wherein, second classifies as and obtains the toxin protein title identified, and the 3rd classifies the protein specific name as.
Obtain the important toxin protein of golden Portugal Pseudomonas of evaluation in the plain ejection preparation in table 1 gold Portugal
Protein sequence number Protein name Kind
gi|2914575 Chain G，Alpha-Hemolysin [Staphylococcus aureus]
gi|9931632 serine protease-like exoprotein A [Staphylococcus aureus]
gi|9931634 serine protease-like exoprotein E [Staphylococcus aureus]
gi|19879322 lipase [Staphylococcus aureus]
gi|21203559 set26 [Staphylococcus aureus subsp.aureus MW2]
gi|21284073 gamma-hemolysin component B [Staphylococcus aureus subsp.aureus MW2]
gi|49483762 putative peptidase [Staphylococcus aureus subsp.aureus MRSA252]
gi|49484059 serine protease [Staphylococcus aureus subsp.aureus MRSA252]
gi|76009542 enterotoxin C2 precursor [Staphylococcus aureus]
gi|82750663 Autolysin [Staphylococcus aureus RF122]
gi|82752016 gamma-hemolysin component C [Staphylococcus aureus RF122]
gi|83681617 fibronectin binding protein B [Staphylococcus aureus]
gi|87160772 V8 protease [Staphylococcus aureus subsp.aureus USA300]
gi|87162036 leukotoxin LukE [Staphylococcus aureus subsp.aureus USA300]
gi|87162123 1-phosphatidylinositol phosphodiesterase [Staphylococcus aureus subsp.aureus USA300]
gi|90585272 Sulfatase [Staphylococcus aureus subsp.aureus JH9]
gi|90585564 Peptidase S1 and S6，chymotrypsin/Hap [Staphylococcus aureus subsp.aureus JH9]
gi|90585851 Glycerophosphoryl diester phosphodiesterase [Staphylococcus aureus subsp.aureus JH9]
Above-mentioned a series of experiment confirm, the high-purity that obtains by the gene engineering method golden staphylococcal enterotoxin SEG that recombinates can absorb by intestinal epithelial cell and enter blood circulation, simultaneously, reorganization enterotoxin G after the absorption still keeps its superantigen activity, can stimulate mouse T lymphocyte significantly to breed, the T lymphocyte of propagation has significant lethal effect to tumor cell.Simultaneously, compare with the plain ejection preparation in existing golden Portugal, the reorganization enterotoxin G for preparing does not contain various golden Portugal Pseudomonas toxin proteins and virulence factor.In sum, reorganization enterotoxin G provided by the invention can be used for the preparation of oral anti-tumor agent.
The sequence that the present invention relates to
＜110〉Zhejiang University
＜120〉recombinant Staphylococcus aureus enterotoxin G oral preparation and application
<160>4
<210>1
<211>705
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(705)
＜223〉contain the recombinant Staphylococcus aureus enterotoxin G of partial prothrombinase restriction enzyme site sequence
<440>1
gga tcc caa ccc gat cct aaa tta gac gaa cta aat aaa gta agt 45
Gly Ser Gln Pro Asp Pro Lys Leu Asp Glu Leu Asn Lys Val Ser
1 5 10 15
gat tat aaa aat aat aag gga act atg ggt aat gta atg aat ctt 90
Asp Tyr Lys Asn Asn Lys Gly Thr Met Gly Asn Val Met Asn Leu
16 20 25 30
tat acg tct cea cct gtt gaa gga aga gga gtt att aat tct aga 135
Tyr Thr Ser Pro Pro Val Glu Gly Arg Gly Val Ile Asn Ser Arg
31 35 40 45
cag ttt tta tct cat gat tta att ttt cea att gag tat aag agt 180
Gln Phe Leu Ser His Asp Leu Ile Phe Pro Ile Glu Tyr Lys Ser
46 50 55 60
tat aat gag gtt aaa act gaa tta gaa aat aca gaa tta gct aac 225
Tyr Asn Glu Val Lys Thr Glu Leu Glu Asn Thr Glu Leu Ala Asn
61 65 70 75
aat tat aaa gat aaa aaa gta gac att ttt ggc gtt cca tat ttt 270
Asn Tyr Lys Asp Lys Lys Val Asp Ile Phe Gly Val Pro Tyr Phe
76 80 85 90
tat aca tgt ata ata cct aaa tct gaa ccg gat ata aac caa aat 315
Tyr Thr Cys Ile Ile Pro Lys Ser Glu Pro Asp Ile Asn Gln Asn
91 95 100 105
ttt gga ggt tgt tgt atg tat ggt ggt ctt aca ttt aat agt tca 360
Phe Gly Gly Cys Cys Met Tyr Gly Gly Leu Thr Phe Asn Ser Ser
106 110 115 120
gaa aat gaa aga gat aaa tta att act gta cag gta aca atc gac 405
Glu Asn Glu Arg Asp Lys Leu Ile Thr Val Gln Val Thr Ile Asp
121 125 130 135
aat aga caa tca ctt gga ttt aca ata act aca aat aag aat atg 450
Asn Arg Gln Ser Leu Gly Phe Thr Ile Thr Thr Asn Lys Asn Met
136 140 145 150
gtt act att cag gaa cta gat tac aaa gca aga cac tgg ctc act 495
Val Thr Ile Gln Glu Leu Asp Tyr Lys Ala Arg His Trp Leu Thr
151 155 160 165
aaa gaa aaa aag cta tac gag ttt gat tgt tct gcg ttt gaa tct 540
Lys Glu Lys Lys Leu Tyr Glu Phe Asp Cys Ser Ala Phe Glu Ser
166 170 175 180
gga tat ata aaa ttt act gaa aag aac aat aca agt ttt tgg ttt 585
Gly Tyr Ile Lys Phe Thr Glu Lys Asn Asn Thr Ser Phe Trp Phe
181 185 190 195
gac tta ttt cct aaa aaa gaa cta gta cct ttt gtt cca tat aag 630
Asp Leu Phe Pro Lys Lys Glu Leu Val Pro Phe Val Pro Tyr Lys
196 200 205 210
ttt tta aat att tac gga gat aat aaa gtt gtt gat tct aag agt 675
Phe Leu Asn Ile Tyr Gly Asp Asn Lys Val Val Asp Ser Lys Ser
211 215 220 225
att aaa atg gaa gta ttt ctt aat act cac 705
Ile Lys Met Glu Val Phe Leu Asn Thr His
226 230 235
<210>2
<211>699
<212>DNA
＜213〉staphylococcus aureus (Staphylococcus aureus)
<220>
<221>CDS
<222>(1)...(699)
＜223〉clone's gained Staphylococcus aureus enterotoxin G
<440>2
caa ccc gat cct aaa tta gac gaa cta aat aaa gta agt gat tat 45
Gln Pro Asp Pro Lys Leu Asp Glu Leu Asn Lys Val Ser Asp Tyr
1 5 10 15
aaa aat aat aag gga act atg ggt aat gta atg aat ctt tat acg 90
Lys Asn Asn Lys Gly Thr Met Gly Asn Val Met Asn Leu Tyr Thr
16 20 25 30
tct cca cct gtt gaa gga aga gga gtt att aat tct aga cag ttt 135
Ser Pro Pro Val Glu Gly Arg Gly Val Ile Asn Ser Arg Gln Phe
31 35 40 45
tta tct cat gat tta att ttt cca att gag tat aag agt tat aat 180
Leu Ser His Asp Leu Ile Phe Pro Ile Glu Tyr Lys Ser Tyr Asn
46 50 55 60
gag gtt aaa act gaa tta gaa aat aca gaa tta gct aac aat tat 225
Glu Val Lys Thr Glu Leu Glu Asn Thr Glu Leu Ala Asn Asn Tyr
61 65 70 75
aaa gat aaa aaa gta gac att ttt ggc gtt cca tat ttt tat aca 270
Lys Asp Lys Lys Val Asp Ile Phe Gly Val Pro Tyr Phe Tyr Thr
76 80 85 90
tgt ata ata cct aaa tct gaa ccg gat ata aac caa aat ttt gga 315
Cys Ile Ile Pro Lys Ser Glu Pro Asp Ile Asn Gln Asn Phe Gly
91 95 100 105
ggt tgt tgt atg tat ggt ggt ctt aca ttt aat agt tca gaa aat 360
Gly Cys Cys Met Tyr Gly Gly Leu Thr Phe Asn Ser Ser Glu Asn
106 110 115 120
gaa aga gat aaa tta att act gta cag gta aca atc gac aat aga 405
Glu Arg Asp Lys Leu Ile Thr Val Gln Val Thr Ile Asp Asn Arg
121 125 130 135
caa tca ctt gga ttt aca ata act aca aat aag aat atg gtt act 450
Gln Ser Leu Gly Phe Thr Ile Thr Thr Asn Lys Asn Met Val Thr
136 140 145 150
att cag gaa cta gat tac aaa gca aga cac tgg ctc act aaa gaa 495
Ile Gln Glu Leu Asp Tyr Lys Ala Arg His Trp Leu Thr Lys Glu
151 155 160 165
aaa aag cta tac gag ttt gat tgt tct gcg ttt gaa tct gga tat 540
Lys Lys Leu Tyr Glu Phe Asp Cys Ser Ala Phe Glu Ser Gly Tyr
166 170 175 180
ata aaa ttt act gaa aag aac aat aca agt ttt tgg ttt gac tta 585
Ile Lys Phe Thr Glu Lys Asn Asn Thr Ser Phe Trp Phe Asp Leu
181 185 190 195
ttt cct aaa aaa gaa cta gta cct ttt gtt cca tat aag ttt tta 630
Phe Pro Lys Lys Glu Leu Val Pro Phe Val Pro Tyr Lys Phe Leu
196 200 205 210
aat att tac gga gat aat aaa gtt gtt gat tct aag agt att aaa 675
Asn Ile Tyr Gly Asp Asn Lys Val Val Asp Ser Lys Ser Ile Lys
211 215 220 225
atg gaa gta ttt ctt aat act cac 699
Met Glu Val Phe Leu Asn Thr His
226 230 233
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉amplification contains the used forward primer of Staphylococcus aureus enterotoxin G gene of restriction enzyme site from the staphylococcus aureus gene group
<440>3
gta gga tcc caa ccc gat cct aaa tta gac
<210>4
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉amplification contains the used downstream primer of Staphylococcus aureus enterotoxin G gene of restriction enzyme site from the staphylococcus aureus gene group
<440>4
gga ctc gag tca gtg agt att aag aaa tac

Claims (5)

1. recombinant Staphylococcus aureus enterotoxin G oral preparation, described recombinant Staphylococcus aureus enterotoxin G belongs to superantigen albumen, it is characterized in that: SEQ ID NO.1 is the individual heavily aminoacid sequence of this group Staphylococcus aureus enterotoxin G, that express this recombinant Staphylococcus aureus enterotoxin G is recombiant plasmid pGEX-4T-1-SEG, this plasmid is formed through recombination to construct by the nucleotide sequence of pGEX-4T-1 and SEQ ID NO.2, and this oral formulations also contains preparation allowable pharmaceutical excipients or carrier.
2. recombinant Staphylococcus aureus enterotoxin G oral preparation according to claim 1 is characterized in that described recombinant Staphylococcus aureus enterotoxin G obtains by following steps:
(1) design has the upstream and downstream primer that is used to clone Staphylococcus aureus enterotoxin G mature peptide sequence of BamH I and Xho I restriction enzyme site respectively:
SEQ ID NO.3:gta Gga tccCaa ccc gat cct aaa tta gac, the line part is a BamH I restriction enzyme site,
SEQ ID NO.4:gga Ctc gagTca gtg agt att aag aaa tac line part is an Xho I restriction enzyme site;
(2) be template with the staphylococcus aureus gene group, adopt pcr amplification to obtain the genetic fragment of the golden staphylococcal enterotoxin G albumen mature peptide of coding, it is connected into multiple clone site on the pGEM-T plasmid vector, make up the pGEM-T-SEG recombiant plasmid, the gene order that confirms to have the genetic fragment of sequence of SEQ ID NO.2 and the coding same protein among the GenBank data base by order-checking is in full accord;
(3) be template with the pGEM-T-SEG plasmid, pcr amplification obtains the gene order that two ends have the coding gold staphylococcal enterotoxin G albumen mature peptide of restriction enzyme site, with the said gene fragment with restriction endonuclease BamH I with after Xho I carries out enzyme action with handle with identical restriction endonuclease after the pGEX-4T-1 plasmid be connected construction expression plasmid pGEX-4T-1-SEG;
(4) the pGEX-4T-1-SEG expression plasmid is converted into e. coli bl21, the reorganization GST-SEG fusion rotein of abduction delivering band GST label, the centrifugal collection supernatant of results thalline and broken back adopts affinity chromatograph, ion chromatography, gel chromatography to obtain the golden staphylococcal enterotoxin G of reorganization successively.
3. recombinant Staphylococcus aureus enterotoxin G oral preparation according to claim 1 is characterized in that: the administering mode of described medicine is selected oral administration, gastrointestinal administration or parenteral route external administration for use.
4. recombinant Staphylococcus aureus enterotoxin G oral preparation according to claim 1 is characterized in that: drug excipient that this oral formulations contains or carrier comprise acceptable component and adjuvant in the composition that prevents or suppress intestinal Chymotrypsin proteolytic activity, the composition that prevents or suppress intestinal trypsin proteolytic activity, the composition that prevents or suppress the pepsin proteolytic activity, absorption enhancer and the biological product.
5. the application of recombinant Staphylococcus aureus enterotoxin G oral preparation according to claim 1 in preparation treatment malignant tumor and other severe complication medicines.
CNA2008100624758A 2008-06-12 2008-06-12 Recombinant Staphylococcus aureus enterotoxin G oral preparation and use Pending CN101322842A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017009364A1 (en) * 2015-07-14 2017-01-19 Swecure Ab Reducing the risk of developing of atopic dermatitis by use of superantigen

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017009364A1 (en) * 2015-07-14 2017-01-19 Swecure Ab Reducing the risk of developing of atopic dermatitis by use of superantigen

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