CN101293092A - Recombined staphylococcus aureus enterotoxin I oral preparation and application thereof - Google Patents

Abstract

The invention provides a recombinant staphylococcal enterotoxin I oral preparation, the recombinant staphylococcal enterotoxin I has SEQ ID NO.1 amino acid sequence, and the oral preparation further comprises a pharmaceutical allowable drug excipient or a carrier. The oral preparation proves that the protein can enter the systemic blood circulation by penetrating epithelial cells on small intestine with the form of complete molecules and maintain the super-antigen activity for promoting the spleen lymphocyte proliferation and inhibiting the growth of tumor cells, as well as the application in the preparation of drugs for treating malignant tumors and other serious complications by the Caco-2 monolayer cell transmembrane transport test.

Description

Recombined staphylococcus aureus enterotoxin I oral preparation and application

Technical field

The invention belongs to biological engineering, relate to recombined staphylococcus aureus enterotoxin I (StaphylococcalEnterotoxin I, preparation SEI) and with the preparation of the oral formulations of the main effective ingredient of SEI and the application in oncotherapy.Specifically, utilize gene engineering method to prepare high-purity recombined staphylococcus aureus enterotoxin I 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 recombined staphylococcus aureus enterotoxin I oral preparation, described recombined staphylococcus aureus enterotoxin I is the superantigen albumen that utilizes gene engineering method to prepare, aminoacid sequence with SEQ ID NO.1, the express recombinant staphylococcus aureus enterotoxin 1 be recombiant plasmid pGEX-4T-1-SEI, 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 recombined staphylococcus aureus enterotoxin I and associated protein goods thereof, and this oral formulations also contains preparation allowable pharmaceutical excipients or carrier.

The present invention by Caco-2 cell monolayer transmembrane transport test confirmed this albumen can see through with the form of complete molecule intestinal epithelial cell enter the systemic blood circulation and can keep its short spleen lymphocyte proliferation and and the superantigen activity that suppresses growth of tumour cell, 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 recombined staphylococcus aureus enterotoxin I 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.

Recombined staphylococcus aureus enterotoxin I of the present invention obtains by following steps:

(1) preparation of recombined staphylococcus aureus enterotoxin I and purification:

(1) design has the upstream and downstream primer that is used to clone staphylococcus aureus enterotoxin 1 mature peptide sequence of BamH I and Xho I restriction enzyme site respectively:

SEQ ID NO.3:gta Gga tccCaa ggt gat att ggt gta ggt (the line part is a BamH I restriction enzyme site),

SEQ ID NO.4:gga Ctc gagTta gtt act ate tac ata tga (the line part is an Xho I restriction enzyme site);

(2) be template with staphylococcus aureus (FRI 100) genome, adopt pcr amplification to obtain the genetic fragment of the golden staphylococcal enterotoxin I albumen mature peptide of coding, it is connected into multiple clone site on the pGEM-T plasmid vector, successfully makes up the pGEM-T-SEI recombiant plasmid.The gene order (AY920267) of the coding same protein among the gene fragment order (seeing SEQ ID NO.2) that confirms the coding gold staphylococcal enterotoxin I 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-SEI plasmid, pcr amplification obtains the gene order that two ends have the coding gold staphylococcal enterotoxin I 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-SEI;

(4) the pGEX-4T-1-SEI expression plasmid is converted into e. coli bl21 (DE3), the reorganization GST-SEI 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 I of high-purity (＞98%) successively.This purified product is promptly got the recombined staphylococcus aureus enterotoxin I lyophilized powder through desalination, lyophilization.

The golden staphylococcal enterotoxin of recombinating is striden film experiment: set up monolayer Caco-2 cell transmembrane transhipment model, that detects the reorganization enterotoxin 1 strides the film effect.Confirmed that the golden staphylococcal enterotoxin I 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 I 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 I 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 I and associated protein goods thereof:

With recombinate golden staphylococcal enterotoxin I 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.

Another purpose of the present invention provides the application of recombined staphylococcus aureus enterotoxin I in preparation treatment malignant tumor and other severe complication medicines.

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.The Chinese patent (publication number CN1962692, CN1900119, CN101066993, CN101037478, CN101045924) of my laboratory application 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 I that recombinates by engineered method, prove that simultaneously the golden staphylococcal enterotoxin I 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 I 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 reorganization enterotoxin 1 albumen that obtains 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, be used for human body immunity improving power, treatment malignant tumor and other severe complications.

The characteristics of the inventive method are: (1) provides the golden staphylococcal enterotoxin I 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 I 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-SEI that contains coding staphylococcus aureus enterotoxin 1 mature peptide gene (sei), 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-SEI fusion rotein (4) that has the GST label and use anion-exchange chromatography, gel permeation chromatography has obtained highly purified reorganization SEI albumen (purity＞98%) to making with extra care purification by affinity purification and through the reorganization SEI albumen of enzyme action.

The inventive method utilizes engineered method to obtain highly purified recombined staphylococcus aureus enterotoxin I.The recombinate expression intensity height of golden staphylococcal enterotoxin I accounts for about the 10-20% of whole bacterial protein, and is solubility expression.Reorganization GST-SEI fusion rotein is behind the thrombin enzyme action, and resulting reorganization enterotoxin 1 is compared with corresponding natural intestine toxin protein I mature peptide, only has more 2 aminoacid (Gly-Ser) at the N end.The inventive method adopts the complete reorganization gold staphylococcal enterotoxin I 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 1 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 I 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 I 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 (serineprotease), Enolase (enolase), dihydrolipoamide dehydrogenase three kinds of enzyme content such as (dihydrolipoamidedehydrogenase) 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 1 that obtains by the present invention does not contain above-mentioned each eka-gold Portugal Pseudomonas toxin protein and virulence factor, causes the probability of related side effects to reduce greatly in clinical use from now on.The present invention obtains the high-purity golden staphylococcal enterotoxin I that recombinates by engineered method, prove that simultaneously the golden staphylococcal enterotoxin I 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 I 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 1 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 I 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 SEI 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 SEI mature peptide.

Fig. 3 is reorganization pGEX-4T-1-SEI plasmid double digestion agarose gel electrophoresis figure.

Fig. 4 is reorganization SEI expression plasmid pGEX-4T-1-SEI sketch map, and " SEI " of sign is the gene order on position of coding SEI albumen mature peptide.

Fig. 5 is reorganization SEI abduction delivering electrophoretogram in BL21 (DE3) escherichia coli.

Fig. 6 is the golden staphylococcal enterotoxin I purification electrophoretogram of reorganization.

Fig. 7 is the anion chromatography collection of illustrative plates of the reorganization gold staphylococcal enterotoxin I (SEI) behind affinitive layer purification and the enzyme action.

Fig. 8 is the gel chromatography collection of illustrative plates through the gold staphylococcal enterotoxin I of the reorganization behind the anion-exchange chromatography purification (SEI).

Fig. 9 for the Caco-2 cell monolayer transport experiment of the golden staphylococcal enterotoxin I of reorganization (SEI) time-relationship between quality figure (A side to B side).

Figure 10 for the Caco-2 cell monolayer transport experiment of the golden staphylococcal enterotoxin I of reorganization (SEI) 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 gold staphylococcal enterotoxin I of Caco-2 cell monolayer (the A side is to the B side).

Figure 12 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 gold staphylococcal enterotoxin I of Caco-2 cell monolayer (the B side is to the A side).

Figure 13 sees through the reorganization SEI and the proliferation function (48h) of the golden staphylococcal enterotoxin SEC2 standard substance of reorganization to the ICR mouse spleen lymphocyte of Caco-2 cell monolayer for through behind the 24hr, wherein compares with the blank group: ^{* *}: P＜0.001; ^*: P＜0.01.

Figure 14 is that the ICR mouse spleen lymphocyte that stimulates of the reorganization SEI through seeing through the Caco-2 cell monolayer and the golden staphylococcal enterotoxin SEC2 standard substance of reorganization is compared with the blank group the chronic marrow originality of adriamycin-resistant leukaemia's (K562-AD) inhibitory action (48h) respectively: ^{* *}: P＜0.001.

The specific embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.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 SEI albumen mature peptide gene order of restriction enzyme site:

Design following primer sequence:

SEQ ID NO.3:gta Gga tccCaa ggt gat att ggt gta ggt (the line part is a BamH I restriction enzyme site)

SEQ ID NO.4:gga Ctc gagTta gtt act atc tac ata tga (the line part is Xho I restriction enzyme site) is a template with staphylococcus aureus (FRI 100) genome, and golden staphylococcal enterotoxin SEI mature peptide genetic fragment sei is used to increase;

The pcr amplification condition is as follows, and amplification obtains coding SEI 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 agargel 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 SEI albumen mature peptide gene of restriction enzyme site.

Embodiment 2: the structure of recombiant plasmid pGEM-T-SEI that contains the coding enterotoxin SEI albumen mature peptide gene sei of restriction enzyme site:

The gene fragment clone of the coding SEI 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-SEI recombiant plasmid, above-mentioned recombinant plasmid transformed is increased to bacillus coli DH 5 alpha.Extract above-mentioned reorganization pGEM-T-SEI plasmid, sample presentation order-checking after enzyme action is identified, sequencing result shows the gene order of coding SEI 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 1 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 (AAX11331) 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-SEI recombinant expression plasmid of the coding SEI mature peptide gene of being with restriction enzyme site:

With BamH I and Xho I respectively enzyme action by the pGEM-T-SEI recombiant plasmid and the pGEX-4T-1 plasmid of the genetic fragment of the coding SEI albumen mature peptide that contains restriction enzyme site that obtains among the embodiment 2.Reclaim the genetic fragment of the coding SEI 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-SEI.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, wherein swimming lane 1: nucleic acid Marker, swimming lane 2: do not pass through the reorganization pGEX-4T-1-SEI plasmid of enzyme action, swimming lane 3: reorganization pGEX-4T-1-SEI plasmid BamH I and Xho I double digestion product.PGEX-4T-1-SEI recombinant expression plasmid sketch map is seen accompanying drawing 4.The detailed sequence of genes of interest on the pGEX-4T-1-SEI plasmid seen SEQ IDNO.2.

Embodiment 4: the structure of reorganization GST-SEI expressing fusion protein bacterial strain

From the bacillus coli DH 5 alpha that contains the pGEX-4T-1-SEI 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-SEI fusion rotein.

Embodiment 5: reorganization GST-SEI Expression of Fusion Protein

It is dull and stereotyped that the engineering bacteria liquid that will contain the pGEX-4T-1-SEI 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 is cultivated in the 37C jolting, 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-SEI fusion rotein

The pretreatment: get by among the embodiment 5 through the inductive bacterium liquid of IPTG in 4 ℃, 10000rpm 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 12000rpm, 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-SEI 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-SEI fusion rotein:

Get by among the embodiment 6 through pretreated GST-SEI 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-SEI fusion protein molecule amount that preliminary purification obtains, electrophoresis result is seen Figure of description 6, swimming lane 1 among the figure: protein Marker, swimming lane 2: through the GST-SEI fusion rotein solution example behind the affinity purification, GST behind the swimming lane 3:GST-SEI fusion rotein process thrombin enzyme action and reorganization SEI albumen mixed protein solution example, swimming lane 4: through the reorganization SEI solution example that the anion chromatography purification obtains, swimming lane 5: the solution example that contains high-purity reorganization SEI that obtains through gel chromatography again behind the anion chromatography.

Embodiment 8: the enzyme action of reorganization GST-SEI fusion rotein

Above-mentioned collection component by the reorganization GST-SEI 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 SEI, and restriction enzyme digestion and electrophoresis the results are shown in Figure of description 6.

Embodiment 9: the refining purification of reorganization SEI

To be replaced as 0.05mol/L Tris (pH8.54) and carry out anion-exchange chromatography behind the buffer solution by mixed protein solution (comprising reorganization SEI, 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 mode of linear gradient elution, improve salt ion intensity eluting gradually, eluent is 0.05mol/L Tris, 1mol/L NaCl (pH8.54) collects first eluting peak, is the reorganization SEI albumen (purity＞95%) of higher degree.To concentrate through the reorganization SEI solution component that ion chromatography obtains, utilize the Sephacryl-200 gel chromatography column that reorganization SEI albumen is made with extra care purification, flow velocity 0.75ml/min collects uv absorption summit corresponding solution component, is high-purity reorganization SEI (purity＞98%).The chromatography collection of illustrative plates is seen Figure of description 7,8, utilizes SDS-PAGE to detect, and shows the single band of the golden staphylococcal enterotoxin I of reorganization for about 27kD position, and purification result is seen Figure of description 6.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 SEI.Solid line is a ultraviolet absorption value among Fig. 8, and this peak is highly purified reorganization SEI.

Embodiment 10: the recombinate preservation of golden staphylococcal enterotoxin I of high-purity

After the high-purity reorganization enterotoxin 1 solution that obtains among the embodiment 9 was replaced as ammonium formate solution, freezing draining promptly was prepared into reorganization enterotoxin 1 lyophilized powder ,-20 ℃ of preservations

Embodiment 11: the film of striding of the golden staphylococcal enterotoxin I that recombinates is measured

Get high-purity reorganization SEI 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 SEI 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 SEI 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 SEI (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 SEI albumen of Caco-2 cell monolayer and the content of HRP.

(1) the BA-ELISA method is measured SEI concentration

After 96 hole ELISA Plate are spent the night with mouse anti SEI 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 SEI series concentration standard solution behind the plate, hatch 2hr for 37 ℃.Discard aforesaid liquid, wash with PBS and add anti-SEI 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 SEI content, wherein each three multiple holes of each sample in the reorganization SEI 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 SEI.The Caco-2 cell monolayer transport experiment of reorganization SEI the results are shown in Figure of description 9,10.Solid line is reorganization SEI 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 SEI of Caco-2 cell monolayer.Solid line is reorganization SEI 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 SEI of Caco-2 cell monolayer.

Embodiment 12: the integrity detection that sees through the reorganization gold staphylococcal enterotoxin I of Caco-2 cell monolayer

Utilize Western Blot method to detect through the proteic integrity of reorganization SEI 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-SEI 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 SEI 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 SEI albumen of promptly recombinating can see through the Caco-2 cell monolayer with complete molecular forms, confirms that reorganization SEI 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 SEI that sees through monolayer Caco-2 cell

Adopt mtt assay, with the positive contrast of mitogen ConA, observation is striden reorganization gold staphylococcal enterotoxin I 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 SEI 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 SEI 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 SEI 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 SEI 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 SEI 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 SEI 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 SEI 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 SEI 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 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]

[Staphylococcus?aureus

gi|49483762 putative?peptidase subsp.aureus?MRSA252]

[Staphylococcus?aureus

gi|49484059 serine?protease 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?S1and?S6，chymotrypsin/Hap [Staphylococcus?aureus?subsp.aureus?JH9]

gi|90585851 Glycerophosphoryl?diester [Staphylococcus?aureus?subsp.aureus?JH9]

phosphodiesterase

Above-mentioned a series of experiment confirm, the high-purity that obtains by the gene engineering method golden staphylococcal enterotoxin SEI that recombinates can absorb by intestinal epithelial cell and enter blood circulation, simultaneously, reorganization enterotoxin 1 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 1 for preparing does not contain various golden Portugal Pseudomonas toxin proteins and virulence factor.In sum, reorganization enterotoxin 1 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〉recombined staphylococcus aureus enterotoxin I oral preparation and application

<160>4

<210>1

<211>660

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(660)

＜223〉contain the recombined staphylococcus aureus enterotoxin I of partial prothrombinase restriction enzyme site sequence

<440>1

gga?tcc?caa?ggt?gat?att?ggt?gta?ggt?aac?tta?aga?aat?ttc?tat 45

Gly?Ser?Gln?Gly?Asp?Ile?Gly?Val?Gly?Asn?Leu?Arg?Asn?Phe?Tyr

1 5 10 15

aca?aaa?cat?gat?tat?ata?gat?tta?aaa?ggc?gtc?aca?gat?aaa?aac 90

Thr?Lys?His?Asp?Tyr?Ile?Asp?Leu?Lys?Gly?Val?Thr?Asp?Lys?Asn

16 20 25 30

cta?cct?att?gca?aat?caa?ctc?gaa?ttt?tca?aca?ggt?acc?aat?gat 135

Leu?Pro?Ile?Ala?Asn?Gln?Leu?Glu?Phe?Ser?Thr?Gly?Thr?Asn?Asp

31 35 40 45

ttg?atc?tca?gaa?tct?aat?aat?tgg?gac?gaa?ata?agt?aaa?ttt?aaa 180

Leu?Ile?Ser?Glu?Ser?Asn?Asn?Trp?Asp?Glu?Ile?Ser?Lys?Phe?Lys

46 50 55 60

gga?aag?aaa?ctg?gat?att?ttt?ggc?att?gat?tat?aat?ggt?cct?tgt 225

Gly?Lys?Lys?Leu?Asp?Ile?Phe?Gly?Ile?Asp?Tyr?Asn?Gly?Pro?Cys

61 65 70 75

aaa?tct?aaa?tac?atg?tat?gga?ggg?gcc?act?tta?tca?gga?caa?tac 270

Lys?Ser?Lys?Tyr?Met?Tyr?Gly?Gly?Ala?Thr?Leu?Ser?Gly?Gln?Tyr

76 80 85 90

tta?aat?tct?gct?aga?aaa?atc?cct?att?aat?ctt?tgg?gtt?aat?ggc 315

Leu?Asn?Ser?Ala?Arg?Lys?Ile?Pro?Ile?Asn?Leu?Trp?Val?Asn?Gly

91 95 100 105

aaa?cat?aaa?aca?att?tct?act?gac?aaa?ata?gca?act?aat?aaa?aaa 360

Lys?His?Lys?Thr?Ile?Ser?Thr?Asp?Lys?Ile?Ala?Thr?Asn?Lys?Lys

106 110 115 120

cta?gta?aca?gct?caa?gaa?att?gat?gtt?aaa?tta?aga?aga?tat?ctt 405

Leu?Val?Thr?Ala?Gln?Glu?Ile?Asp?Val?Lys?Leu?Arg?Arg?Tyr?Leu

121 125 130 135

caa?gaa?gaa?tac?aat?ata?tat?ggt?cat?aat?aac?act?ggt?aaa?ggc 450

Gln?Glu?Glu?Tyr?Asn?Ile?Tyr?Gly?His?Asn?Asn?Thr?Gly?Lys?Gly

136 140 145 150

aaa?gaa?tat?gga?tat?aaa?tct?aaa?ttt?tat?tca?ggt?ttt?aat?aat 495

Lys?Glu?Tyr?Gly?Tyr?Lys?Ser?Lys?Phe?Tyr?Ser?Gly?Phe?Asn?Asn

151 155 160 165

ggg?aaa?gtt?tta?ttt?cat?tta?aat?aat?gaa?aaa?tca?ttt?tca?tat 540

Gly?Lys?Val?Leu?Phe?His?Leu?Asn?Asn?Glu?Lys?Ser?Phe?Ser?Tyr

166 170 175 180

gat?ttg?ttt?tat?aca?gga?gat?gga?ctg?cct?gta?agt?ttt?ttg?aaa 585

Asp?Leu?Phe?Tyr?Thr?Gly?Asp?Gly?Leu?Pro?Val?Ser?Phe?Leu?Lys

181 185 190 195

att?tat?gaa?gat?aat?aaa?ata?ata?gaa?tct?gaa?aaa?ttt?cat?ctt 630

Ile?Tyr?Glu?Asp?Asn?Lys?Ile?Ile?Glu?Ser?Glu?Lys?Phe?His?Leu

196 200 205 210

gat?gtc?gaa?ata?tca?tat?gta?gat?agt?aac 660

Asp?Val?Glu?Ile?Ser?Tyr?Val?Asp?Ser?Asn

211 215 220

<210>2

<211>654

<212>DNA

＜213〉staphylococcus aureus (Staphylococcus aureus)

<220>

<221>CDS

<222>(1)...(654)

＜223〉clone's gained staphylococcus aureus enterotoxin 1

<440>2

caa?ggt?gat?att?ggt?gta?ggt?aac?tta?aga?aat?ttc?tat?aca?aaa 45

Gln?Gly?Asp?Ile?Gly?Val?Gly?Asn?Leu?Arg?Asn?Phe?Tyr?Thr?Lys

1 5 10 15

cat?gat?tat?ata?gat?tta?aaa?ggc?gtc?aca?gat?aaa?aac?cta?cct 90

His?Asp?Tyr?Ile?Asp?Leu?Lys?Gly?Val?Thr?Asp?Lys?Asn?Leu?Pro

16 20 25 30

att?gca?aat?caa?ctc?gaa?ttt?tca?aca?ggt?acc?aat?gat?ttg?atc 135

Ile?Ala?Asn?Gln?Leu?Glu?Phe?Ser?Thr?Gly?Thr?Asn?Asp?Leu?Ile

31 35 40 45

tca?gaa?tct?aat?aat?tgg?gac?gaa?ata?agt?aaa?ttt?aaa?gga?aag 180

Ser?Glu?Ser?Asn?Asn?Trp?Asp?Glu?Ile?Ser?Lys?Phe?Lys?Gly?Lys

46 50 55 60

aaa?ctg?gat?att?ttt?ggc?att?gat?tat?aat?ggt?cct?tgt?aaa?tct 225

Lys?Leu?Asp?Ile?Phe?Gly?Ile?Asp?Tyr?Asn?Gly?Pro?Cys?Lys?Ser

61 65 70 75

aaa?tac?atg?tat?gga?ggg?gcc?act?tta?tca?gga?caa?tac?tta?aat 270

Lys?Tyr?Met?Tyr?Gly?Gly?Ala?Thr?Leu?Ser?Gly?Gln?Tyr?Leu?Asn

76 80 85 90

tct?gct?aga?aaa?atc?cct?att?aat?ctt?tgg?gtt?aat?ggc?aaa?cat 315

Ser?Ala?Arg?Lys?Ile?Pro?Ile?Asn?Leu?Trp?Val?Asn?Gly?Lys?His

91 95 100 105

aaa?aca?att?tct?act?gac?aaa?ata?gca?act?aat?aaa?aaa?cta?gta 360

Lys?Thr?Ile?Ser?Thr?Asp?Lys?Ile?Ala?Thr?Asn?Lys?Lys?Leu?Val

106 110 115 120

aca?gct?caa?gaa?att?gat?gtt?aaa?tta?aga?aga?tat?ctt?caa?gaa 405

Thr?Ala?Gln?Glu?Ile?Asp?Val?Lys?Leu?Arg?Art?Tyr?Leu?Gln?Glu

121 125 130 135

gaa?tac?aat?ata?tat?ggt?cat?aat?aac?act?ggt?aaa?ggc?aaa?gaa 450

Glu?Tyr?Asn?Ile?Tyr?Gly?His?Asn?Asn?Thr?Gly?Lys?Gly?Lys?Glu

136 140 145 150

tat?gga?tat?aaa?tct?aaa?ttt?tat?tca?ggt?ttt?aat?aat?ggg?aaa 495

Tyr?Gly?Tyr?Lys?Ser?Lys?Phe?Tyr?Ser?Gly?Phe?Asn?Asn?Gly?Lys

151 155 160 165

gtt?tta?ttt?cat?tta?aat?aat?gaa?aaa?tca?ttt?tca?tat?gat?ttg 540

Val?Leu?Phe?His?Leu?Asn?Asn?Glu?Lys?Ser?Phe?Ser?Tyr?Asp?Leu

166 170 175 180

ttt?tat?aca?gga?gat?gga?ctg?cct?gta?agt?ttt?ttg?aaa?att?tat 585

Phe?Tyr?Thr?Gly?Asp?Gly?Leu?Pro?Val?Ser?Phe?Leu?Lys?Ile?Tyr

181 185 190 195

gaa?gat?aat?aaa?ata?ata?gaa?tct?gaa?aaa?ttt?cat?ctt?gat?gtc 630

Glu?Asp?Asn?Lys?Ile?Ile?Glu?Ser?Glu?Lys?Phe?His?Leu?Asp?Val

196 200 205 210

gaa?ata?tca?tat?gta?gat?agt?aac 654

Glu?Ile?Ser?Tyr?Val?Asp?Ser?Asn

211 215 218

<210>3

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplification contains the used forward primer of staphylococcus aureus enterotoxin 1 gene of restriction enzyme site from the staphylococcus aureus gene group

<440>3

gta?gga?tcc?caa?ggt?gat?att?ggt?gta?ggt

<210>4

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplification contains the used downstream primer of staphylococcus aureus enterotoxin 1 gene of restriction enzyme site from the staphylococcus aureus gene group

<440>4

gga?ctc?gag?tta?gtt?act?atc?tac?ata?tga

Claims (5)

1. recombined staphylococcus aureus enterotoxin I oral preparation, described recombined staphylococcus aureus enterotoxin I belongs to superantigen albumen, it is characterized in that: SEQ ID NO.1 is the aminoacid sequence of this recombined staphylococcus aureus enterotoxin I, that express this recombined staphylococcus aureus enterotoxin I is recombiant plasmid pGEX-4T-1-SEI, 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. recombined staphylococcus aureus enterotoxin I oral preparation according to claim 1 is characterized in that described recombined staphylococcus aureus enterotoxin I obtains by following steps:

(1) design has the upstream and downstream primer that is used to clone staphylococcus aureus enterotoxin 1 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, the 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 I albumen mature peptide of coding, it is connected into multiple clone site on the pGEM-T plasmid vector, make up the pGEM-T-SEI 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-SEI plasmid, pcr amplification obtains the gene order that two ends have the coding gold staphylococcal enterotoxin I 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-SEI;

(4) the pGEX-4T-1-SEI expression plasmid is converted into e. coli bl21, the reorganization GST-SEI 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 I of reorganization successively.

3. recombined staphylococcus aureus enterotoxin I 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. recombined staphylococcus aureus enterotoxin I 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 recombined staphylococcus aureus enterotoxin I oral preparation according to claim 1 in preparation treatment malignant tumor and other severe complication medicines.

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