CN1900119A

CN1900119A - Staphylococcus aureus enterotoxin 1 and preparation and use

Info

Publication number: CN1900119A
Application number: CN 200610052649
Authority: CN
Inventors: 丁丁; 李丹曦; 潘映秋; 陈枢青
Original assignee: Zhejiang University ZJU
Current assignee: Hangzhou Minsheng Pharmaceutical Co Ltd
Priority date: 2006-07-26
Filing date: 2006-07-26
Publication date: 2007-01-24

Abstract

The present invention provides a kind of recombinant staphylococcus aureus enterotoxin I prepared through genetic engineering process, and the protein is one kind of super antigen with the amino acid sequence of SEQ ID No. 1. The recombinant plasmid is reconstructed with pGEX-4T-1 and the nucleotide sequence of SEQ ID No. 2. Extracorporeal experiment shows that the prepared high purity recombinant SEI protein can excite the splenic lymphopoiesis of mouse effectively in dosage depending relationship and the excited splenic lymphocyte of the mouse has obvious tumor cell killing effect. Compared with available staphylococcus aureus enterotoxin C as the main effective component in the staphylococcus aureus filtrate preparation, the recombinant SEI protein has even high super antigen activity and may be prepared into super antigen preparation for inhibiting tumor growth effectively.

Description

Staphylococcus aureus enterotoxin 1 and preparation and application

Technical field

The invention belongs to biotechnology, relate to a kind of staphylococcus aureus enterotoxin 1 (StaphylococcalEnterotoxin I, preparation SEI) and be used to stimulate lymphopoiesis, suppress growth of tumour cell.Specifically, be used to come from gene and the reorganization of a kind of plasmid vector of the coding SEI of streptococcus aureus exactly, and it is converted into suitable host expresses, obtain highly purified reorganization SEI albumen by affinity purification, and utilize its superantigen activity to be applied to lymphopoiesis and tumour cell inhibition, and carry out specific activity with the present Staphylococcus aureus enterotoxin C (SEC) of the main effective constituent of golden Portugal's bacterium filtrate preparation of use clinically of conduct.

Background technology

1989, the notion of superantigen is proposed by Sweden scientist White, it is a class has the protein of powerful stimulatory function by what bacterium, virus, parasite produced to lymphocyte and since its to the lymphocytic activation capability of T be common antigen 2000-50000 doubly, so be called superantigen.(Staphylococcal Enterotoxin SE) is a kind of superantigen of broad research in the present worldwide to Staphylococcus aureus enterotoxin.The most deep A type Staphylococcus aureus enterotoxin (the StaphylococcalEnterotoxin A that mainly comprises of research wherein, SEA), Type B Staphylococcus aureus enterotoxin (Staphylococcal EnterotoxinB, SEB), C2 type Staphylococcus aureus enterotoxin (Staphylococcal Enterotoxin C2, SEC2) etc.

Different with common antigen, the enterotoxin superantigen at first is incorporated into the antigen of the surperficial II class major histocompatibility complex (MHC) of antigen presenting cell (APC) in conjunction with the groove outside with complete molecule, then combine, form a large amount of activated T cells behind SE-MHC-TCR three molecular complexes with the V β district of the antigen receptor molecule (TCR) of T cell surface.But the activated T cells direct killing is expressed the tumour cell of MHC-II quasi-molecule thus, and also can produce indirect lethal effect to the tumour cell of not expressing the MHC-II quasi-molecule.Simultaneously, by SE activated CD4 ⁺The T cell can be secreted a large amount of cytokines, as IL-2, IFN-γ, TNF-α etc., tumour cell is had the intensive lethal effect.The factors such as IL-2 also can activate the NK cell, make its performance natural killer effect, and cytokines such as IFN-γ, TNF-α also can promote the expression of the MHC-II quasi-molecule of tumour cell, strengthen the identification of T cell.Therefore, can produce direct or indirect efficiently lethal effect to tumour cell and tumor tissues through the SE activated T cells.Confirm efficiently to induce human bladder cancer cell's strain RT112 cell and RT4 natural death of cerebral cells as people such as Perabo through SEB stimulated peripheral mononuclear cells (PBMC).Experiment shows that the SEA activated T cell makes it proliferation function and be dose-dependence in every mouse 0.1-100 μ g scope in the body that people such as Hedlund carry out, and injects to occur maximum effect in back 1 day, and multiplication effect was kept 4 days.For improving target and the specificity in oncotherapy, people are at different tumor cell surface specific antigenss, prepared the monoclonal antibody-superantigen fusion rotein of multiple related antigen or with monoclonal antibody and superantigen coupling, strengthened the target of institute's activated T cells greatly to tumour cell and tumor tissues, make its killing tumor cell specifically, suppress tumor growth.In addition, people also utilize the part of the acceptor of tumor cell surface overexpression to prepare part-superantigen fusion rotein, have improved thus activated T cells effectively to the target of tumor tissues.In addition, with superantigen gene transfection tumour cell, the tumour cell that superantigen is modified has also obtained the effect that gratifying opposing tumour cell is attacked once more as knurl seedling immune animal then.

In China, the ancillary drug for the treatment of as the anti-chemotherapy of tumour for the filter preparation of staphylococcus aureus of main effective constituent with Staphylococcus aureus enterotoxin C (SEC) is applied to clinical existing nearly ten days, a large amount of clinical data proofs uses the immunological competence of back tumour patient to obtain significant raising, and patient's appetite, sleep and overall health of patients have all obtained being clearly better.As Luo Feng etc. 35 routine shallow table metastatic tumo(u)rs are used golden Portugal bacterium filtrate preparation and carry out topical therapeutic, treat that total effective rate reaches 82.9% after one month.Tang Wei etc. by early stage oral carcinoma is adopted the treatment of local golden Portugal bacterium filtrate preparation and to late period oral carcinoma use golden Portugal bacterium filtrate preparation combined chemotherapy, find that it can make big amount lymphocyte and fibrous tissue accumulate near the cancer nests, and can observe tumour cell significantly minimizing even the disappearance of some cases tumour cell.Wu Jie etc. are applied to esophageal carcinoma therapy with golden Portugal bacterium filtrate preparation as the ancillary drug of chemotherapy, and the result shows and compare with control group that obviously leukocyte increasing alleviates gastrointestinal reaction.

Although be that the golden Portugal bacterium filtrate preparation of main effective constituent has obtained widely and uses at present with SEC, but owing to exist amounts of protein and polypeptide class impurity in the preparation, and main effective constituent enterotoxin relative content is extremely low, makes a part of tumour patient similar side reaction to a certain degree occur in the clinical use of said preparation.Reported have 28% and 24% tumour patient side reactions such as heating and local pain to occur using golden Portugal bacterium filtrate preparation and unite in the carboplatin treatment malignant pleural effusion process respectively as, Li Hong etc.Observed major side effects in the process of the nasopharyngeal carcinoma patient being used the treatment of golden Portugal bacterium filtrate preparation combined radiotherapy such as Liu Xiuying be in, low heating, account for 5% of treatment group patient sum.The Li Jia congruence has also been reported the patient and has been treated the major side effects that occurs in the pernicious hydrothorax process for heating using golden Portugal bacterium filtrate preparation combination with cisplatin, accounts for 65.5% of treatment group patient sum.Though these side reactions are temporary substantially, symptom can spontaneous remission or is eliminated by symptomatic treatment, but still the treatment and the rehabilitation of tumour patient caused bad influence.Therefore, the problem that in clinical application, runs into for present golden Portugal bacterium filtrate preparation, need the highly purified enterotoxin of preparation and set up strict controlled quality standard progressively to overcome solution, in the hope of reducing the toxic side effect that in use produces and strengthening its tumor suppression curative effect.At present, the someone utilizes genetic engineering means to prepare the Staphylococcus aureus enterotoxin superantigen, as Jiang Yongqiang etc. with SEA, SEB, SEC1 gene fragment clone to pBV220, in intestinal bacteria, obtain to express through heat-inducible, but purification step has all adopted the method for ion-exchange in these methods, exists absorption to select weakness such as poor specificity.Xu Mingkai etc. express the SEC2 gene clone to pET-28a, but the recombinant products of expressing increases by 36 amino acid than native protein, so the possibility of antigenic determinant and its lytic activity change is all bigger.

Summary of the invention

An object of the present invention is to provide a kind of recombined staphylococcus aureus enterotoxin I that utilizes genetic engineering means to prepare (Staphylococcal Enterotoxin I, SEI), this albumen belongs to a kind of superantigen, has the aminoacid sequence of SEQ ID NO.1.The present invention utilize its superantigen activity be applied to external short spleen lymphocyte proliferation and and extracorporeal suppression tumor cell growth, and with Staphylococcus aureus enterotoxin C (Staphylococcal Enterotoxin C SEC) carries out the superantigen specific activity.

Another object of the present invention provides the preparation method of recombined staphylococcus aureus enterotoxin I (SEI), the present invention makes up a kind of recombinant plasmid that can be used for expressing staphylococcus aureus enterotoxin 1, this plasmid is formed through recombination to construct by the nucleotide sequence of pGEX-4T-1 and SEQ ID NO.2, and plasmid is seen Figure of description 6.

The inventive method specifically realizes by following steps:

(1) at first designs a pair of upstream and downstream primer: SEQ ID NO.3:5 '-caa tca taa ctt agt aaa ggaaat gcc-3 ' (primer is drawn in the upstream), SEQ ID NO.4:5 '-acg tac aca tta gcc cta gag act t-3 ' (downstream primer), with streptococcus aureus (FRI 100) genome is template, adopt pcr amplification to obtain the proteic gene fragment of coding SEI, utilize T-A clone that it is connected into multiple clone site to the pGEM-T plasmid vector, successfully make up the pGEM-T-SEI recombinant plasmid.The gene order (AY920267) of the coding same protein in the proteic gene fragment order of coding SEI (seeing SEQ ID NO.2) that confirm to obtain by order-checking and the GenBank database is in full accord.

(2) design has the upstream and downstream primer of BamH I and Xho I restriction enzyme site respectively: SEQ ID NO.5:5 '-gta Gga tccCaa ggt gat att ggt gta ggt-3 ' (upstream primer, the line part is a BamH I restriction enzyme site), SEQ ID NO.6:5 '-gga Ctc gagTta gtt act atc tac ata tga-3 ' (downstream primer, the line part is an Xho I restriction enzyme site), with the pGEM-T-SEI plasmid is template, adopt pcr amplification to obtain the proteic gene order of coding enterotoxin SEI that two ends have restriction enzyme site, this gene fragment is cut the back and is connected successful construction expression plasmid pGEX-4T-1-SEI with the pGEX-4T-1 plasmid after the identical restriction endonuclease processing with corresponding restriction endonuclease.

(3) the pGEX-4T-1-SEI expression plasmid is converted into e. coli bl21 (DE3), the GST-SEI fusion rotein of abduction delivering band GST label.Centrifugal and the collection supernatant in results thalline and broken back.With the affine filler thorough mixing of thalline supernatant liquor with energy specific combination GST, with elutriant wash-out GST-SEI fusion rotein and collection, with fusion rotein and zymoplasm fully reaction under conditions suitable of collecting, post reaction mixture once more with can specific combination the affine filler thorough mixing of GST, separation is also collected purified SEI albumen, the sampling electrophoresis detection.Energy characteristic ground is in conjunction with the affine filler of GST, and preferred coupling has the Glutathione Sepharose 4Fast Flow of gsh.After obtaining electrophoretically pure purified product after testing, this purified product is promptly got the SEI lyophilized powder of recombinating through desalination, lyophilize.

The initial albumen that obtains utilizes affine this fusion rotein of filler purifying earlier for having the fusion rotein GST-SEI of GST (Thiadiazolidine isomerase) label, utilizes affine filler purification of Recombinant SEI albumen once more after further removing this label.

The initial GST-SEI fusion rotein that obtains accounts for the 15-25% of total bacterial protein content.

Another purpose of the present invention provides recombined staphylococcus aureus enterotoxin I to be stimulated lymphopoiesis in preparation, suppresses the application in the growth of tumour cell medicine.

After preparing electrophoretically pure SEI albumen by previous methods, adopt mtt assay, with the positive contrast of mitogen ConA, set up suitable external model to observe reorganization SEI albumen to the proliferation function of ICR mouse spleen lymphocyte and be subjected to mouse spleen lymphocyte that SEI stimulates proliferation lethal effect to tumour cell.Using this model, to observe the superantigen of Staphylococcus aureus enterotoxin C (SEC) simultaneously active and compare with the proteic activity of reorganization SEI, and the result shows that reorganization SEI albumen has typical superantigen activity, and stronger than the effect of SEC.

The characteristics of the inventive method are: (1) provides a kind of highly purified reorganization SEI albumen, and utilize external mice spleen lymphocytes proliferation experiment and tumour cell to suppress this recombinant protein of experiment confirm and have typical superantigen activity, and stronger than SEC.This recombinant protein has the GST label before not by the zymoplasm cutting, be fit to affinity purification, this protein amino acid sequence after Thrombin treatment and corresponding nucleotide sequence are seen SEQ ID NO.1, compare with corresponding native protein, and this recombinant protein has increased by 2 amino-acid residues at the N end; (2) provide a kind of recombinant expression plasmid pGEX-4T-1-SEI of the SEI of containing gene, 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, it contains strong promoter, induces down at inductor to efficiently express the GST-SEI fusion rotein that has the GST label; (3) use the engineering bacteria contain the expression plasmid that the nucleotide construction by pGEX-4T-1 and SEQ ID NO.2 forms, expression product has the GST label, and use contain can specific combination GST affine filler come this product of purifying; (4) provide a kind of engineering bacteria, this bacterial strain contains expression plasmid pGEX-4T-1-SEI, can efficiently express the GST-SEI fusion rotein that has the GST label under suitable inductor effect.(5) be applicable to that efficient production has the exploitation of active high purity enterotoxin of superantigen and superantigen preparation, is used for tumour patient clinical rehabilitation and treatment.

The inventive method efficiently expresses reorganization SEI, and expression intensity accounts for about the 15-25% of whole bacterial protein, and is solubility expression, is convenient to the later separation purifying.Fusion rotein is after the efficient cutting of zymoplasm, and resulting recombinant protein is compared with corresponding native protein, only has more 2 amino acid at the N end.The inventive method adopts affinity chromatography that target protein is carried out purifying, it is simple to have step, the fireballing characteristics of purifying are compared from golden Portugal bacterium culturing filtrate separation and Extraction and are utilized ion exchange chromatography or method such as sieve chromatography can obtain highly purified reorganization SEI albumen.This albumen of external application stimulates mice spleen lymphocytes proliferation and suppresses growth of tumour cell, and the result proves that this target protein has typical superantigen activity.In view of the main effective constituent of the golden Portugal bacterium filtrate preparation of using in clinical acquisition is SEC, the present invention is stronger than SEC by relatively confirming the proteic superantigen activity of reorganization SEI, and therefore this reorganization SEI albumen is expected to develop and becomes clinical rehabilitation and the treatment that a kind of new superantigen preparation is used for tumour patient.

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 operation instructions that supplier advised according to the normal experiment method.

Description of drawings

Fig. 1 is pcr amplification gained coding SEI gene agarose gel electrophoresis figure, and wherein swimming lane 1: nucleic acid Marker, swimming lane 2: coding SEI protein gene PCR product.

Fig. 2 is surveyed coding SEI gene order sequencing result figure after by pcr amplification.

Fig. 3 is reorganization pGEM-T-SEI plasmid double digestion agarose gel electrophoresis figure, and wherein swimming lane 1: nucleic acid Marker, swimming lane 2: reorganization pGEM-T-SEI plasmid, swimming lane three: reorganization pGEM-T-SEI plasmid Nco I and Pst I double digestion product.

Fig. 4 contains the coding SEI albumen mature peptide gene agarose gel electrophoresis figure of restriction enzyme site for the pcr amplification gained, and wherein swimming lane 1: nucleic acid Marker, swimming lane 2: the coding SEI albumen mature peptide gene PCR product that contains restriction enzyme site.

Fig. 5 is reorganization pGEX-4T-1-SEI plasmid double digestion agarose gel electrophoresis figure, and wherein swimming lane 1: nucleic acid Marker, swimming lane 2: reorganization pGEX-4T-1-SEI plasmid, swimming lane three: reorganization pGEX-4T-1-SEI plasmid BamH I and Xho I double digestion product.

Fig. 6 is reorganization SEI expression plasmid pGEX-4T-1-SEI synoptic diagram, and " sei " of sign is coding SEI protein gene sequence on position.

Fig. 7 is reorganization SEI abduction delivering electrophorogram in BL21 (DE3) intestinal bacteria, and wherein swimming lane 1: protein Marker, swimming lane 2: through the bacterial protein of abduction delivering.

Fig. 8 is reorganization SEI purifying electrophorogram, and wherein swimming lane 1: protein Marker, swimming lane 2: the reorganization SEI albumen behind the purifying, swimming lane 3: the GST-SEI fusion rotein behind the purifying, GST, SEI mixing solutions after the swimming lane 4:Thrombin cutting.

Fig. 9 for reorganization SEI and SEC to the proliferation function (48h) of ICR mouse spleen lymphocyte, compare with the blank group: ^*: P＜0.01.

Figure 10 for reorganization SEI and SEC to the chronic marrow originality of adriamycin-resistant leukemia cell's (K562-AD) restraining effect (48h), compare with the blank group: ^{* *}: P＜0.001.

Embodiment

Following examples provide realization the preferred embodiments of the invention.It will be appreciated by persons skilled in the art that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.Disclosed technology is represented the technology found by the inventor in the following examples, and these technology can effectively be implemented the present invention.But, it will be understood by those skilled in the art that under the prerequisite that does not exceed design of the present invention, can carry out multiple change, still can obtain similar result these specific embodiments.

The gene clone of embodiment 1:SEI and pGEM-T-SEI construction of recombinant plasmid

The pcr amplification of the proteic full-length gene order of coding SEI: design following a pair of primer sequence:

SEQ ID NO.3:(upstream primer) 5 '-caa tca taa ctt agt aaa gga aat gcc-3 '

SEQ ID NO.4:(downstream primer) 5 '-acg tac aca tta gcc cta gag act t-3 '

With streptococcus aureus (FRI 100) genomic templates, carry out pcr amplification according to following condition, amplification obtains the dna fragmentation of 852bp, PCR as a result gel electrophoresis figure referring to Fig. 1.

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?FRI?100，10ng/μL)： 1μL

Taq?Polymerase(5U/μL)： 1μL

The PCR program:

1,95 ℃ of pre-sex change 3min

2,95 ℃ of sex change 30s, 55 ℃ of annealing 60s, 72 ℃ are extended 1min

3, according to step 2 circulation 34 times

4,72 ℃ are extended 10min

The pGEM-T-SEI construction of recombinant plasmid that contains the proteic full-length gene order of coding SEI: the SEI full-length gene of pcr amplification gained is cloned into the pGEM-T plasmid, is converted in the bacillus coli DH 5 alpha and increases.Extract this recombinant plasmid, cut through Nco I and Pst I enzyme and identify that the back sample presentation checks order, the result shows that the sequence (AY920267) of coding same protein of proteic full length gene sequence of the coding SEI of acquisition and GenBank database is in full accord.Sequencing result is referring to Fig. 2.The pGEM-T-SEI plasmid enzyme restriction is identified referring to Fig. 3.

The pcr amplification that contains the coding SEI mature peptide gene order of restriction enzyme site: design following a pair of primer sequence:

SEQ ID NO.5 (upstream primer, the line part is a BamH I restriction enzyme site): 5 '-gta Gga tccCaaggt gat att ggt gta ggt-3 ',

SEQ ID NO.6 (downstream primer, the line part is an Xho I restriction enzyme site): 5 '-gga Ctc gagTta gttact atc tac ata tga-3 ',

As template, carry out pcr amplification according to following condition with the pGEM-T-SEI recombinant plasmid that contains the SEI full-length gene, amplification obtains the dna fragmentation of 675bp, and PCR result is referring to Fig. 4.

The PCR system:

H ₂O： 70μL

Buffer(10×)： 10μL

Mg ²⁺(25mmol/L)： 8μL

Primer-up(25μmol/L)： 4μL

Primer-down(25μmol/L)： 4μL

dNTP(20mmol/L)： 2μL

Template(pGEM-T-SEI)： 1μL

Taq?Polymerase(5U/μL)： 1μL

The PCR program:

1,95 ℃ of pre-sex change 3min

2,95 ℃ of sex change 30s, 55 ℃ of annealing 60s, 72 ℃ are extended 1min

3, according to step 2 circulation 30 times

4,72 ℃ are extended 10min

The structure that contains the pGEX-4T-1-SEI recombinant expression plasmid of coding SEI mature peptide gene: with BamHI and Xho I respectively enzyme cut the gene fragment and the pGEX-4T-1 plasmid of the coding SEI albumen mature peptide that contains restriction enzyme site that obtains in the above-mentioned steps.The gene fragment of the coding SEI mature peptide that contains restriction enzyme site after reclaiming enzyme respectively and cutting and the big fragment of pGEX-4T-1 plasmid enzyme restriction product, and both are connected, made up expression plasmid pGEX-4T-1-SEI.This recombinant plasmid is changed over to bacillus coli DH 5 alpha amplification and extracts plasmid, cut evaluation through BamH I and Xho I enzyme, the result shows that target gene fragment inserted vector plasmid, and electrophoresis result is referring to Fig. 5.PGEX-4T-1-SEI plasmid synoptic diagram is referring to Fig. 6.The detailed sequence of goal gene on the pGEX-4T-1-SEI plasmid seen SEQ ID NO.1.

Embodiment 2: the expression of reorganization SEI

The structure of SEI expression strain: from the bacillus coli DH 5 alpha that contains the pGEX-4T-1-SEI recombinant plasmid, extract this plasmid, be converted in the e. coli bl21 (DE3), by the antibiotics resistance screening positive clone, the engineering bacterial strain that acquisition can great expression GST-SEI fusion rotein.

The expression of fusion rotein GST-SEI: above-mentioned engineering bacteria list colony inoculation is contained in the LB substratum of penbritin in 5mL, and 37 ℃ of shaking culture 6h are as seed liquor.The inoculum size of this seed liquor with 1-5% is inoculated in the 2 * YT substratum that contains penbritin, 37 ℃ of shaking culture 4h, the volume ratio of pressing 0.01%-0.1% adds 0.1mol/L IPTG abduction delivering 5h.

Embodiment 3: the purifying of reorganization SEI

The pretreatment: 4 ℃ of 10000rpm are centrifugal through IPTG inductive bacterium liquid, abandon the supernatant collecting precipitation.Precipitation is resuspended with the PBS of 1/10 amount of original bacteria liquid volume, and suspension is put in the FRENCH cytoclasis instrument, and broken down in 700psi, it is thick that suspension is.Ultrasonication 2min is continued with Ultrasonic Cell Disruptor in the back makes nucleolysis, and the cellular lysate fluid viscosity reduces.The Triton-100 of ultrasonic back adding 20% in cellular lysate liquid is 1% to final concentration, abundant mixing, and ice bath leaves standstill 30min.Leave standstill finish after with 4 ℃ of centrifugal 30min of 12000rpm of cellular lysate liquid, it is stand-by to get the supernatant liquor cryopreservation.Supernatant liquor sampling carrying out SDS-PAGE detects, and at molecular weight 50kD place denseer band is arranged, and this is the GST-SEI fusion rotein of abduction delivering.Quantity One image analysis software shows that this band concentration accounts for the 15-25% of total protein concentration greatly.Cellular lysate liquid eggs white appliances swimming result is referring to Fig. 7 after inducing.

The purifying of GST-SEI fusion rotein: get 1mL through pretreated protein solution, be added to behind 0.45 μ m membrane filtration in the 1mL Glutathione Sepharose 4Fast Flow post of pre-equilibration, mixing gently is in conjunction with flowing to end liquid behind the 30min.Wash pillar 3 times with PBS, each 10mL flows to end liquid.Xiang Zhuzhong adds 0.5mL reduced glutathion elutriant, mixing gently.Collect solution 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, and the result shows that protein concentration is normal distribution in each eluant component.Each is collected component check purity and concentration with SDS-PAGE, the result shows that the GST-SEI fusion rotein that purifying obtains is single band, the about 50kD of molecular weight, and electrophoresis result is referring to Fig. 8.

The cutting of fusion rotein: the GST-SEI fusion rotein collection component that above-mentioned purifying is obtained merges, and dialyses through desalting column or with the dialysis band of 10000 molecular weight cut-offs, solution is replaced as contain 1.5mol/LNaCl, 2.5mmol/LCaCl ₂Zymoplasm buffered soln and remove gsh in the solution simultaneously.Every 1mL protein solution adds 20mg/mL zymoplasm 2 μ L, 25 ℃ of reaction 24h, and the sampling electrophoresis detects the endonuclease reaction degree, and enzyme is cut and be the results are shown in Figure 8.

The purifying of reorganization SEI: reacted protein solution is added in the GlutathioneSepharose 4Fast Flow pillar of pre-equilibration, mixing gently leaves standstill and liquid is flow to end behind the 20min and collect.SDS-PAGE detects purity of protein and concentration in the solution.The result shows that the GST label of fusion rotein removes, and single band is arranged near the 26KD molecular weight, is the SEI albumen of purifying, and freeze-drying after the protein solution desalination behind the purifying is preserved.SEI purifying protein electrophoresis result is referring to Fig. 8.

Embodiment 4:MTT method is measured the proliferation function of reorganization SEI to the ICR mouse spleen lymphocyte

Splenic lymphocyte with the ICR mouse is a target cell, and it is suspended in the RPMIM1640 nutrient solution that contains 10% calf serum, and adjusting cell concn is 5 * 10 ⁶Individual/mL, every hole 100 μ L are laid in 96 orifice plates, if zeroing hole (only containing substratum), blank well (splenic lymphocyte+substratum), positive control hole (substratum+splenic lymphocyte+Con A) and experimental port (SEI of substratum+splenic lymphocyte+three a different concns gradient) are established four multiple holes for every kind.Wherein, Con A final concentration is 10 μ g/mL, and the SEI final concentration is respectively 1 μ g/mL, 0.1 μ g/mL and 0.01 μ g/mL.Treating to add in the gaging hole MTT solution in 10 μ L/ holes in 44h behind the bed board, behind the careful piping and druming mixing, after continuing in the incubator to cultivate 4h, carefully absorb supernatant, and add the DMSO in 120 μ L/ holes, place 10min in the incubator behind the piping and druming hydrotropy, (570nm is for measuring wavelength for microplate reader double wave regular way, 630nm is a reference wavelength) measure each hole OD570nm-OD630nm value, mapping analysis effect enterotoxin is to lymphocytic proliferation function, and does statistical study with student t check.

The absorbancy in each hole is compared with control group, and positive control Con A and 0.01-1 μ g/mL SEI ICR mouse spleen lymphocyte behind effect 48h all has extremely significantly increase, and (P＜0.01 is simultaneously along with the increase action effect of drug level also more remarkable (referring to Fig. 9).Detect the proliferation function of SEC to the ICR mouse spleen lymphocyte by same experimental regime, through comparing, SEI is stronger than SEC to the effect of ICR mice spleen lymphocytes proliferation in reorganization.

Embodiment 5:MTT method is measured the external antitumor activity of reorganization enterotoxin SEI

If zeroing hole (only containing substratum), tumour cell control wells (substratum+tumour cell), lymphocyte background release aperture [contains blank well (substratum+splenic lymphocyte), positive control hole (substratum+splenic lymphocyte+Con A) and experimental port (SEI of substratum+splenic lymphocyte+four a different concns gradient)] and the tumor-inhibiting action hole [contain blank well (substratum+splenic lymphocyte+tumour cell), positive control hole (substratum+splenic lymphocyte+tumour cell+Con A) and experimental port (SEI of substratum+splenic lymphocyte+tumour cell+four a different concns gradient)], establish three multiple holes for every kind.Wherein, Con A final concentration is 10 μ g/mL, and the SEI final concentration is respectively 1 μ g/mL, 0.1 μ g/mL, 0.01 μ g/mL and 0.001 μ g/mL, in the background release aperture 5 * 10 ⁶Individual/mL splenic lymphocyte adds with 100 μ L/ holes, in the function of tumor hole 5 * 10 ⁶Individual/mL splenic lymphocyte and 2.5 * 10 ⁵Individual/chronic marrow the originality of mL adriamycin-resistant leukemia cell's (K562-AD) mixing suspension joins in the experimental port with 100 μ L/ holes, at last by diluting medicine to requirement concentration with 10%FCS RPMI 1640 substratum, 50 μ L/ holes add each hole, and carefully blow and beat mixing.

44h 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 4h, the careful supernatant of absorbing, and add the DMSO in 120 μ L/ holes, and place 10min in the incubator behind the piping and druming hydrotropy, (570nm is for measuring wavelength for microplate reader double wave regular way, 630nm is a reference wavelength) measure each hole OD570nm-OD630nm value, only contain the zeroing hole zeroing of substratum.Be calculated as follows tumour inhibiting rate: tumour inhibiting rate (%)=100-[(tumor-inhibiting action hole-lymphocyte background release aperture)/the tumour cell control wells] * 100, the mapping analysis enterotoxin is to the restraining effect of tumour cell, and does statistical study with student t check.

Mtt assay is measured the external antitumor activity experiment of SEI (each three mouse), SEI does target cell with K562-AD, compare with the knurl blank hole that presses down of not dosing, the enterotoxin 1 of positive control Con A and 0.001-1mg/L tumour inhibiting rate behind effect 48h all has extremely significantly and increases (P＜0.001), simultaneously along with the increase action effect of drug level also more remarkable (referring to Figure 10).Detect SEC to the effect of K562-AD cell inhibiting by same experimental regime, through comparing, effect is better than SEC to reorganization SEI to the K562-AD cell inhibiting.

The sequence that the present invention relates to

＜110〉Zhejiang University

＜120〉staphylococcus aureus enterotoxin 1 and preparation and application

<160>6

<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?ctt?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〉streptococcus 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?ctt?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?Arg?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>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the used upstream primer of amplification staphylococcus aureus enterotoxin 1 gene from the staphylococcus aureus gene group

<440>3

caa?tca?taa?ctt?agt?aaa?gga?aat?gcc

<210>4

<211>25

<212>DNA

＜213〉artificial sequence

<220>

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

<440>4

acg?tac?aca?tta?gcc?cta?gag?act?t

<210>5

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉add the used upstream primer of restriction enzyme site to staphylococcus aureus enterotoxin 1 gene two ends

<440>5

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

<210>6

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉add the used downstream primer of restriction enzyme site to staphylococcus aureus enterotoxin 1 gene two ends

<440>6

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

Claims

1. staphylococcus aureus enterotoxin 1, this albumen is a kind of superantigen, has the aminoacid sequence of SEQ ID NO.1.

2. the preparation method of staphylococcus aureus enterotoxin 1 according to claim 1, specifically realize by following steps: (1) with the staphylococcus aureus gene group as template, carry out pcr amplification according to ordinary method, the target gene fragment sequence that confirm to obtain by gene sequencing and the gene order (AY920267) of the coding same protein in the GenBank database are in full accord, and the proteic gene order of SEI of will encoding is connected into the pGEM-T carrier; (2) add BamH I and Xho I restriction enzyme site and utilize subclone technique construction pGEX-4T-1-SEI recombinant plasmid at these gene two ends, after changing this plasmid over to intestinal bacteria, adopt ordinary method IPTG inducible protein to express " adopt ordinary method IPTG inducible protein to express; (3) utilizing can be specifically in conjunction with the affinity purification filler purification of Recombinant SEI albumen of GST, and the demonstration of electrophoresis qualification result has obtained highly purified reorganization SEI albumen; It is characterized in that:

The upstream and downstream primer that step (1) is used for amplification coding enterotoxin SEI gene is:

SEQ?ID?NO.3：5’-caa?tca?taa?ctt?agt?aaa?gga?aat?gcc-3’，

SEQ?ID?NO.4：5’-acg?tac?aca?tta?gcc?cta?gag?act?t-3’；

Step (2) the upstream and downstream primer that contains the coding enterotoxin SEI mature peptide gene that has BamH I and Xho I restriction enzyme site that is used to increase is: SEQ ID NO.5:5 ' gta Gga tccCaa ggt gat att ggt gtaggt-3 ', the line part is a BamH I restriction enzyme site; SEQ ID NO.6:5 ' gga Ctc gagTta gtt act atctac ata tga-3 ', the line part is an Xho I restriction enzyme site; The pGEX-4T-1-SEI recombinant plasmid is to be formed through recombination to construct by the nucleotide sequence of pGEX-4T-1 and SEQ ID NO.2;

The host that step (3) is fit to plasmid pGEX-4T-1-SEI prokaryotic expression system expressing protein selects e. coli bl21 for use.

3. the preparation method of staphylococcus aureus enterotoxin 1 according to claim 2, it is characterized in that: the initial albumen that obtains is the fusion rotein GST-SEI that has the Thiadiazolidine isomerase label, utilize affine this fusion rotein of filler purifying earlier, utilize affine filler purification of Recombinant SEI albumen once more after further removing this label.

4. the preparation method of staphylococcus aureus enterotoxin 1 according to claim 2 is characterized in that: the initial GST-SEI fusion rotein that obtains accounts for the 15-25% of total bacterial protein content.

5. the application of staphylococcus aureus enterotoxin 1 according to claim 1 in preparation stimulation lymphopoiesis medicine.

6. the application of staphylococcus aureus enterotoxin 1 according to claim 1 in preparation inhibition growth of tumour cell medicine.