CN101597612B - SEC2 mutant gene with increased activity of super-antigen and preparation method thereof - Google Patents
SEC2 mutant gene with increased activity of super-antigen and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a gene engineering technology, in particular to an SEC2 mutant gene with increased activity of super-antigen and a preparation method thereof. The SEC2 mutant gene is provided with a base sequence in a sequence table SEQ ID NO:1 and also provided with an amino acid sequence in a sequence table SEQ ID NO:2. The invention establishes the SEC2 mutant gene with remarkably enhanced super-antigen activity and antitumor effect for the first time by using a gene targeted mutagenesis technology and successfully realizes the soluble expression in escherichia coli. The invention can provide the gene engineering strain directly used for producing the mutant protein of the super-antigen.
Description
Technical field
The present invention relates to genetic engineering technique, is SEC2 mutator gene of a kind of superantigen increased activity and preparation method thereof specifically.
Background technology
Superantigen (superantigen, SAg) be one group of protein molecule by bacterium or encoding viral, it does not need the processing of antigen presenting cell (APC), and MHC II molecule direct with intact protein and on the cytolemma combines the formation mixture, (1～10ng/ml) can stimulate the T cell proliferation that has specificity V β in a large number, thereby causes extremely strong organism immune response and produce certain anti-tumor activity at extremely low concentration.Therefore, superantigen is expected to be developed to a kind of effective new type antineoplastic medicine as a kind of ideal immunomodulator and synergistic agent, is used for oncotherapy.
(Staphylococcal enterotoxins SEs) is the representative bacterial exotoxin of a class to Staphylococcus aureus enterotoxin.Because its extremely strong t cell activation function becomes a quasi-representative microorganism superantigen, is subjected to people's extensive attention.In recent years, people have carried out many bases and clinical study work to Staphylococcus aureus enterotoxin (SE) at aspects such as immunologic function and antitumor application, obtained challenging result, but it mainly concentrates on SEA, SEB etc.And SEC2 is also fewer to its research at anti-tumor aspect both at home and abroad as a member in the Staphylococcus aureus enterotoxin family.I first the gene of SEC2 has been carried out clone, order-checking and has carried out significant trial aspect the targent fused protein research, for the research and development of further carrying out Enteromycin C 2 (SEC2) molecular modification and targent fused protein thereof are laid a good foundation.
Existing studies show that, because Staphylococcus aureus enterotoxin belongs to a kind of bacterial endotoxin, therefore under the finite concentration condition, can cause food poisoning and toxin syndromes, thereby cause symptoms such as fever, vomiting, shock, limit the dosage use range of Staphylococcus aureus enterotoxin in clinical antineoplaston to a certain extent, influenced result of treatment.In addition, in recent years studies show that superantigen SEA and SEB all can produce immunosuppressive effect in vivo with in the experiment in vitro, and the use of high dosage and repeat administration all can cause the inactivation and the apoptosis of T cell.And the nearest SEC that studies show that also can induce the generation immunosuppressive effect, and immunosuppressive effect is certain dependence with dosage and time, and the SEC of high dosage can cause immunosuppression, causes T lymphocyte incapability, thereby causes anti-tumor activity to reduce.In a word, activity of endotoxin that golden staphylococcal enterotoxin had and immunosuppressive effect have limited its dosage use range in clinical antineoplaston to a certain extent, have influenced the effect of anti-tumor immunotherapy.Therefore, necessary superantigen activity and the anti-tumour effect that fundamentally improves enterotoxin by genetic engineering means, make it under the extremely low concentration condition, can produce powerful antineoplastic immune effect, remedy to a certain extent in clinical anti-tumor immunotherapy by the caused fringing effect of enterotoxin itself.
Because superantigen inductive immunological effect realizes by interacting with MHC II molecule and TCR-V β, therefore improve the superantigen activity and just become possibility by change superantigen and above both avidity.Yet, because some healthy tissues of human body also can be expressed MHC II molecule, therefore superantigen inductive cytotoxicity also can produce certain damage to this class normal cell usually, is not a feasible approach obviously so improve superantigen active by the avidity that strengthens enterotoxin and MHC II molecule.And improve the superantigen activity by the avidity that changes enterotoxin superantigen and TCR, and then the enhancing anti-tumour effect then is possible.
Summary of the invention
In order further to improve the superantigen and the anti-tumor activity of Staphylococcus aureus enterotoxin, remedy its existing defective in clinical antitumor application to a certain extent, the invention provides SEC2 mutator gene of a kind of superantigen increased activity and preparation method thereof
For achieving the above object, the technical solution used in the present invention is:
The SEC2 mutator gene of superantigen increased activity has the base sequence among the sequence table SEQ ID NO:1.
The proteins encoded of the SEC2 mutator gene of superantigen increased activity has the aminoacid sequence among the sequence table SEQ ID NO:2.
The preparation method of the SEC2 mutator gene of superantigen increased activity:
1) the SEC2 mutator gene of preparation superantigen increased activity: with recombinant plasmid pET-28-TM-sec2 is template, adopt respectively primer to 1 and primer carry out the overlapping pcr amplification in the left and right sides to 2, being template with the overlapping PCR product in the left and right sides then, is that primer carries out overlapping PCR with primer to 3; Then being template again with the amplified production, is that primer carries out pcr amplification again with primer to 4, promptly gets the mutator gene SEC2 (T20L/G22E) of superantigen increased activity;
Primer to 1 is R-Mutant, 5 '-TATTTTCCATCAAACCAGTAAACTCACTTG-3 ' and TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ';
Primer is F-Mutant to 2,5 '-GTTTACTGGTTTGATGGAAAATATGAAATAT-3 '; And SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is TM-F to 3,5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is SEC2-F to 4,5 '-CGGAATTCGAGAGTCAACCAGA-3 ' and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
2) the SEC2 mutain of preparation superantigen increased activity: said mutation gene SEC2 (T20L/G22E) is cloned into the pGEM-T carrier, subclone is to expression vector pET-28a behind EcoR I and Xho I double digestion, and transformed into escherichia coli BL21 (DE3), by the IPTG abduction delivering, obtain the superantigen mutant protein of solubility, and obtain the pure product of SEC2 mutain of superantigen increased activity through separation and purification.
Described pET-28-TM-sec2 recombinant plasmid is to be that template is carried out the left side pcr amplification by primer to 1 with the TM sequence, be that template is carried out the right side pcr amplification by primer to 2 with the sec2 sequence, then be that template is carried out overlapping pcr amplification with primer to 3 with left and right sides pcr amplification product, product through enzyme cut rear clone to the pET-28a expression vector promptly;
Primer is TM-F:5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and TM-SEC2-R 5 '-TGACTCTCGGATCCCATCGTGTACTTCCG-3 to 1;
Primer to 2 is: 5 '-ACACGATGGGATCCGAGAGTCAACCAGAC-3 ', and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is to 3:TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGA GTTATCCATTCTTTGTTG-3 '.
The present invention has following advantage:
1. the present invention utilizes the site-directed point mutation technology, made up the active and remarkable enhanced SEC2 of the anti-tumour effect mutain of superantigen first, and success realizes solubility expression in intestinal bacteria.The present invention can provide and be directly used in the engineering strain of producing this superantigen mutant protein.
2. the active enhancing of superantigen mutant protein is by improving wild SEC2 albumen the avidity of TCR to be realized among the present invention, therefore can not increase the damage that normal cell caused of some being expressed MHC II molecule.
3. the heterogenous expression of SEC2 (T20L/G22E) gene provides a kind of novel method for preparing this superantigen mutant protein among the present invention, has the output height, and purifying is convenient, and activity stabilized characteristics.
4. the SEC2 among the present invention (T20L/G22E) mutain obtains by the site-directed point mutation technology, its 20 and 22 amino acids that relate to TXi Baoshouti (TCR) binding site in to SEC2 albumen are replaced, thereby make 20 Threonine (Threonine, T) and 22 glycine (Glycine, G) become leucine (Leucine respectively, L) and L-glutamic acid (Glutamicacid, E), obtain to make the active mutain that improves of superantigen with expectation, thereby improve the clinical antitumous effect of superantigen SEC2 by changing enterotoxin and TCR avidity.
Description of drawings
Fig. 1 cuts the checking collection of illustrative plates for the enzyme of the pcr amplification of SEC2 of the present invention (T20L/G22E) mutating protein gene and corresponding recombinant plasmid and (wherein 1 represents DL2000 marker; 2 represent the PCR product in left side, mutational site (T20L/G22E); 3 represent the PCR product on right side, mutational site (T20L/G22E); 4 representative overlapping pcr amplification product TM-SEC2 (T20L/G22E); 5 represent the pcr amplification product of purpose mutator gene SEC2 (T20L/G22E); 6 representative reorganization pGEM-SEC2 (T20L/G22E) electropherogram spectrums; The electrophorogram of 7 representative reorganization pGEM-SEC2 (T20L/G22E) behind EcoR I and Xho I double digestion; 8 representative reorganization pET-28-SEC2 (T20L/G22E) electrophorograms; The electrophorogram of 9 representative reorganization pET-28-SEC2 (T20L/G22E) behind EcoR I and Xho I double digestion; 10 represent λ-Eco T14 DNA marker).
Fig. 2 (wherein 1 represents the empty bacterium contrast through IPTG inductive BL21 (DE3) for the expression and the purifying electrophoretogram of SEC2 of the present invention (T20L/G22E) mutain; The BL21 that contain pET-28a empty carrier (DE3) the whole protein contrast of 2 representatives behind the IPTG abduction delivering; 3 represent the whole protein collection of illustrative plates of target protein behind the IPTG abduction delivering; 4 represent the inclusion body whole protein collection of illustrative plates of target protein behind the IPTG abduction delivering; 5 represent the solubility supernatant of target protein behind the IPTG abduction delivering; SEC2 (T20L/G22E) pure protein of 6 and 7 representatives behind the Ni affinity purification; M represents molecular weight of albumen marker).
Fig. 3 for the active test experience of the superantigen of superantigen mutant protein SEC2 (T20L/G22E) (mice spleen lymphocytes proliferation experiment) as a result figure (wherein X-coordinate is that the wild albumen of SEC2 contrasts and concentration use range and the BSA negative control of SEC2 (T20L/G22E).Ordinate zou is corresponding value added index).
Fig. 4 detects (with mouse fibroma cell L929 is target cell, and splenic lymphocyte is the effector cell) figure (the wherein negative contrast bovine serum albumin of ordinate zou (BSA), SEC2 (T20L/G22E) and positive control SEC2 among A and the B figure as a result for the antitumor activity of superantigen mutant protein SEC2 of the present invention (T20L/G22E); X-coordinate is an inhibition rate of tumor cell.Each albumen was to the indirect inhibition effect of L929 oncocyte when A figure representative added splenic lymphocyte, and B figure representative does not add under the splenic lymphocyte condition (no effect cell) each albumen to the direct inhibition effect of L929 oncocyte).
Embodiment
The SEC2 mutator gene of superantigen increased activity has the base sequence (referring to sequence table 1) among the sequence table SEQ ID NO:1.The proteins encoded of its mutator gene has the aminoacid sequence (referring to sequence table 2) among the sequence table SEQ ID NO:2.
The SEC2 mutator gene of superantigen increased activity:
001?gagagtcaac?cagaccctac?gccagatgag?ttgcacaaat?caagtgagtt
051?tactggtttg?atggaaaata?tgaaatattt?atatgatgat?cattatgtat
101?cagcaactaa?agttatgtct?gtagataaat?ttttggcaca?tgatttaatt
151?tataacatta?gtgataaaaa?actaaaaaat?tatgacaaag?tgaaaacaga
201?gttattaaat?gaagatttag?caaagaagta?caaagatgaa?gtagttgatg
251?tgtatggatc?aaattactat?gtaaactgct?atttttcatc?caaagataat
301?gtaggtaaag?ttacaggtgg?taaaacttgt?atgtatggag?gaataacaaa
351?acatgaagga?aaccactttg?ataatgggaa?cttacaaaat?gtacttataa
401?gagtttatga?aaataaaaga?aacacaattt?cttttgaagt?gcaaactgat
451?aagaaaagtg?taacagctca?agaactagac?ataaaagcta?ggaatttttt
501?aattaataaa?aaaaatttgt?atgagtttaa?cagttcacca?tatgaaacag
551?gatatataaa?atttattgaa?aataacggca?atactttttg?gtatgatatg
601?atgcctgcac?caggcgataa?gtttgaccaa?tctaaatatt?taatgatgta
651?caacgacaat?aaaacggttg?attctaaaag?tgtgaagata?gaagtccacc
701?ttacaacaaa?gaatgga
The information of SEQ ID NO.1
(a) sequence signature:
* length: 717 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The proteins encoded of the SEC2 mutator gene of superantigen increased activity
Superantigen mutant protein SEC2 (T20L/G22E) has the amino acid series among the sequence table SEQ ID NO:2:
001?esqpdptpde?lhksseftgl?menmkylydd?hyvsatkvms?vdkflahdli
051?ynisdkklkn?ydkvktelln?edlakkykde?vvdvygsnyy?vncyfsskdn
101?vgkvtggktc?myggitkheg?nhfdngnlqn?vlirvyenkr?ntisfevqtd
151?kksvtaqeld?ikarnflink?knlyefnssp?yetgyikfie?nngntfwydm
201?mpapgdkfdq?skylmmyndn?ktvdsksvki?evhlttkng
The information (referring to sequence table) of SEQ ID NO.2
(a) sequence signature:
* length: 239 amino acid
* type: peptide chain
* chain: strand
* structure: secondary structure mainly is made of α spiral and βZhe Die, and wherein 93 and 110 amino acids constitute a typical halfcystine ring structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
1) structure of pET-28-TM-sec2 plasmid DNA template:
Design synthetic pcr primer thing is by overlapping PCR method amplification TM-sec2 sequence
1. the pcr amplification of TM sequence on the left of
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 5 μ l, dNTP 4uL, each 20pmol of upstream and downstream primer, pCVN/Her2DNA template 100ng, pfu archaeal dna polymerase 2U, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 60 ℃, 30 seconds; 72 ℃, 30 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 5 minutes.
Amplimer is TM-F:5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and TM-SEC2-R 5 '-TGACTCTCGGATCCCATCGTGTACTTCCG-3 '.
2. the pcr amplification of right side sec2 sequence
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 5 μ l, dNTP 4uL, each 20pmo l of upstream and downstream primer, the golden bacterium genomic dna template 100ng of Portugal, pfu archaeal dna polymerase 2U, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 60 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 5 minutes.
Amplimer is 5 '-ACACGATGGGATCCGAGAGTCAACCAGAC-3 ', and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 '.
3. the overlapping PCR of TM-SEC2 gene is synthetic
The left side TM sequence and the right side sec2 sequence that obtain with above-mentioned amplification are overlapping pcr template, are that primer is right with TM-F and SEC2-R, by the synthetic TM-SEC2 gene of overlapping pcr amplification.
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 10 μ l, dNTP 8uL, each 40pmol of upstream and downstream primer, the overlapping PCR product of left and right sides be 50ng, Pfu archaeal dna polymerase 4U respectively, aseptic ultrapure water polishing volume to 100 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 120 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 10 minutes; 4 ℃ of preservations.
Amplimer is TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGA GTTATCCATTCTTTGTTG-3 '.
4. the structure of pET-28-TM-sec2 plasmid DNA
The TM-SEC2 gene product that above-mentioned amplification is obtained is with EcoRI, Xho I double digestion, and enzyme is cut product and reclaimed through PCR product purification test kit, after connect into expression vector through the pET-28a of same double digestion, be built into expression vector pET-28-TM-SEC2.Transformed E .coli DH5 α competent cell, by enzyme cut the checking and dna sequencing identify correct recombinant clone.
2) extraction of pET-28-TM-sec2 plasmid DNA template:
The above-mentioned bacillus coli DH 5 alpha colony inoculation that contains plasmid pET-28-TM-sec2 of picking is in 5ml liquid LB substratum, and 37 ℃ of shaking table incubated overnight are got the centrifugal collection thalline of culture of 3ml.Extract plasmid pET-28-TM-sec2, and in-20 ℃ of freezing preservations.(the extraction operation of plasmid DNA is pressed F. Ao Sibai, R. Brunt, R.E. James Kingston, D.D. Moore, J.G. Sai Deman, J.A. Smith, K. Si Telaer " fine works molecular biology experiment guide " USA New York John Wiley; The nineteen ninety-five third edition P39-40 of Sons press).
3) amplification of SEC2 (T20L/G22E) gene:
Design synthetic pcr primer thing, by the gene segment of overlapping PCR method amplification SEC2 (T20L/G22E):
1. overlapping pcr amplification product on the left of
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 5 μ l, dNTP 4uL, each 20pmol of upstream and downstream primer, pET-28-TM-sec2100ng, Pfu archaeal dna polymerase 2U, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 50 ℃, 30 seconds; 72 ℃, 60 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 10 minutes.
Amplimer is R-Mutant, 5 '-TATTTTCCATCAAACCAGTAAACTCACTTG-3 ', and TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 '.
2. the overlapping pcr amplification product in right side
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 5 μ l, dNTP 4uL, each 20pmol of upstream and downstream primer, pET-28-TM-sec2100ng, Pfu archaeal dna polymerase 2U, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 90 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 10 minutes.
Amplimer is F-Mutant, 5 '-GTTTACTGGTTTGATGGAAAATATGAAATAT-3 '; And SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 '.
3. the overlapping PCR of TM-SEC2 (T20L/G22E) gene is synthetic
With the overlapping PCR product in above-mentioned left side and right side is template, is that primer is right with TM-F and SEC2-R, by synthetic TM-SEC2 (T20L/G22E) gene of overlapping pcr amplification.
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 10 μ l, dNTP 8uL, each 40pmol of upstream and downstream primer, the overlapping PCR product of left and right sides be 50ng, Pfu archaeal dna polymerase 4U respectively, aseptic ultrapure water polishing volume to 100 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 120 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 10 minutes; 4 ℃ of preservations.
Amplimer is TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGA GTTATCCATTCTTTGTTG-3 '.
4. the pcr amplification of SEC2 (T20L/G22E) gene
With above-mentioned TM-SEC2 (T20L/G22E) gene product is template, is that primer is right with SEC2-F and SEC2-R, by the full gene of the synthetic SEC2 (T2OL/G22E) of pcr amplification.
The PCR reaction system is: 10 * Pfu dna polymerase reaction damping fluid, 10 μ l, dNTP 8uL, each 40pmol of upstream and downstream primer, TM-SEC2 (T20L/G22E) product 50ng, Pfu archaeal dna polymerase 4U, aseptic ultrapure water polishing volume to 100 μ l.
The PCR reaction conditions is: 95 ℃ of fs, 5 minutes; 94 ℃ of subordinate phase, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 90 seconds; Totally 30 circulations; 72 ℃ of phase IIIs, 10 minutes; Amplified production is the mutator gene SEC2 (T2OL/G22E) of superantigen increased activity, referring to sequence table 1.
Amplimer is SEC2-F, 5 '-CGGAATTCGAGAGTCAACCAGA-3 ' and SEC2-R, 5 '-TCGC TCGAGTTATCCATTCTTTGTTG-3 '.
4) evaluation of SEC2 (T20L/G22E) gene: get SEC2 (T2OL/G22E) gene product that above-mentioned pcr amplification obtains and under the 120V voltage conditions, carry out electrophoretic analysis and separate through 1.5% sepharose, downcut the purpose band of corresponding size, reclaim the test kit working instructions by vast Tyke, Beijing DNA of biotech company glue and carry out the glue recovery; Reclaim product and be connected, be built into recombinant cloning vector pGEM-T-SEC2 (T20L/G22E) with the pGEM-T cloning vector.The product pGEM-T of Promega company test kit working instructions are pressed in ligation.Connect product transformed into escherichia coli DH5 α competent cell, (conversion operation is pressed F. Ao Sibai, the R. Brunt, R.E. James Kingston, D.D. Moore, J.G. Sai Deman, J.A. Smith, K. Si Telaer " fine works molecular biology experiment guide " USA New York JohnWiley; The nineteen ninety-five third edition P 39-40 of Sons press), measure dna sequence dna by the pcr amplification screening positive clone and with the terminal cessation method of the two deoxidations of Sanger.Synthetic and the construction of recombinant plasmid result of SEC2 (T20L/G22E) gene is referring to Fig. 1.
As shown in Figure 1, successfully increasing by overlapping PCR method has obtained total length SEC2 (T20L/G22E) gene, and size conforms to theoretical value.It is all correct that recombinant cloning vector that makes up and expression vector are cut checking through enzyme.
The expression of superantigen mutant protein SEC2 (T20L/G22E)
1) structure of superantigen mutant protein expression vector: with EcoRI, Xho I double digestion, corresponding enzyme is cut gene product and is reclaimed the test kit recovery with DNA glue with gene clone carrier pGEM-T-SEC2 (T20L/G22E) plasmid DNA.Above-mentioned recovery product being connected into the expression vector through the pET-28a of same double digestion, be built into expression vector pET-28-SEC2 (T20L/G22E) with the T4 dna ligase.Transformed E .coli BL21 (DE3) competent cell, by enzyme cut the checking and dna sequencing identify correct recombinant clone.
2) abduction delivering of superantigen mutant protein and purifying: inoculation has transformed the single bacterium colony of BL21 (DE3) of recombinant expression vector pET-28-SEC2 (T20L/G00E) in the liquid LB substratum that contains 50 μ g/ml kantlex, the activation culture of spending the night, with the inoculum size switching next stage of 1: 100 (volume ratio), 37 ℃ of shaking tables are cultured to OD
600Be 0.6, add the IPTG of final concentration 1.0mmol/L, 30 ℃ of abduction deliverings 5 hours.Then, be resuspended in level pad (the 50mM NaH of 1/5 volume with the centrifugal collection thalline of the speed of 5000rpm/min
2PO
4, 0.5M NaCl, 10mM imidazole pH8.0), is splined on the good Ni affinity column of pre-balance with 0.5ml/min speed; To contain 10 column volumes of level pad washing of 40mM imidazoles, the foreign protein of flush away non-specific binding; At last with elution buffer (50mM NaH
2PO
4, 0.5M NaCl, the 250mM imidazoles pH8.0) carries out wash-out, collects under the ultraviolet condition eluted product when the absorption peak of 480nm, is superantigen mutant protein SEC2 (T20L/G22E), and to eluted product with the PBS desalination of dialysing.Identify the expression and the purification effect (referring to Fig. 2) of target protein by the SDS-PAGE electrophoresis.As shown in Figure 2, target protein SEC2 (T20L/G22E) mainly exists with soluble form behind the IPTG abduction delivering, has obtained the mutain of higher degree by affinity chromatography, can be used for further biologic activity experiment.
Stimulate the mice spleen lymphocytes proliferation activity to detect: to get SPF level mouse inbred lines BALB/c, put to death the back by cervical vertebra and under aseptic condition, get its spleen, cross 200 eye mesh screens gently after the crushing.To cross the cell suspension centrifugal 5min collecting cell precipitation under the 1000rpm/min rotating speed behind the screen cloth then,, leave standstill behind the 5min in the centrifugal 5min of 1000rpm/min with 5mL erythrocyte cracked liquid re-suspended cell.Clean cell 1-2 time with the RPMI-1640 substratum that does not contain serum again, regulate cell concn with the RPMI-1640 nutrient solution that contains 10% calf serum at last, with 8 * 10
5The ce11s/ hole is added in 96 orifice plates.With purified superantigen mutant protein SEC2 of the present invention (T20L/G22E) and the wild albumen of SEC2 respectively with 1,10,100,1000, the final concentration of 10000ng/mL adds each hole, each concentration is done three repetitions.The hole if lymphocyte accrues, and with bovine serum albumin (BSA) as negative control.After cultivating 72h, add the MTT liquid in 20 μ l/ holes.Continue to cultivate 4h, 1500r/min abandons the supernatant nutrient solution after centrifugal 5 minutes, and collecting cell adds dimethyl sulfoxide (DMSO) (DMSO) 120u l/ hole, behind the dissolving 15min, measures the light absorption value in each hole on the microplate reader with 570nm.The T ability of cell proliferation is with proliferation index (Proliferation Index, PI) expression, the PI=experimental port light absorption value lymphocyte hole light absorption value (referring to Fig. 3) that accrues.As shown in Figure 3, from 1ng/ml to 10000ng/ml, compare with the wild albumen of SEC2, the superantigen activity of SEC2 (T20L/G22E) mutain is significantly increased, and the BSA negative control does not have tangible hormesis to mouse spleen lymphocyte.
Described RPMI-1640 nutrient solution is the nutritive medium commonly used that is used for human or animal's cell strain growth, is produced by U.S. Gibco company; To be tetrazolium bromide (MTT) be dissolved in phosphate buffered saline buffer (NaCl 8.0g, Na with the concentration of 5mg/ml to MTT liquid
2HPO
41.44g, KH
2PO
40.24g KCl 0.2g is dissolved in the 1000ml deionized water, pH 7.4) preparation forms, and 4 ℃ of preservations behind the filtering with microporous membrane of 0.22 μ m.
The proteic anti-tumor activity of SEC2 (T20L/G22E) detects: mouse fibroma cell L929 is gone down to posterity cultivate back trypsinase-EDTA Digestive system digestion, being diluted to concentration with 1640 substratum that contain 10%FBS is 2 * 10
5The single cell suspension of cells/mL is then with 1 * 10
4Cultivate at 96 orifice plates in the cells/ hole.To separate the mouse spleen lymphocyte of acquisition with 2 * 10 by method among the embodiment 3
5The ce11s/ hole joins in 96 orifice plates of the above-mentioned L929 of containing cell, makes effect target ratio reach 20: 1 (cell quantity ratio).Then with the superantigen mutant protein SEC2 (T20L/G22E) of purifying of the present invention respectively with 100,1000, the final concentration of 10000ng/mL adds each hole.As negative control, the wild albumen of SEC2 is as positive control with BSA, and establishes tumour cell control wells, lymphocyte nature release aperture, blank hole and zeroing hole, and each concentration is established 3 multiple holes, and condition is cultivated 72h routinely, adds the MTT liquid in 20ul/ hole.Continue to cultivate 4h, the 1000r/min centrifugal collecting cell adds dimethyl sulfoxide (DMSO) (DMSO) 120ul/ hole, behind the dissolving 15min, measures the light absorption value in each hole on the microplate reader with 570nm.Tumor suppression knurl calculation formula is as follows: 100[(albumen experimental port light absorption value lymphocyte nature release aperture light absorption value) (tumour cell control wells light absorption value blank hole light absorption value)] * 100% (referring to Fig. 4).Shown in 4B figure, under the situation that does not add effector cell's (splenic lymphocyte), SEC2 positive control albumen and SEC2 (T20L/G22E) mutain all can not produce tumor killing effect, do not have obvious difference with negative control BSA, prove that SEC2 and SEC2 (T20L/G22E) are all at the cell-mediated tumor-inhibiting action that produces down of T.Shown in 4A figure, under situation, compare with the wild albumen of SEC2 with splenic lymphocyte action effect cell, from 1ng/ml to 10000ng/ml, SEC2 (T20L/G22E) mutain has all shown remarkable enhanced antitumous effect.Show that the alternative SEC2 of SEC2 (T20L/G22E) mutain is used for further clinical antitumor research.
SEQUENCE?LISTING
＜110〉Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences
＜120〉SEC2 mutator gene of a kind of superantigen increased activity and preparation method thereof
<130>
<160> 2
<170> PatentIn?version?3.1
<210> 1
<211> 717
<212> DNA
<213> Staphylococcus?aureus
<220>
<221> CDS
<222> (1)..(717)
<223>
<400> 1
gag?agt?caa?cca?gac?cct?acg?cca?gat?gag?ttg?cac?aaa?tca?agt?gag 48
Glu?Ser?Gln?Pro?Asp?Pro?Thr?Pro?Asp?Glu?Leu?His?Lys?Ser?Ser?Glu
1 5 10 15
ttt?act?ggt?ttg?atg?gaa?aat?atg?aaa?tat?tta?tat?gat?gat?cat?tat 96
Phe?Thr?Gly?Leu?Met?Glu?Asn?Met?Lys?Tyr?Leu?Tyr?Asp?Asp?His?Tyr
20 25 30
gta?tca?gca?act?aaa?gtt?atg?tct?gta?gat?aaa?ttt?ttg?gca?cat?gat 144
Val?Ser?Ala?Thr?Lys?Val?Met?Ser?Val?Asp?Lys?Phe?Leu?Ala?His?Asp
35 40 45
tta?att?tat?aac?att?agt?gat?aaa?aaa?cta?aaa?aat?tat?gac?aaa?gtg 192
Leu?Ile?Tyr?Asn?Ile?Ser?Asp?Lys?Lys?Leu?Lys?Asn?Tyr?Asp?Lys?Val
50 55 60
aaa?aca?gag?tta?tta?aat?gaa?gat?tta?gca?aag?aag?tac?aaa?gat?gaa 240
Lys?Thr?Glu?Leu?Leu?Asn?Glu?Asp?Leu?Ala?Lys?Lys?Tyr?Lys?Asp?Glu
65 70 75 80
gta?gtt?gat?gtg?tat?gga?tca?aat?tac?tat?gta?aac?tgc?tat?ttt?tca 288
Val?Val?Asp?Val?Tyr?Gly?Ser?Asn?Tyr?Tyr?Val?Asn?Cys?Tyr?Phe?Ser
85 90 95
tcc?aaa?gat?aat?gta?ggt?aaa?gtt?aca?ggt?ggt?aaa?act?tgt?atg?tat 336
Ser?Lys?Asp?Asn?Val?Gly?Lys?Val?Thr?Gly?Gly?Lys?Thr?Cys?Met?Tyr
100 105 110
gga?gga?ata?aca?aaa?cat?gaa?gga?aac?cac?ttt?gat?aat?ggg?aac?tta 384
Gly?Gly?Ile?Thr?Lys?His?Glu?Gly?Asn?His?Phe?Asp?Asn?Gly?Asn?Leu
115 120 125
caa?aat?gta?ctt?ata?aga?gtt?tat?gaa?aat?aaa?aga?aac?aca?att?tct 432
Gln?Asn?Val?Leu?Ile?Arg?Val?Tyr?Glu?Asn?Lys?Arg?Asn?Thr?Ile?Ser
130 135 140
ttt?gaa?gtg?caa?act?gat?aag?aaa?agt?gta?aca?gct?caa?gaa?cta?gac 480
Phe?Glu?Val?Gln?Thr?Asp?Lys?Lys?Ser?Val?Thr?Ala?Gln?Glu?Leu?Asp
145 150 155 160
ata?aaa?gct?agg?aat?ttt?tta?att?aat?aaa?aaa?aat?ttg?tat?gag?ttt 528
Ile?Lys?Ala?Arg?Asn?Phe?Leu?Ile?Asn?Lys?Lys?Asn?Leu?Tyr?Glu?Phe
165 170 175
aac?agt?tca?cca?tat?gaa?aca?gga?tat?ata?aaa?ttt?att?gaa?aat?aac 576
Asn?Ser?Ser?Pro?Tyr?Glu?Thr?Gly?Tyr?Ile?Lys?Phe?Ile?Glu?Asn?Asn
180 185 190
ggc?aat?act?ttt?tgg?tat?gat?atg?atg?cct?gca?cca?ggc?gat?aag?ttt 624
Gly?Asn?Thr?Phe?Trp?Tyr?Asp?Met?Met?Pro?Ala?Pro?Gly?Asp?Lys?Phe
195 200 205
gac?caa?tct?aaa?tat?tta?atg?atg?tac?aac?gac?aat?aaa?acg?gtt?gat 672
Asp?Gln?Ser?Lys?Tyr?Leu?Met?Met?Tyr?Asn?Asp?Asn?Lys?Thr?Val?Asp
210 215 220
tct?aaa?agt?gtg?aag?ata?gaa?gtc?cac?ctt?aca?aca?aag?aat?gga 717
Ser?Lys?Ser?Val?Lys?Ile?Glu?Val?His?Leu?Thr?Thr?Lys?Asn?Gly
225 230 235
<210> 2
<211> 239
<212> PRT
<213> Staphylococcus?aureus
<400> 2
Glu?Ser?Gln?Pro?Asp?Pro?Thr?Pro?Asp?Glu?Leu?His?Lys?Ser?Ser?Glu
1 5 10 15
Phe?Thr?Gly?Leu?Met?Glu?Asn?Met?Lys?Tyr?Leu?Tyr?Asp?Asp?His?Tyr
20 25 30
Val?Ser?Ala?Thr?Lys?Val?Met?Ser?Val?Asp?Lys?Phe?Leu?Ala?His?Asp
35 40 45
Leu?Ile?Tyr?Asn?Ile?Ser?Asp?Lys?Lys?Leu?Lys?Asn?Tyr?Asp?Lys?Val
50 55 60
Lys?Thr?Glu?Leu?Leu?Asn?Glu?Asp?Leu?Ala?Lys?Lys?Tyr?Lys?Asp?Glu
65 70 75 80
Val?Val?Asp?Val?Tyr?Gly?Ser?Asn?Tyr?Tyr?Val?Asn?Cys?Tyr?Phe?Ser
85 90 95
Ser?Lys?Asp?Asn?Val?Gly?Lys?Val?Thr?Gly?Gly?Lys?Thr?Cys?Met?Tyr
100 105 110
Gly?Gly?Ile?Thr?Lys?His?Glu?Gly?Asn?His?Phe?Asp?Asn?Gly?Asn?Leu
115 120 125
Gln?Asn?Val?Leu?Ile?Arg?Val?Tyr?Glu?Asn?Lys?Arg?Asn?Thr?Ile?Ser
130 135 140
Phe?Glu?Val?Gln?Thr?Asp?Lys?Lys?Ser?Val?Thr?Ala?Gln?Glu?Leu?Asp
145 150 155 160
Ile?Lys?Ala?Arg?Asn?Phe?Leu?Ile?Asn?Lys?Lys?Asn?Leu?Tyr?Glu?Phe
165 170 175
Asn?Ser?Ser?Pro?Tyr?Glu?Thr?Gly?Tyr?Ile?Lys?Phe?Ile?Glu?Asn?Asn
180 185 190
Gly?Asn?Thr?Phe?Trp?Tyr?Asp?Met?Met?Pr0Ala?Pro?Gly?Asp?Lys?Phe
195 200 205
Asp?Gln?Ser?Lys?Tyr?Leu?Met?Met?Tyr?Asn?Asp?Asn?Lys?Thr?Val?Asp
210 215 220
Ser?Lys?Ser?Val?Lys?Ile?Glu?Val?His?Leu?Thr?Thr?Lys?Asn?Gly
225 230 235
Claims (3)
1. the SEC2 mutator gene of a superantigen increased activity, it is characterized in that: the SEC2 mutator gene of superantigen increased activity is the base sequence among the sequence table SEQ ID NO:1.
2. the proteins encoded of the SEC2 mutator gene of a superantigen increased activity, it is characterized in that: the proteins encoded of the SEC2 mutator gene of superantigen increased activity is the aminoacid sequence among the sequence table SEQ ID NO:2; And make up as follows:
1) the SEC2 mutator gene of preparation superantigen increased activity: with recombinant plasmid pET-28-TM-sec2 is template, adopt respectively primer to 1 and primer carry out the overlapping pcr amplification in the left and right sides to 2, being template with the overlapping PCR product in the left and right sides then, is that primer carries out overlapping PCR with primer to 3; Then being template again with the amplified production, is that primer carries out pcr amplification again with primer to 4, promptly gets the mutator gene SEC2 T20L/G22E of superantigen increased activity;
Primer to 1 is R-Mutant, 5 '-TATTTTCCATCAAACCAGTAAACTCACTTG-3 ' and TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ';
Primer is F-Mutant to 2,5 '-GTTTACTGGTTTGATGGAAAATATGAAATAT-3 '; And SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is TM-F to 3,5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is SEC2-F to 4,5 '-CGGAATTCGAGAGTCAACCAGA-3 ' and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
2) the SEC2 mutain of preparation superantigen increased activity: SEC2T20L/G22E is cloned into the pGEM-T carrier with the said mutation gene, subclone is to expression vector pET-28a behind EcoR I and Xho I double digestion, and transformed into escherichia coli BL21 (DE3), by the IPTG abduction delivering, obtain the superantigen mutant protein of solubility, and obtain the pure product of SEC2 mutain of superantigen increased activity through separation and purification.
3. preparation method by the SEC2 mutator gene of the described superantigen increased activity of claim 1 is characterized in that:
1) the SEC2 mutator gene of preparation superantigen increased activity: with recombinant plasmid pET-28-TM-sec2 is template, adopt respectively primer to 1 and primer carry out the overlapping pcr amplification in the left and right sides to 2, being template with the overlapping PCR product in the left and right sides then, is that primer carries out overlapping PCR with primer to 3; Then being template again with the amplified production, is that primer carries out pcr amplification again with primer to 4, promptly gets the mutator gene SEC2 T20L/G22E of superantigen increased activity;
Primer to 1 is R-Mutant, 5 '-TATTTTCCATCAAACCAGTAAACTCACTTG-3 ' and TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ';
Primer is F-Mutant to 2,5 '-GTTTACTGGTTTGATGGAAAATATGAAATAT-3 '; And SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is TM-F to 3,5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is SEC2-F to 4,5 '-CGGAATTCGAGAGTCAACCAGA-3 ' and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Described pET-28-TM-sec2 recombinant plasmid is to be that template is carried out the left side pcr amplification by primer to 1 with the TM sequence, be that template is carried out the right side pcr amplification by primer to 2 with the sec2 sequence again, then be that template is carried out overlapping pcr amplification with primer to 3 with left and right sides pcr amplification product, product through enzyme cut rear clone to the pET-28a expression vector promptly;
Primer is TM-F:5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and TM-SEC2-R 5 '-TGACTCTCGGATCCCATCGTGTACTTCCG-3 to 1;
Primer to 2 is: 5 '-ACACGATGGGATCCGAGAGTCAACCAGAC-3 ', and SEC2-R, 5 '-TCGCTCGAGTTATCCATTCTTTGTTG-3 ';
Primer is to 3:TM-F, 5 '-CGGAATTCGCCGAGCAGAGAGC-3 ' and SEC2-R, 5 '-TCGCTCGA GTTATCCATTCTTTGTTG-3 '.
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| US20040236082A1 (en) * | 2001-04-13 | 2004-11-25 | Marshall Matthew J. | Modified staphylococcal enterotoxins and expression systems therefore |
| WO2005121342A1 (en) * | 2004-06-14 | 2005-12-22 | National University Corporation Hirosaki University | Recombinant enterotoxin c and vaccine with the use of the same |
| CN1891718A (en) * | 2005-07-06 | 2007-01-10 | 中国科学院沈阳应用生态研究所 | Fusion immunotoxin ML-L-SEC2 and gene and its preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20040236082A1 (en) * | 2001-04-13 | 2004-11-25 | Marshall Matthew J. | Modified staphylococcal enterotoxins and expression systems therefore |
| WO2005121342A1 (en) * | 2004-06-14 | 2005-12-22 | National University Corporation Hirosaki University | Recombinant enterotoxin c and vaccine with the use of the same |
| CN1891718A (en) * | 2005-07-06 | 2007-01-10 | 中国科学院沈阳应用生态研究所 | Fusion immunotoxin ML-L-SEC2 and gene and its preparation |
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