CN101113165B - SEC2 super antigen gene engineering peptide and encoding gene and heterogenous expression method thereof - Google Patents
SEC2 super antigen gene engineering peptide and encoding gene and heterogenous expression method thereof Download PDFInfo
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Abstract
The invention relates to gene cloning and heterogenous expression in gene engineering peptide, in particular to a series of gene engineering peptide with SEC2 superantigen activity, and the preparation of coding gene and heterogeneous expression thereof. The SEC2-1 to SEC2-14 respectively has animo-acid sequences in SEQ ID No.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and SEQ ID No.28. The cloned nucleotide sequences can be expressed in Escherichia coli BL21 (DE3) and the corresponding protein SEC2-1 to SEC2-14 being expressed are proved to have biological activity of super antigen.
Description
Technical field
The present invention relates to the gene clone and the heterogenous expression of gene engineering peptides, is the preparation and the heterogenous expression of active gene engineering peptides of a series of SEC2 of having superantigens and encoding sox thereof specifically.
Background technology
Superantigen (superantigen SAg) is one group of protein molecule by bacterium or encoding viral, can extremely low concentration (1～10ng/mL) stimulates most of T hyperplasia, has superpower enhancing body immunoreation function and certain anti-tumor activity.Therefore, superantigen is a kind of fabulous immunomodulator and synergistic agent, is expected to be developed to the potential new type antineoplastic medicine, is used for oncotherapy.
(Staphylococcal enterotoxins SEs) is one type of representative mikrobe extracellular toxin to Staphylococcus aureus enterotoxin.Because its extremely strong t cell activation function becomes a quasi-representative mikrobe superantigen, receives people's extensive attention.In recent years, people are to Staphylococcus aureus enterotoxin (SE) structure, immunologic function and antitumor etc. carried out a large amount of research work, wherein mainly concentrate on to SEB etc. the research aspect.I first the gene of SEC2 has been carried out clone, order-checking and has obtained initiative achievement aspect the targent fused protein research in the world, 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.
Experimental oncology research shows that utilizing SEC2 to have the incomparable advantage of other medicines: SEC2 as the superantigen medicine of oncotherapy can resist digestion and heat-resisting and only need use very little dosage just can cause t cell response and tangible tumor inhibitory effect.However, also not exclusively fully up to expectations in clinical use.1) can cause toxic side effect such as being accompanied by vomiting and serious diarrhoea as potential stomach toxin; 2) the SEC2 superantigen lacks target property to the tumour cell action site, when the enhancing body immunologic function suppresses growth of tumour cell, possibly produce CDCC to the human normal cell; Excessive or the misapplication of dosage also can produce immunological tolerance; 3) as potential clinical development medicine, superantigen SEC2 molecular weight excessive (being about 27.6kD) is not easy to intravenous injection and absorption of human body.These issues limit its application clinically.Therefore, how to avoid unfavorable sememe factor, make full use of its powerful immunologic function, for visit plain SEC2 antitumor in addition antiviral in effect, have very important significance.
To the toxic effect that is faced in the superantigen SEC2 practical clinical, lack problems such as target property and molecular weight be bigger than normal; The present invention is according to staphylococcus aureus gene group, information biology scheduling theory; Utilize modern molecular biology technique to realize the directional transformation of SEC2 protein molecular; To obtain a series of active gene engineering polypeptides of superantigen that have; This superantigen gene engineering peptides through genetic modification is for enlarging its clinical application range, improves curative effect, reduces toxic side effect and have important practice significance, has the potential development prospect.
Summary of the invention
The present invention relates to SEC2 superantigen gene engineering peptides and encoding sox thereof and heterogenetic expression method; It is a series of active gene engineering peptides of superantigen that have; Utilize the present invention under manually operated condition, to obtain the highly purified active gene engineering peptides of superantigen that has in a large number.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
The SEC2 superantigen gene engineering peptides is respectively one of following arbitrary amino acid sequence,
SEC2-1 has aminoacid sequence among the sequence table SEQ ID NO:2;
SEC2-2 has aminoacid sequence among the sequence table SEQ ID NO:4;
SEC2-3 has aminoacid sequence among the sequence table SEQ ID NO:6;
SEC2-4 has aminoacid sequence among the sequence table SEQ ID NO:8;
SEC2-5 has aminoacid sequence among the sequence table SEQ ID NO:10;
SEC2-6 has aminoacid sequence among the sequence table SEQ ID NO:12;
SEC2-7 has aminoacid sequence among the sequence table SEQ ID NO:11;
SEC2-8 has aminoacid sequence among the sequence table SEQ ID NO:16;
SEC2-9 has aminoacid sequence among the sequence table SEQ ID NO:18;
SEC2-10 has aminoacid sequence among the sequence table SEQ ID NO:20;
SEC2-11 has aminoacid sequence among the sequence table SEQ ID NO:22;
SEC2-12 has amino based acid sequence among the sequence table SEQ ID NO:24;
SEC2-13 has aminoacid sequence among the sequence table SEQ ID NO:26;
Or SEC2-14 has aminoacid sequence among the sequence table SEQ ID NO:28.
Said its encoding sox because of the engineering peptide corresponds respectively to one of following nucleotide sequences,
The gene that SEC2-1 is corresponding has base sequence among the sequence table SEQ ID NO:1;
The gene that SEC2-2 is corresponding has base sequence among the sequence table SEQ ID NO:3;
The gene that SEC2-3 is corresponding has base sequence among the sequence table SEQ ID NO:5;
The gene that SEC2-4 is corresponding has base sequence among the sequence table SEQ ID NO:7;
The gene that SEC2-5 is corresponding has base sequence among the sequence table SEQ ID NO:9;
The gene that SEC2-6 is corresponding has base sequence among the sequence table SEQ ID NO:11;
The gene that SEC2-7 is corresponding has base sequence among the sequence table SEQ ID NO:13;
The gene that SEC2-8 is corresponding has base sequence among the sequence table SEQ ID NO:15;
The gene that SEC2-9 is corresponding has base sequence among the sequence table SEQ ID NO:17;
The gene that SEC2-10 is corresponding has base sequence among the sequence table SEQ ID NO:19;
The gene that SEC2-11 is corresponding has base sequence among the sequence table SEQ ID NO:21;
The gene that SEC2-12 is corresponding has base sequence among the sequence table SEQ ID NO:23;
The gene that SEC2-13 is corresponding has base sequence among the sequence table SEQ ID NO:25;
Or the corresponding gene of SEC2-14 has base sequence among the sequence table SEQ ID NO:27.
The heterogenetic expression method of SEC2 superantigen gene engineering peptides:
Nucleotide sequence sec2-1, sec2-2, sec2-3, sec2-4, sec2-5, sec2-6, sec2-7, sec2-8, sec2-9, sec2-10, sec2-11, sed-12, sec2-13 or the sec2-14 of these a series of SEC2 superantigen gene engineering peptides of coding are cloned into expression vector pET-28a respectively; With e. coli bl21 (DE3) is the host bacterium; Be built into the genetic engineering bacterium of heterogenous expression superantigen active gene engineering peptide, heterogenous expression has the active SEC2-1 of superantigen, SEC2-2, SEC2-3, SEC2-4, SEC2-5, SEC2-6, SEC2-7, SEC2-8, SEC2-9, SEC2-10, SEC2-11, SEC2-12, SEC2-13 or SEC2-14 gene engineering peptides in substratum.
The cloning process of the nucleotide sequence of said coding SEC2 superantigen gene engineering peptides is following,
With staphylococcus aureus gene group DNA is template, adopts primer sec2-1-F:5 ' gAATTCgAgAgTCAACCAgACCCTA 3 ' and sec2-1-R:5 ' gCCTCgAgTTATTTTATgTCTAgTTC 3 ' to amplify the sec2-1 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-2-F:5 ' gAATTCgAgAgTCAACCAgACCCTAC 3 ' and see2-2-R:5 ' gCTCgAgTTAgTggTTTCCTTCATg 3 ' to amplify the sec2-2 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-3-F:5 ' gAATTCgAgAgTCAACCAgA 3 ' to amplify the sec2-3 gene fragment with sec2-3-R:gCCTCgAgTTATCCATACATACAAg 3 ' through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-4-F:5 ' GAATTCGAGAGTCAACCAGACCCTA 3 ' and sec2-4-R:5 ' gCTCgAgTTATTTTgTTgTTATTCCTCCA 3 ' to amplify the sec2-4 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-5-F:5 ' gAATTCggTACgATgggTAATATg 3 ' and sec2-5-R:5 ' CTCgAgTTATCCATTCTTTgTTgTAAggT 3 ' to amplify the sec2-5 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-6-F:5 ' gAATTCggTACgATgggTAATATg 3 ' and sec2-6-R:5 ' CTCgAgTTATTTTgTTATTCCTCCAT 3 ' to amplify the sec2-6 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-7-F:5 ' gAATTCggTACgATgggTAATATg 3 ' and sec2-7-R:5 ' CgCTCgAgTTATCCATACATACAAg 3 ' to amplify the sec2-7 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-8-F:5 ' gAATTgCAAAgAAgTACAAAgATg 3 ' and sec2-8-R:5 ' CgCTCgAgTTATTTTATgTCTAgTTC 3 ' to amplify the sec2-8 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-9-F:5 ' ggAATTCgAgAgTCAACCAgACCCTA 3 ' and sec2-9-R:5 ' CgCTCgAgTTATCCATACACATCAAC 3 ' to amplify the sec2-9 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-10-F:5 ' gAATTCgAgAgTCAACCAgAC 3 ' and sec2-0-R:5 ' CgCTCgAgTTAATCTTTgTACTTC 3 ' to amplify the sec2-10 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-11-F:5 ' gAATTCgAgAgTCAACCAgACCCTA 3 ' and sec2-11-R:5 ' CCCgCTCgAgTTTAGTTTACATAgTAAT 3 ' to amplify the sec2-11 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-12-F:5 ' gAATTCAAATCAAgTgAgTTTACTg 3 ' and sec2-12-R:5 ' CgCTCgAgTTAgTTTACATAgTAAT 3 ' to amplify the sec2-12 gene fragment through round pcr;
With staphylococcus aureus gene group DNA is template, adopts primer sec2-13-F:5 ' gAATTCggTACgATgggTAATAT 3 ' and sec2-13-R:5 ' CCgCTCgAgTTagTTTACATAgTAA 3 ' to amplify the sec2-13 gene fragment through round pcr;
Or; With staphylococcus aureus gene group DNA is template, adopts primer sec2-14-F:5 ' gAATTCgAgAgTCAACCAgACCCTA 3 ' and sec2-14-R:5 ' CgCTCgAgTTATAATAACTCTgTTTTCAC 3 ' to amplify the sec2-14 gene fragment through round pcr.
The present invention has following advantage:
1. the present invention is the nucleotide sequence of artificial constructed a series of coding superantigen gene engineering peptides, knows the composition of its gene complete sequence first.
2. the present invention utilizes round pcr, clones the nucleotide gene of a series of coding superantigen gene engineering peptides, and at expression in escherichia coli, a series of superantigen gene engineering peptides of expression have the BA of superantigen through check; Use the present invention, the engineering strain that directly is used to produce these a series of superantigen gene engineering peptides can be provided.
3. the heterogenous expression of gene of the present invention provides a kind of novel method of preparation superantigen antineoplastic biologic preparation, has the output height, produces and stablizes, and makes things convenient for the characteristics of purifying.
Description of drawings
Fig. 1 is the pcr amplification agarose gel electrophoresis figure of sec2-1, sec2-2, sec2-3, sec2-4, sec2-5 and sec2-6 gene; Wherein: 1 is the DL2000DNA molecular weight standard, and 2-7 is the pcr amplification product of sec2-1, sec2-2, sec2-3, sec2-4, sec2-5 and sec2-6 gene;
Fig. 2 is the pcr amplification agarose gel electrophoresis figure of sec2-7, sec2-8, sec2-9, sec2-10, sec2-11, sec2-12, sec2-13 and sec2-14 gene; Wherein: 1 is DL2000 dna molecular amount standard, and 2-9 is the pcr amplification product of s sec2-7, sec2-8, sec2-9, sec2-10, sec2-11, sec2-12, see2-13 and sec2-14 gene;
Fig. 3 is the active test experience of the superantigen of superantigen gene engineering peptides SEC2-1, SEC2-2, SEC2-3, SEC2-4, SEC2-5, SEC2-6, SEC2-7, SEC2-8, SEC2-9, SEC2-10, SEC2-11, SEC2-12, SEC2-13 and SEC2-14 (PMNC PBMC proliferation experiment) figure as a result.The negative contrast bovine serum albumin of X-coordinate (BSA), serial superantigen gene engineering peptides and positive control Enteromycin C 2; Ordinate zou is the reading value of ELIASA under OD570nm.
Embodiment
Embodiment 1
(1)
(A) superantigen gene engineering peptides SEC2-1 has the amino acid series among the sequence table SEQ ID NO:2:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?Iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?nc?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?t?c?m?y?g?g?I?t?k?h?e?g?n?h?f?d?n?g?n?l?q?n?v?l?I?r?v?y?e?nk?r?n?t?I?s?f?e?v?q?t?d?k?k?s?v?t?a?q?e?l?d?I?k
The information (referring to sequence table) of SEQ ID NO.2
(a) sequence signature;
*Length: 162 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 4 complete α spirals and 8 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68,81-87,108-118 form four βZhe Die structures respectively, and 129-154 amino acids residue forms three βZhe Dies.Except 7-11; Outside three βZhe Dies that two α spirals in 13-17 position and residue 129-154 form; Other α spirals and βZhe Die form an independent structures territory in three-dimensional structure; Constitute " β-bucket " shape structure, and 118 and two Zn atom binding site points of 122 hyte propylhomoserin residues formation.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-1 of coding SEC2-1 has nucleotide sequence among the sequence table SEQ ID NO:1.G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?CA?C?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?AT?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?GC?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?TA?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?GT?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?CT?G?C?T?A?T?T?T?T?T?C?A?T?C?C?A?A?A?G?A?T?A?A?T?G?T?A?G?G?T?A?A?A?G?TT?A?C?A?G?G?T?G?G?T?A?A?A?A?C?T?T?G?T?A?T?G?T?A?T?G?G?A?G?G?A?A?T?AA?C?A?A?A?A?C?A?T?G?A?A?G?G?A?A?A?C?C?A?C?T?T?T?G?A?T?A?A?T?G?G?G?AA?C?T?T?A?C?A?A?A?A?T?G?T?A?C?T?T?A?T?A?A?G?A?G?T?T?T?A?T?G?A?A?A?A?TA?A?A?A?G?A?A?A?C?A?C?A?A?T?T?T?C?T?T?T?T?G?A?A?G?T?G?C?A?A?A?C?T?GA?T?A?A?G?A?A?A?A?G?T?G?T?A?A?C?A?G?C?T?C?A?A?G?A?A?C?T?A?G?A?C?A?TA?A?A?A
The information (referring to sequence table) of SEQ ID NO.1
(a) sequence signature:
*Length: 486 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)
(B) superantigen gene engineering peptides SEC2-2 has the amino acid series among the sequence table SEQ ID NO:4:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?nc?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?t?c?m?y?g?g?I?t?k?h?e?g?n?h
The information (referring to sequence table) of SEQ ID NO.4
(a) sequence signature:
*Length: 122 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is folded by 4 complete α spirals and 5 β and is constituted.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68,81-87,108-118 form four βZhe Die structures respectively.Except 7-11, outside two the α spirals in 13-17 position, other α spirals and βZhe Die form an independent structures territory in three-dimensional structure, constitute " β-bucket " shape structure, and 118 and two Zn atom binding site points of 122 hyte propylhomoserin residues formation.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-2 of coding SEC2-2 has nucleotide sequence among the sequence table SEQ ID NO:3.
G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?C?A?C?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?A?TA?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?G?CA?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?A?CA?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?T?AA?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?A?AA?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?G?TA?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?C?TG?C?T?A?T?T?T?T?T?G?A?T?C?C?A?A?A?G?A?T?A?A?T?G?T?A?G?G?T?A?A?A?G?T?TA?C?A?G?G?T?G?G?T?A?A?A?A?C?T?T?G?T?A?T?G?T?A?T?G?G?A?G?G?A?A?T?A?AC?A?A?A?A?C?A?T?G?A?A?G?G?A?A?A?C?C?A?C
The information (referring to sequence table) of SEQ ID NO.3
(a) sequence signature:
*Length: 366 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)
(C) superantigen gene engineering peptides SEC2-3 has the amino acid series among the sequence table SEQ ID NO:6:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?nc?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?t?c?m?y?g
The information (referring to sequence table) of SEQ ID NO.6
(a) sequence signature:
*Length: 113 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure constitutes by 4 complete α spirals with 4 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68,81-87 form four β stack structures respectively.Except 7-11, outside two the α spirals in 13-17 position, other α spirals and βZhe Die form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-3 of coding SEC2-3 has nucleotide sequence among the sequence table SEQ ID NO:5.
G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?C?AC?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?A?T?A?TG?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?G?C?A?A?CT?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?AT?T?T?A?A?T?TT?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?A?A?A?AT?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?T?G?A?A?GA?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?G?G?T?A?G?T?T?G?A?TG?T?G?T?A?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?C?T?G?C?T?A?T?T?T?TT?C?A?T?C?C?A?A?A?G?A?T?A?A?TG?T?A?G?G?T?A?A?A?G?T?T?A?C?A?G?G?T?G?GT?A?A?A?A?C?T?T?G?T?A?T?G?T?A?T?G?G?G?A
The information (referring to sequence table) of SEQ ID NO.5
(a) sequence signature:
*Length: 339 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)
(D) superantigen gene engineering peptides SEC2-4 has the amino acid series among the sequence table SEQ ID NO:8:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?i?y?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?nc?y?f?s?s?k?d?nv?g?k?v?t?g?g?k?t?c?m?y?g?g?I?t?k
The information (referring to sequence table) of SEQ ID NO.8
(a) sequence signature:
*Length: 117 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 4 complete α spirals and 5 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68,81-87,108-117 form five βZhe Die structures respectively.Remove sub-7-11, outside two the α spirals in 13-17 position, other α spirals and βZhe Die form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-4 of coding SEC2-4 has nucleotide sequence among the sequence table SEQ ID NO:7.
G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?CA?C?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?AT?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?GC?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?AC?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?TA?A?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?AA?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?GT?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?CT?G?C?T?A?T?T?T?T?T?C?A?T?C?C?A?A?A?G?A?T?A?A?T?G?T?A?G?G?T?A?A?A?G?T?T?A?C?A?G?G?T?G?G?T?A?A?A?A?C?T?T?G?T?A?T?G?T?A?T?G?G?A?G?G?A?A?T?AA?C?A?A?A?A
The information (referring to sequence table) of SEQ ID NO.7
(a) sequence signature:
*Length: 351 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)
(E) superantigen gene engineering peptides SEC2-5 has the amino acid series among the sequence table SEQ ID NO:10:
g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?a?h?d?l?i?y?n?I?s?d?k?k?l?k?n?y?d?kv?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n?c?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?tc?m?y?g?g?I?t?k?h?e?g?n?h?f?d?n?g?n?l?q?n?v?l?I?r?v?y?e?n?k?r?n?t?I?s?f?e?v?q?t?d?k?k?s?v?t?aq?e?l?d?I?k?a?r?n?f?l?I?n?k?k?n?l?y?e?f?n?s?s?p?y?e?t?g?y?I?k?f?l?e?n?n?g?n?t?f?w?y?d?m?m?pa?p?g?d?k?f?d?q?s?k?y?l?m?m?y?n?d?n?k?t?v?d?s?k?s?v?k?I?e?v?h?l?t?t?k?n?g
The information (referring to sequence table) of SEQ ID NO.10
(a) sequence signature:
*Length: 221 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 4 complete α spirals and 12 βZhe Dies.Amino-acid residue 3-11 wherein, 52-60,138-154,190-202 form four complete αLuo Xuanjiegou respectively; Amino-acid residue 14-21,29-35,44-50,63-69,90-110,111-136,162-182, and 204-207,210-219 forms 12 βZhe Die structures respectively.The α spiral and the 111-136 that form of 138-154 position wherein, 162-182, and 204-207; Seven βZhe Dies that 210-219 forms, and 3-11,52-60; Three α spirals that 190-202 forms and and 14-21,29-35,44-50; 63-69, the formed βZhe Die of 90-100 forms two independent structures territories respectively in three-dimensional structure.And 100 and two Zn atom binding site points of 104 hyte propylhomoserin residues formation.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-5 of coding SEC2-5 has nucleotide sequence among the sequence table SEQ ID NO:9.G?G?T?A?C?G?A?T?G?G?G?T?A?A?T?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?AT?C?A?T?T?A?T?G?T?A?T?C?A?G?C?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?GA?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?TA?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?AA?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?AG?T?A?C?A?A?A?G?A?T?G?A?A?G?T?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?AA?A?T?T?A?C?T?A?T?G?T?A?A?A?C?T?G?C?T?A?T?T?T?T?T?C?A?T?C?C?A?A?A?G?AT?A?A?T?G?T?A?G?G?T?A?A?A?G?T?T?A?C?A?G?G?T?G?G?T?A?A?A?A?C?T?T?G?TA?T?G?T?A?T?G?G?A?G?G?A?A?T?A?A?C?A?A?A?A?C?A?T?G?A?A?G?G?A?A?A?C?CA?C?T?T?T?G?A?T?A?A?T?G?G?G?A?A?C?T?T?A?C?A?A?A?A?T?G?T?A?C?T?T?A?T?AA?G?A?G?T?T?T?A?T?G?A?A?A?A?T?A?A?A?A?G?A?A?A?C?A?C?A?A?T?T?T?C?T?T?TT?G?A?A?G?T?G?C?A?A?A?C?T?G?A?T?A?A?G?A?A?A?A?G?T?G?T?A?A?C?A?G?C?TC?A?A?G?A?A?C?T?A?G?A?C?A?T?A?A?A?A?G?C?T?A?G?G?A?A?T?T?T?T?T?T?A?AT?T?A?A?T?A?A?A?A?A?A?A?A?T?T?T?G?T?A?T?G?A?G?T?T?T?A?A?C?A?G?T?T?C?AC?C?A?T?A?T?G?A?A?A?C?A?G?G?A?T?A?T?A?T?A?A?A?A?T?T?T?A?T?T?G?A?A?AA?T?A?A?C?G?G?C?A?A?T?A?C?T?T?T?T?T?G?G?T?A?T?G?A?T?A?T?G?A?T?G?C?C?TG?C?A?C?C?A?G?G?C?G?A?T?A?A?G?T?T?T?G?A?C?C?A?A?T?C?T?A?A?A?T?A?T?T?TA?A?T?G?A?T?G?T?A?C?A?A?C?G?A?G?A?A?T?A?A?A?A?C?G?G?T?T?G?A?T?T?C?TA?A?A?A?G?T?G?T?G?A?A?G?A?T?A?G?A?A?G?T?C?C?A?C?C?T?T?A?C?A?A?C?A?AA?G?A?A?T?G?G?A
The information (referring to sequence table) of SEQ ID NO.9
(a) sequence signature:
*Length: 663 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)
(F) superantigen gene engineering peptides SEC2-6 has the amino acid series among the sequence table SEQ ID NO:12:
g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?a?h?d?l?Iy?n?I?s?d?k?k?l?k?n?y?d?kv?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n?c?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?tc?m?y?g?g?I?t?k
The information (referring to sequence table) of SEQ ID NO.12
(a) sequence signature:
*Length: 99 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 2 complete α spirals and 5 βZhe Dies.Amino-acid residue 3-11 wherein, 52-60 forms two complete αLuo Xuanjiegou respectively; Amino-acid residue 14-21,29-35,44-50,63-69,90-99 form five βZhe Die structures respectively.All α spirals and βZhe Die form an independent structures territory in three-dimensional structure, form " β-bucket " shape structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-6 of coding SEC2-6 has nucleotide sequence among the sequence table SEQ ID NO:11.G?G?T?A?C?G?A?T?G?G?G?T?A?A?T?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?AT?C?A?T?T?A?T?G?T?A?T?C?A?G?C?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?GA?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?TA?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?AA?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?AG?T?A?C?A?A?A?G?A?T?G?A?A?G?T?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?AA?A?T?T?A?C?T?A?T?G?T?A?A?A?C?T?G?C?T?A?T?T?T?T?T?C?A?T?C?C?A?A?A?G?AT?A?A?T?G?T?A?G?G?T?A?A?A?G?T?T?A?C?A?G?G?T?G?G?T?A?A?A?A?C?T?T?G?TA?T?G?T?A?T?G?G?A?G?G?A?A?T?A?A?C?A?A?A?A
The information (referring to sequence table) of SEQ ID NO.11
(a) sequence signature:
*Length: 297 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)
(G) superantigen gene engineering peptides SEC2-7 has the amino acid series of sequence table SEQ ID NO:14:
g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?a?h?d?l?i?y?n?I?s?d?k?k?l?k?n?y?d?kv?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n?c?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?tc?m?y?g
The information (referring to sequence table) of SEQ ID NO.14
(a) sequence signature:
*Length: 95 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 2 complete α spirals and 4 βZhe Dies.Amino-acid residue 3-11 wherein, 52-60 forms two complete αLuo Xuanjiegou respectively; Amino-acid residue 14-21,29-35,44-50,63-69 form four βZhe Die structures respectively.Two α spirals and four βZhe Dies form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-7 of coding SEC2-7 has nucleotide sequence among the sequence table SEQ ID NO:13.G?G?T?A?C?G?A?T?G?G?G?T?A?A?T?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?AT?C?A?T?T?A?T?G?T?A?T?C?A?G?C?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?GA?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?TA?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?AA?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?AG?T?A?C?A?A?A?G?A?T?G?A?A?G?T?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?AA?A?T?T?A?C?T?A?T?G?T?A?A?A?C?T?G?C?T?A?T?T?T?T?T?C?A?T?C?C?A?A?A?G?AT?A?A?T?G?T?A?G?G?T?A?A?A?G?T?T?A?C?A?G?G?T?G?G?T?A?A?A?A?C?T?T?G?TA?T?G?T?A?T?G?G?A
The information (referring to sequence table) of SEQ ID NO.13
(a) sequence signature:
*Length: 285 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)
(H) superantigen gene engineering peptides SEC2-8 has the amino acid series among the sequence table SEQ ID NO:16:
a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n?c?y?f?s?s?k?d?n?v?g?k?v?t?g?g?k?t?c?m?y?g?g?I?t?k?he?g?n?h?f?d?n?g?n?l?q?n?v?l?I?r?v?y?e?n?k?r?n?t?I?s?f?e?v?q?t?d?k?k?s?v?t?a?q?e?l?d?I?k
The information (referring to sequence table) of SEQ ID NO.16
(a) sequence signature:
*Length: 89 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 2 α spirals and 5 βZhe Dies.Amino-acid residue 1-5 wherein, 83-89 forms two αLuo Xuanjiegou respectively; Amino-acid residue 8-14,35-45,56-64,66-77,79-81 form five βZhe Die structures respectively.Two α spirals and five βZhe Dies form an independent structures territory in three-dimensional structure.And 45 and two Zn atom binding site points of 49 hyte propylhomoserin residues formation.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-8 of coding SEC2-8 has nucleotide sequence among the sequence table SEQ ID NO:15.G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?G?T?A?G?T?T?G?A?T?G?T?G?TA?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?C?T?G?C?T?A?T?T?T?T?T?C?AT?C?C?A?A?A?G?A?T?A?A?T?G?T?A?G?G?T?A?A?A?G?T?T?A?C?A?G?G?T?G?G?T?AA?A?A?C?T?T?G?T?A?T?G?T?A?T?G?G?A?G?G?A?A?T?A?A?C?A?A?A?A?C?A?T?G?A?A?G?G?A?A?A?C?C?A?C?T?T?T?G?A?T?A?A?T?G?G?G?A?A?C?T?T?A?C?A?A?A?A?TG?T?A?C?T?T?A?T?A?A?G?A?G?T?T?T?A?T?G?A?A?A?A?T?A?A?A?A?G?A?A?A?C?A?CA?A?T?T?T?C?T?T?T?T?G?A?A?G?T?G?C?A?A?A?C?T?G?A?T?A?A?G?A?A?A?A?G?T?GT?A?A?C?A?G?C?T?C?A?A?G?A?A?C?T?A?G?A?C?A?T?A?A?A?A
The information of SEQ ID NO.15 (referring to the preface table)
(a) sequence signature:
*Length: 267 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)
(I) superantigen gene engineering peptides SEC2-9 has the amino acid series among the sequence table SEQ ID NO:18:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?Iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?I?n?e?d?I?a?k?k?y?k?d?e?v?v?d?v?y?g
The information (referring to sequence table) of SEQ ID NO.18
(a) sequence signature:
*Length: 86 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 4 complete α spirals and 4 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68,81-86 form four βZhe Die structures respectively.Remove 7-11, outside two α spirals that 13-17 forms, other two α spirals and four βZhe Dies form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-9 of coding SEC2-9 has nucleotide sequence among the sequence table SEQ ID NO:17.G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?CA?C?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?AT?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?GC?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?AC?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?TA?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?AA?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?GT?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A
The information (referring to sequence table) of SEQ ID NO.17
(a) sequence signature:
*Length: 258 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)
(J) superantigen gene engineering peptides SEC2-10 has the amino acid series among the sequence table SEQ ID NO:20:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d
The information (referring to sequence table) of SEQ ID NO.20
(a) sequence signature:
*Length: 79 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 4 complete α spirals and 3 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68 forms three βZhe Die structures respectively.Remove 7-11, outside two α spirals that 13-17 forms, other two α spirals and four βZhe Dies are forming an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-10 of coding SEC2-10 has nucleotide sequence among the sequence table SEQ ID NO:19.
G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?C?A?C?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?AT?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?GC?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?AC?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?TA?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?AA?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T
The information (referring to sequence table) of SEQ ID NO.19
(a) sequence signature:
*Length: 237 base pairs
*Type: nucleic acid
*Chain: two strands
*Topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: golden yellow grape ball coccus (Staphylococcus aureus)
(K) superantigen gene engineering peptides SEC2-11 has the amino acid series among the sequence table SEQ ID NO:22:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n
The information (referring to sequence table) of SEQ ID NO.22
(a) sequence signature:
*Length: 92 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 4 complete α spirals and 4 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29,70-78 forms four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68,81-86 form four βZhe Die structures respectively.Remove 7-11, outside two α spirals that 13-17 forms, other two α spirals and four βZhe Dies form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-1 of coding SEC2-11 has nucleotide sequence among the sequence table SEQ ID NO:21.
G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?C?A?G?A?T?G?A?G?T?T?G?CA?C?A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?AT?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?GC?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A A?T?T?T?T?T?G?G?C?AC?A?T?G?A?T?T T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?TA?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?AA?A?T?G?A?A?G?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?GT?A?G?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?C
The information (referring to sequence table) of SEQ ID NO.21
(a) sequence signature:
*Length: 276 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)
(L) superantigen gene engineering peptides SEC2-12 has the amino acid series among the sequence table SEQ ID NO:24:
k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?a?h?d?l?i?y?n?I?s?d?k?k?lk?n?y?dk?v?k?t?e?l?I?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n
The information (referring to sequence table) of SEQ ID NO.24
(a) sequence signature:
*Length: 80 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 3 complete α spirals and 4 βZhe Dies.Amino-acid residue 1-5 wherein, 9-17,58-66 forms three complete αLuo Xuanjiegou respectively; Amino-acid residue 20-27,35-41,50-56,69-74 form four βZhe Die structures respectively.Except that the α spiral that 1-5 forms, other two α spirals and four βZhe Dies form an independent structures territory in three-dimensional structure.
(b) divide subclass: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
Coding sign indicating number SEC2-12 gene sec2-12, have nucleotide sequence among the sequence table SEQ ID NO:23.
A?A?A?T?C?A?A?G?T?G?A?G?T?T?T?A?C?T?G?G?T?A?C?G?A?T?G?G?G?T?A?A?T?A?TG?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?T?T?A?T?G?T?A?T?C?A?G?C?A?AC?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?G?A?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?TG?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?AA?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?TG?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?A?G?T?A?C?A?A?A?G?A?T?G?A?A?G?T?A?GT?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?A?A?A?T?T?A?C?T?A?T?G?T?A?A?A?C
The information (referring to sequence table) of SEQ ID NO.23
(a) sequence signature:
*Length: 240 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)
(M) superantigen gene engineering peptides SEC2-13 has the amino acid series of sequence table SEQ ID NO:26:
g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?a?h?d?l?i?y?n?l?I?s?d?k?k?l?k?n?y?d?kv?k?t?e?lI?n?e?d?l?a?k?k?y?k?d?e?v?v?d?v?y?g?s?n?y?y?v?n
The information (referring to sequence table) of SEQ ID NO.26
(a) sequence signature:
*Length: 74 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 2 complete α spirals and 4 βZhe Dies.Amino-acid residue 3-11 wherein, 70-78 forms four complete αLuo Xuanjiegou respectively: amino-acid residue 14-21,29-35,44-50,63-68 form four βZhe Die structures respectively.Two α spirals and four βZhe Dies form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-13 of coding SEC2-13 has nucleotide sequence among the sequence table SEQ ID NO:25.
G?G?T?A?C?G?A?T?G?G?G?T?A?A?T?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?AT?C?A?T?T?A?T?G?T?A?T?C?A?G?C?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?GA?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?C?A?T?TA?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?A?A?A?A?T?T?A?T?G?A?C?A?A?A?G?T?G?AA?A?A?C?A?G?A?G?T?T?A?T?T?A?A?A?T?G?A?A?G?A?T?T?T?A?G?C?A?A?A?G?A?AG?T?A?C?A?A?A?G?A?T?G?A?A?G?T?A?C?T?T?G?A?T?G?T?G?T?A?T?G?G?A?T?C?AA?A?T?T?A?C?T?A?T?G?T?A?A?A?C
The information (referring to sequence table) of SEQ ID NO.25
(a) sequence signature:
*Length: 222 territory bases are right
*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)
(N) superantigen gene engineering peptides SEC2-14 has the amino acid series among the sequence table SEQ ID NO:28:
e?s?q?p?d?p?t?p?d?e?l?h?k?s?s?e?f?t?g?t?m?g?n?m?k?y?l?y?d?d?h?y?v?s?a?t?k?v?m?s?v?d?k?f?l?ah?d?l?Iy?n?I?s?d?k?k?l?k?n?y?d?k?v?k?t?e?l?l
The information (referring to sequence table) of SEQ ID NO.28
(a) sequence signature:
*Length: 69 amino acid
*Type: peptide chain
*Chain: strand
*Structure: secondary structure is made up of 3 complete α spirals and 3 βZhe Dies.Amino-acid residue 7-11 wherein, 13-17,21-29 form four complete αLuo Xuanjiegou respectively; Amino-acid residue 32-39,47-53,62-68 form three βZhe Die structures respectively.Remove 7-11, outside two α spirals that 13-17 forms, other α spiral and three βZhe Dies form an independent structures territory in three-dimensional structure.
(b) molecule type: ripe peptide chain
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus (Staphylococcus aureus)
The gene sec2-14 of coding SEC2-14 has nucleotide sequence among the sequence table SEQ ID NO:27.
G?A?G?A?G?T?C?A?A?C?C?A?G?A?C?C?C?T?A?C?G?C?A?G?A?T?G?A?G?TT?C?G?C?A?C?A?A?A?T?C?A?A?G?T?G?A?G?G?T?T?T?A?C?T?G?G?T?A?C?G?A?TG?G?G?T?A?A?T?A?T?G?A?A?A?T?A?T?T?T?A?T?A?T?G?A?T?G?A?T?C?A?TT?A?T?G?T?A?T?C?A?G?C?A?A?C?T?A?A?A?G?T?T?A?T?G?T?C?T?G?T?A?GA?T?A?A?A?T?T?T?T?T?G?G?C?A?C?A?T?G?A?T?T?T?A?A?T?T?T?A?T?A?A?CA?T?T?A?G?T?G?A?T?A?A?A?A?A?A?C?T?A?A?A?A?A?A?T?T?A?T?G?A?C?AA?A?G?T?G?A?A?A?A?C?A?G?A?G?T?T?A?T?T?A
The information (referring to sequence table) of SEQ ID NO.27
(a) sequence signature:
*Length: 207 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)
(2) pcr amplification of full gene sec2-1, sec2-2, sec2-3, sec2-4, sec2-5, sec2-6, sec2-7, sec2-8, sec2-9, sec2-10, sec2-11, sec2-12, sec2-13 and the sec2-14 of coding superantigen gene engineering peptides
1) extraction of staphylococcus aureus gene group DNA
The single bacterium colony of inoculation streptococcus aureus is in 5ml liquid LB substratum, and 37 ℃ of shaking table incubated overnight are got the centrifugal collection thalline of culture of 1.5ml.(F. Ao Sibai is pressed in the extracting genome DNA operation to extract staphylococcus aureus gene group DNA; 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 " the USA New York nineteen ninety-five third edition P39-40 of John Wiley Sons press).
2) PCR design of primers and reaction conditions:
Design synthetic pcr primer thing be used for the increasing gene segment of sec2-1, sec2-2, sec2-3, sec2-4, sec2-5, sec2-6, sec2-7, sec2-8, sed-9, sec2-10, sec2-11, sec2-12, sec2-13 and sec2-14 respectively, primer sequence such as above-mentioned summary of the invention are outer said;
The PCR reaction system is: 10 * Pyrobest buffer5 μ l, dNTP250 μ mol, 0.02%BSA2 μ l, each 25pmol of upstream and downstream primer, template staphylococcus aureus gene group DNA0.1 μ g, ExTaq archaeal dna polymerase 2U, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions of each gene segment is:
95 ℃ of fs, 5 minutes;
94 ℃ of subordinate phase, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 60 seconds (remove the sec1-5 segment, the sec1-5 segment adopted 90 seconds); Totally 30 circulations;
72 ℃ of phase IIIs, 10 minutes;
3) evaluation of SEC2 gene: each segment pcr amplification product is through 1.2% agarose gel electrophoresis analysis and separation (referring to accompanying drawing 1, shown in 2); Downcut the purpose band of corresponding size, reclaim the test kit working instructions by the clean biochemical technology of Hangzhou Wei Te ltd glue and carry out the glue recovery; Reclaim product and be connected with the pGEM-T cloning vector respectively, be built into gene clone carrier pGEM-T-sec2-1, pGEM-T-sec2-2, pGEM-T-sec2-3, pGEM-T-sec2-4, pGEM-T-sec2-5, pGEM-T-sec2-6, pGEM-T-sec2-7, pGEM-T-sec2-8, pEM-T-sec2-9, pGEM-T-sec2-10, pGEM-T-sec2-11, pGEM-T-sec2-12, pGEM-T-sec2-13 and the pGEM-T-sec2-14 of a series of superantigen gene engineering peptides.Promega Company products pGEM-T 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. screening positive clone and measure dna sequence dna Si Telaer " fine works molecular biology experiment guide " the USA New York nineteen ninety-five third edition P39-40 of John Wiley Sons press), with the terminal cessation method of the two deoxidations of Sanger.
The expression of superantigen gene engineering peptides
1) structure of superantigen gene engineering peptides expression vector: respectively with gene clone carrier pGEM-T-sec2-1, pGEM--sec2-2, pGEM-T-sec2-3, pGEM-T-sec2-4, pGEM-T-sec2-5, pGEM-T-sec2-6, pGEM-T-sec2-7, pGEM-T-sec2-8, pGEM-T-sec2-9, pGEM-T-sec2-10, pGEM-T-sec2-11, pGEM-T-sec2-12, pGEM-T-sec2-13 and pGEM-T-sec2-14 DNA with EcoRI, XhoI double digestion, glue reclaims the gene fragment that test kit reclaims corresponding superantigen gene engineering peptides.Connect into expression vector respectively with the T4 dna ligase, be built into expression vector pET-28a-sec2-1, pET-28a-sec2-2, pET-28a-sec2-3, pET-28a-sec2-4, pET-28a-sec2-5, pET-28a-sec2-6, pET-28a-sec2-7, pET-28a-sec2-8, pET-28a-sec2-9, pET-28a-sec2-10, pET-28a-sec2-11, pET-28a-sec2-12, pET-28a-sec2-13 and pET-28a-sec2-14 through the pET-28a of same double digestion.Transformed E .coli BL21 (DE3) competent cell is identified correct recombinant clone through the terminal cessation method order-checking of the two deoxidations of Sanger.
2) abduction delivering of superantigen gene engineering peptides and purifying: respectively inoculation transformed each expression vector plasmid the single bacterium colony of BL21 (DE3) in the liquid LB substratum that contains 40 μ g/ml kantlex wherein; The activation culture of spending the night; With 1% inoculum size switching next stage, 37 ℃ of shaking tables are cultured to OD
600Be 0.6, add the IPTG of final concentration 1.0mmol/L, 30 ℃ of abduction deliverings 6 hours.
Centrifugal collection thalline respectively, (20mM imidazole pH7.9), is splined on the good Ni affinity column of pre-balance with 0.5ml/min speed for 20mMTirs-HCl, 0.5M NaCl to be resuspended in the level pad of 1/5 volume; To contain 10 column volumes of level pad washing of 50mM imidazole, the foreign protein of flush away non-specific binding; At last with elution buffer (20mMTirs-HCl; 0.5M NaCl; 200mM imidazole, pH7.9) purpose product both each superantigen gene engineering peptides SEC2-1, SEC2-2, SEC2-3, SEC2-4, SEC2-5, SEC2-6, SEC2-7, SEC2-8, SEC2-9, SEC2-10, SEC2-11, SEC2-12, SEC2-13 and SEC2-14 under the wash-out.
Eluted product concentrates through the dialysis method desalination.
The superantigen of superantigen gene engineering peptides is active
After getting the anti-freezing of fresh normal human peripheral, density gradient centrifugation separates PMNC.Regulate cell concn with the RPMI-1640 nutrient solution that contains 10% calf serum, with 1 * 10
5Cells/wdll is added in 96 orifice plates; Respectively each purified superantigen gene engineering peptides SEC2-1, SEC2-2, SEC2-3, SEC2-4, SEC2-5, SEC2-6, SEC2-7, SEC2-8, SEC2-9, SEC2-10, SEC2-11, SEC2-12, SEC2-13 and SEC2-14 final concentration 50ng/ml are added and be equipped with the hole; With BSA as negative control; The SEC2 standard substance are answered holes for 3 every kind as positive control.Cultivate 96h by normal condition, add the MTT liquid of 50ul/wd1l.Continue to cultivate 4h, the 1000r/min centrifugal collecting cell adds DMSO120ul/well, behind the dissolving 15min, measures the light absorption value (shown in accompanying drawing 3, superantigen gene engineering peptides stimulates the PMNC proliferation experiment) in each hole on the ELIASA with 570nm.
Experimental result shows that the superantigen gene engineering peptides among the present invention of trace can produce propagation by effective stimulation human peripheral blood mononuclearcell (PBMC).Its stimulation ability has been compared with the SEC2 standard substance significantly and has been reduced.Wherein SEC2-1, SEC2-4, SEC2-5 and SEC2-8 do not reduce than SEC2 because of destroying superantigen major function district and molecular weight to some extent, are equal to demonstrate the PBMC suitable with the SEC2 standard substance ability that stimulates proliferation under the mass concentration.A series of superantigen gene engineering peptides among this experimental result explanation the present invention all have certain superantigen biological activity; Its powerful stimulated in vitro PBMC multiplication capacity demonstrates the serial superantigen gene engineering peptides that utilizes among the present invention as the new antitumoral biotechnological formulation, has huge potentiality to be exploited.
Claims (3)
1.SEC2 superantigen gene engineering peptides is characterized in that: be aminoacid sequence among the sequence table SEQ ID NO:2.
2. the encoding sox of the said gene engineering peptides of claim 1 is characterized in that: be nucleotide sequence among the sequence table SEQ ID NO:1.
3. the heterogenetic expression method of the said SEC2 superantigen gene engineering peptides of claim 1 is characterized in that:
The nucleotide sequence sec2 of the said SEC2 superantigen gene engineering peptides of coding is cloned into expression vector pET-28a; With the e. coli bl21 is the host bacterium; Be built into the genetic engineering bacterium of heterogenous expression superantigen active gene engineering peptide, heterogenous expression has the active SEC2 gene engineering peptides of superantigen in substratum;
The cloning process of the nucleotide sequence of said coding SEC2 superantigen gene engineering peptides is following,
With staphylococcus aureus gene group DNA is template, adopts primer sec2-F:5 ' gAATTCgAgAgTCAACCAgACCCTA3 ' and sec2-R:5 ' gCCTCgAgTTATTTTATgTCTAgTTC 3 ' to amplify the sec2 gene fragment through round pcr.
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