CN101314616B - Hemorrhagic bacillus coli of intestine 0157:H7 shiga toxin IIB epitope peptide and uses thereof - Google Patents
Hemorrhagic bacillus coli of intestine 0157:H7 shiga toxin IIB epitope peptide and uses thereof Download PDFInfo
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Abstract
The invention belongs to the pharmaceutical biotechnology field, and relates to enterohemorrhagic E.coli O157:H7 Shiga toxin IIB epitope peptide and the application thereof. Four preferred polypeptides are derived from B-cell epitope of the subunit (Stx2A1) of EHEC O157:H7 Shiga toxin IIB epitope peptide. The product can be applied for preparing pharmaceuticals for the diagnosis and treatment of EHEC O157 infection and complications thereof.
Description
Technical field
The present invention relates to the medical biotechnology field, relate in particular to enterorrhagia Bacillus coil 0157: H7 shiga toxin IIB epitope peptide and application thereof.
Background technology
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is that important New Development infectious diseases common to human beings and animals is former, and the food poisoning of its initiation comprises that all over the world all there is large-scale outbreak of epidemic in China.The malicious spinach incident that involves 26 states in U.S.'s outburst was exactly by due to this fungi pollution in 2006, at the beginning of the year ends to 2,000 1999, having taken place up to now in the world in east China some areas, the O157:H7 of maximum-norm infects eruption and prevalence, therefore, the infection of this bacterium has become a global public health problem.Because EHEC O157:H7 bacterium is cultivated easily, infectivity is strong, the route of transmission is various, make the O157 bacterium very likely as the bacteriological weapon and the bio-terrorism war agent of following military struggle.The Center for Disease Control (CDC) is classified EHEC O157 bacterium as category-B bio-terrorism pathogenic agent and is strictly taken precautions against.In addition, the strong virulence factor of EHEC O157:H7 bacterium also might be used for the new bio weapon---the structure of genetic weapon.
Yet it is infected still lack the effectively preventing method at present.Studies have shown that: microbiotic can impel O157 bacterium release lethality shiga toxin, and (Shiga toxin Stx), thereby increases the weight of conditions of patients." enterorrhagia Bacillus coil 0157: H7 infectious diarrhea plan for emergency handling (trying) " the 5th regulation of China national Ministry of Health formulation in 2002: enterorrhagia Bacillus coil 0157: patient H7 and patient suspected ban use of microbiotic.Therefore, still be the needs of biological anti-terrorism no matter from public health, it is extremely urgent to explore new treatment means.
Studies show that: shiga toxin II (Stx2) is the main virulence factor of O157:H7.After Stx2 enters blood circulation, can cause the target tissue organ,, finally cause the generation of hemolytic uremic syndrome (HUS) and thrombus thrombopenia purpura severe complications such as (TTP) as the damage of renal glomerulus and colonic chrotoplast.Therefore, Stx2 is the main target for the treatment of the drug research of O157:H7 bacterium infection at present.
The Stx2 toxin is made up of 1 A subunit (32KDa) and 5 B subunits (7.7KDa/ monomer).Stx2 is by the B subunit and after acceptor Gb3 combines, and after entering endocytoplasmic reticulum A subunit and being cracked into A1 and two fragments of A2, A1 sheet cracked ends endoplasmic reticulum enters tenuigenin effect eukaryote rrna 28SrRNA, causes protein synthesis to stop.This effect can cause the death of the cell of kidney endotheliocyte, intestinal epithelial cells, Vero cell, Hela cell or other any Gb3 of having acceptors.
In view of the structure of Stx2 and its mechanism of causing a disease, mainly concentrate on the research of vaccine and the neutrality antibody of Stx2 at the drug research of Stx2, but still do not have vaccine and the neutrality antibody listing of Stx2 at present both at home and abroad.
In view of above research background the present invention prediction and with become Stx2A1 toxicity subunit on the B cell epitope; and the immunogenicity and the immune protective of B cell epitope of prediction identified B cell epitope peptide of the present invention can be used for the peptide vaccine research that EHEC O157:H7 infects and utilizes the B cell epitope peptide to prepare the infection of special monoclonal antibody diagnosis of Stx2 and treatment O157:H7.
Summary of the invention
An object of the present invention is to provide a kind of from enterorrhagia Bacillus coil 0157: the B cell epitope peptide of H7Stx2A1 toxicity subunit, it has at least a in the following aminoacid sequence:
1) aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or the SEQ ID NO:4;
2) aminoacid sequence that the aminoacid sequence shown in described SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or the SEQ ID NO:4 is still had described aminoacid sequence function through replacement, disappearance or the interpolation of one or several amino-acid residue.
Another object of the present invention provide a kind of encode above-mentioned from enterorrhagia Bacillus coil 0157: the nucleotide sequence of the B cell epitope peptide in the H7Stx2A1 toxicity subunit, it has at least a in the following nucleotide sequence:
1) nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or the SEQ ID NO:8;
2) different with the nucleotide sequence shown in described SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or the SEQ ID NO:8 but have the nucleotide sequence of same-code product.
Another object of the present invention provides above-mentioned from enterorrhagia Bacillus coil 0157: the application of the B cell epitope peptide of H7 shiga toxin II A1 subunit, it can be used for preparing the pharmaceutical composition of prevention or treatment Enterohemorrhagic Escherichia coli (EHEC) infection, or the preparation monoclonal antibody.
The medicine of prevention of the present invention or treatment Enterohemorrhagic Escherichia coli (EHEC) infection, it comprises above-mentioned from enterorrhagia Bacillus coil 0157: the B cell epitope peptide and the pharmaceutically acceptable carrier of H7 shiga toxin II A1 subunit, wherein above-mentioned pharmaceutically acceptable carrier comprise at least a in thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, the absorption carrier.Can be oral after the adjuvant heat-labile toxin is formed pharmaceutical preparation as B cell epitope peptide of the present invention, form the pharmaceutical preparation venoclysis with aluminium adjuvant and penetrate.
Of the present invention another can prevent or treat the vaccine composition of Enterohemorrhagic Escherichia coli (EHEC) infection, it also comprises above-mentioned from enterorrhagia Bacillus coil 0157: the B cell epitope peptide of H7 shiga toxin IIA1 subunit.B cell epitope peptide of the present invention and pharmaceutical preparation thereof can be used for prevention and treatment enterorrhagia Bacillus coil 0157: H7 infect due to hemorrhagic enteritis; Hemolytic uremic syndrome and thrombus thrombopenia purpura.
Thinking of the present invention is as follows: at first based on Stx2 and the interactional crystalline structure of part VITAMIN B4, press the prediction principle of B cell epitope, aminoacid sequence to the Stx2A1 subunit that comprises the toxicity avtive spot is analyzed, the B cell epitope of prediction Stx2A1, and employing DNAStar software analysis is estimated, synthetic B epitope peptide, with synthetic B cell epitope peptide coupling carrier albumen keyhole limpet hemocyanin (keyhole limpethemocyanin, KLH) the big ear rabbit of back immunity Japan is identified its immunogenicity; Dot ELISA and Westernblot identify the specificity of rabbit anti-serum, have obtained 4 B cell epitope peptides; With 4 immune Balb/c mouse of B cell epitope peptides difference; attack poison with natural Stx2; identify the immune protective of 4 B cell epitope peptides; the result shows: P1B cell epitope Toplink stimulates in the body generation and the antibody of Stx2; this peptide section is positioned at Stx2A1 fragment N and holds the 53rd～80 amino acids, comprises the tyrosine on 77 at the toxicity avtive spot of Stx2-the be positioned at Stx2A1 fragment N end.B epitope peptide of the present invention can be used for developing the peptide vaccine of the O157:H7 that people or mouse use, can be used for developing the polypeptide drugs that prevention or treatment EHEC O157:H7 infect.
The present invention is extensive studies through going deep into, based on the proteic crystalline structure of the Stx2 of EHEC O157:H7, be aided with the DNAStar software analysis, 4 the B cell epitope peptides that may induce body to produce humoral immunoresponse(HI) have optionally been synthesized, and its immunological characteristic estimated, find these 4 epitope peptides can be in Balb/c mouse body the humoral immunoresponse(HI) of inducing specific, the antiserum(antisera) of generation all can combine with Stx2 albumen generation specificity.Therefore, these four polypeptide can be used for the design of the multi-epitope peptide vaccine of EHEC O157:H7; 4 polypeptide of the present invention can be used for the vaccine that people and animal use and the exploitation of medicine.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.
Description of drawings
Figure 1A-Figure 1B represents the specificity of dot-ELISA evaluation rabbit anti-serum
A. the synoptic diagram of dot-ELISA envelope antigen; B. dot-ELISA result
1.P1B epitope peptide rabbit anti-serum
2.P2B epitope peptide rabbit anti-serum
3.P3B epitope peptide rabbit anti-serum
4.P4B epitope peptide rabbit anti-serum
5. normal rabbit serum;
6.PBS blank
Fig. 2 represents the specificity of immunoblotting evaluation rabbit anti-serum
1. the immunoblotting of negative rabbit anteserum
2.P1B the immunoblotting of epitope peptide rabbit anti-serum
3.P2B the immunoblotting of epitope peptide rabbit anti-serum
4.P3B the immunoblotting of epitope peptide rabbit anti-serum
5.P4B the immunoblotting of epitope peptide rabbit anti-serum;
Embodiment
Below in conjunction with specific embodiment; further set forth the present invention; be understood that; these embodiment only are used to illustrate the present invention rather than limitation of the present invention; to preparation method's of the present invention simple modifications, the utilization that reaches epitope peptide of the present invention and nucleotide sequence thereof all belongs to the scope of protection of present invention under design prerequisite of the present invention.
Embodiment 1The Screening and Identification of B epi-position:
1. the prediction of epitope peptide and synthetic
At Stx2 and Stx2 and the part VITAMIN B4 crystallographic structural analysis figure (numbering 1R4P and 2GA4) of the structured data library searching EHEC of NCBI O157:H7, network address:
Http:// www.ncbi.nlm.nih.gov/sites/entrez? db=Structure).Based on Stx2 and the interactional three-dimensional arrangement of part VITAMIN B4, according to being exposed to protein surface, contain stretching area and random coil or βZhuan Jiao, and the prediction principle of about 15～30 the B epitope peptides such as amino acid of peptide segment length, choose the amino acid peptide section on the Stx2A1 toxic fragment, adopt DNAStar software that the amino acid peptide section of prediction is carried out hydrophilic (Kyte-Doolittle scheme), surface accessibility (Emini scheme), plasticity-(Karp lus-Schulz scheme), secondary structure (Garnier and Chou-Fasman scheme) and antigenicity parameters such as (Jameson-Wolf schemes) are estimated, if in certain peptide section, antigenic index 〉=1, hydrophilic index 〉=0, amino acid whose surperficial possibility index 〉=1, simultaneously it is inner or neighbouringly have flexible structure again, and then this section is that the possibility of epitope is bigger.After the homology of B cell epitope peptide by BLAST retrieval comparison prediction and people, rabbit, mouse, final 4 possible B cell epitope peptide sequences determining that this institute uses, 4 possible B epitope polypeptides of synthetic (are assisted synthetic by BeiJing ZhongKe Asia Optical, article 4, the essential information of B cell epitope peptide sees Table 1), and difference coupling carrier albumen KLH.
The essential information of the Stx2B epi-position after table 1 prediction is synthetic
2.STX2A1B the cell epitope peptide immunogenicity is identified
2.1 the big ear rabbit of immunity Japan
The big ear rabbit of male Japan, be divided into 5 groups at random, every group two, use 4 B cell epitope peptides (P1-KLH, P2-KLH, P3-KLH, P4-KLH) of coupling KLH to add the subcutaneous multi-point injection immunity of complete freund adjuvant respectively, every injection 1ml, contain antigen peptide 1mg, after this add the incomplete Freund's adjuvant booster immunization every two weeks with same dosage, strengthen altogether 3 times, after two weeks, do not add the same dosage immunity of adjuvant more once, week back bloodletting, indirect ELISA detects antiserum titre, control group PBS substitutes polypeptide and carries out immunity, and immunization method and program are identical.
2.2ELISA detection serum titer
The envelope antigen of indirect ELISA is respectively 4 not polypeptide and the natural Stx2 of coupling KLH.Concrete grammar is as follows:
The bag quilt: is 2 μ g/ml with coating buffer with antigen diluent, adds enzyme plate, 100 μ l/ holes, and 4 ℃ are spent the night, PBS washing 5 times, empty doing; Sealing: add 1%BSA confining liquid 300 μ l/ holes, 4 ℃ are spent the night, and wash 5 times, empty doing, and it is standby to seal 4 ℃ of preservations; The rabbit anteserum dilution: by 1: 100,1: 200,1: 400...... carried out serial doubling dilution to the 10 pipes; Get bag by good enzyme plate, add dilute serum 100 μ l/ holes successively, 37 ℃ of water-baths 30 minutes are washed 5 times, empty doing; Goat anti-rabbit igg antibody working fluid (1: 20000 dilution) the 100 μ l/ holes that add horseradish peroxidase-labeled, 37 ℃ of water-baths 30 minutes washs 4 times, and are empty dried; Add substrate colour developing liquid 100 μ l/ holes, room temperature lucifuge reaction 5～10 minutes; Add stop buffer 100 μ l/ holes, on microplate reader, measure the OD value immediately with the 492nm wavelength; The result judges: OD value is anti-Stx2 antibody positive more than or equal to negative control (preceding 1: the 10 times of dilution of serum of mouse immune) 2.1 times.The maximum dilution multiple that is antibody positive is antibody titer.
The result: the indirect ELISA result shows that 4 synthetic polypeptide all can stimulate the big ear rabbit of Japan to produce the antibody of anti-polypeptide and Stx2, tiring of rabbit anteserum antibody and polypeptide and natural Stx2 reaction is as shown in table 2, and rabbit anteserum before the immunity and the detection of PBS immunity control group serum are negative, and this result shows that 4 B cell epitope polypeptide of the present invention have reactionogenicity and immunogenicity preferably.
Table 2 B cell epitope peptide immunize rabbit serum titer
The specificity of 3 polyclonal antibodies is identified
3.1 dot-ELISA detects the specificity of antibody
Envelope antigen: on nitrocellulose membrane (NC film), add each 2 μ l/ point of 4 antigen peptide, KLH and natural Stx2 respectively, treat its drying at room temperature.Sealing: the NC film of envelope antigen immersed in 5% the skim-milk, put 37 ℃ 1 hour.Washing: discard confining liquid, use TTBS rinsing film 3 times, each 1 minute, NC film following some stream filter paper and sponge.Add 4 kinds of antiserum(antisera)s and negative rabbit anteserum respectively, 2 μ l/ points, put 37 ℃ 15 minutes, TTBS flushing 3 times.The NC film is gone into the goat anti-rabbit igg of HRP mark, and working concentration is 1: 10000, put 37 ℃ 15 minutes, TTBS flushing 3 times.Add DAB colour developing liquid, jog is to colour developing, distilled water rinsing termination reaction.
3.2 immunoblotting detects the specificity of antibody
Get the natural Stx2 that has handled, and set protein molecular weight standard, concentrate glue, carry out vertical SDS-PAGE on 15% separation gel 5%, 80V, 30 minutes, adjustment voltage was 150V, about 1.5 hours.After electrophoresis finishes, take off gel, one another piece places on the nitrocellulose filter with Xylene Brilliant Cyanine G R-250 dye liquor rapid dyeing, and 15V constant voltage electrotransfer spends the night.Nitrocellulose filter after the transfer dyeed in Ponceau S 1 minute, and the labelled protein molecular criteria is sloughed redness with distilled water again.Transfer film is placed confining liquid (TTBS), 37 ℃, 150rpm, the jolting sealing discarded confining liquid in 60 minutes gently, used washings (TTBS) rinsing film 4 times, each 10～15 minutes.The rabbit anti-serum that adding is diluted with washings at 1: 1000,37 ℃, 150rpm, the jolting reaction is 60 minutes gently.TBS rinsing film 4 times, each 10～15 minutes.Adding is with the little rabbit igg of goat-anti (1: 20000) of the HRP mark of TBS dilution, 150rpm, and the jolting reaction is 60 minutes gently.TBS rinsing 4 times, each 10～15 minutes.Add DAB colour developing liquid, jog is to colour developing, distilled water rinsing termination reaction.
Dot-ELISA detected result (shown in Figure 1A and Figure 1B) shows: the rabbit anti-serum of 4 B cell epitope peptide preparations the colour developing spot occurs at corresponding polypeptide antigen, KLH and Stx2 place respectively, positive, and colour developing does not appear in the above-mentioned antigen of normal rabbit serum place, negative, this presentation of results: what the rabbit polyclonal antibody of 4 B cell epitope peptide preparations can be special reacts with B cell epitope peptide, Stx2 and carrier proteins KLH; Adopt immunoblotting that the specificity of 4 kinds of rabbit anti-serums is done to identify that further result (Fig. 2) shows: the A subunit that at the about 32KDa of molecular weight place is natural Stx2 locates, as seen the band of single positive reaction clearly; And be anti-hatching with preimmune serum, no specific reaction band occurs, and 4 special polyclonal antibodies at Stx2A subunit of B cell epitope inducing peptide bodies generation are described.
Embodiment 2Immune mouse challenge test screening protectiveness B cell epitope peptide
6～8 the week ages female Balb/c mouse, be divided into 5 groups at random, every group 5, use 4 B cell epitope peptide (P1-KLH of coupling KLH respectively, P2-KLH, P3-KLH, P4-KLH) subcutaneous multiple spot adds abdominal injection immunity Balb/c mouse, every injection 0.51ml, contain antigen 1 00 μ g, the immunity time is 0,2,4,6,8 weeks, totally 5 times, add the Fu Shi Freund's complete adjuvant the 1st time and add freund 's incomplete adjuvant the 2nd～4 time, last does not add the immunity of the same dosage abdominal injection of adjuvant once, and the 2nd, 3,4,5 immunity back 7d docking blood samplings adopt indirect elisa method to detect immune Balb/c mice serum tire (operation steps is the same); The positive rate of rotation of calculating antibody and geometry titre mean number (GMT) and average increment multiple.The last immunity is every mouse peritoneal injection lethal dose (LD after 10 days
100) natural Stx2,50ng/kg observes the survival rate of respectively organizing mouse in 10 days.Control group PBS substitutes polypeptide and carries out immunity, and immunization method and program are identical.
Table 3 immune serum specific antibody sun rate of rotation
Analysis ELISA detected result (table 3) increases by 4 times with immune front and back antibody titers and is positive commentaries on classics standard, calculates antibody male rotary rate, geometric mean titer (GMT) and the average increment multiple respectively organized after immune 2,3,4,5 times respectively.The positive rate of rotation of P1, P2, P3, P4 group is respectively 93.3%, 93.3%, 73.3% and 66.7% after table 3 data presentation, immunity 2 times, and the positive rate of rotation after the immunity 4 times is respectively 100%, 100%.93.3% and 86.7%.
Table 4 immune serum Stx2 antibody horizontal
As shown in table 4: the GMT of 4 groups of anti-Stx2 antibody is ascendant trend one by one with the increase of immune time, the GMT of P1, P2, P3, P4 is respectively 11380,12211,6948,5171 after the 5th immunity, be respectively the immunity before 113.8 times, 122.1 times, 69.5 times, 51.7 times, and the GMT of PBS control group be 1: 106 only for the immunity before 1.06 times.(table 3～4).
Above result proves that 4 single B cell epitope peptides all can stimulate mouse to produce special, effective humoral immunoresponse(HI).Can be used as the peptide vaccine component.
Stx2 attacks malicious protection test result and shows: mouse peritoneal injects 50ng/kg (LD
100) behind the natural Stx2 of dosage, each is organized mouse and all successively heavier toxicity symptom occurred, shows as the fluffy wrinkle of fur Zhe, dull, the minimizing of ingesting, listless drowsiness of activity.Especially occur with the mouse toxicity symptom of control group, P2, P3, P4 that fast (24h～72h), and be carrying out property and increase the weight of until death, and relatively slow (96h) appears in the mouse toxicity symptom of P1 group, symptom has the part mouse to recover gradually after occurring.Each is organized shown in concrete survival of mouse and the death condition table 5:
Attack the protection effect observation of poison behind table 5 mouse immune
* .P＜0.01vs PBS organizes; * .P＞0.05vs PBS group
By table 5 as seen; the PBS control group mice is attacked in the 144h of poison back all dead; and P1; P2; P3; the part mouse of P4 antigen immune group can tolerate the Stx2 toxin attacks of lethal dose and be survived; survival rate after 10 days is respectively 70%; 20%; 10%; 10%; analyze the P1 group through χ2Jian Yan and significant difference (P＜0.01) is arranged with the PBS control group; P2; P3; P4 immune group and PBS control group there was no significant difference (P＞0.05); illustrate that single P1B cell epitope peptide can stimulate in the body generation and the antibody of Stx2; has stronger immanoprotection action; and single P2; P3; P4B cell epitope peptide protection efficient is poor, but can not get rid of P2; P3; can produce immanoprotection action during P4B cell epitope peptide combined utilization.This result provides strong experimental basis for the component of candidate of utilizing the B cell epitope peptide to prepare Stx2 neutralizing monoclonal antibody and O157:H7 peptide vaccine.
Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the invention; when can doing a little change and improvement, so the present invention's protection domain is as the criterion when looking the claim person of defining.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A
＜120〉enterorrhagia Bacillus coil 0157: H7 shiga toxin IIB epitope peptide and application thereof
<130>
<160>8
<170>PatentIn?version?3.2
<210>1
<211>28
<212>PRT
＜213〉from enterorrhagia Bacillus coil 0157: the aminoacid sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>1
Asp?Ile?Arg?Gly?Leu?Asp?Val?Tyr?Gln?Ala?Arg?Phe?Asp?His?Leu?Arg
1 5 10 15
Leu?Ile?Ile?Glu?Gln?Asn?Asn?Leu?Tyr?Val?Ala?Gly
20 25
<210>2
<211>27
<212>PRT
＜213〉from enterorrhagia Bacillus coil 0157: the aminoacid sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>2
Val?Thr?Val?Thr?Ala?Glu?Ala?Leu?Arg?Phe?Arg?Gln?Ile?Gln?Arg?Glu
1 5 10 15
Phe?Arg?Gln?Ala?Leu?Ser?Glu?Thr?Ala?Pro?Val
20 25
<210>3
<211>22
<212>PRT
＜213〉from enterorrhagia Bacillus coil 0157: the aminoacid sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>3
Leu?Glu?His?Ile?Ser?Gln?Gly?Thr?Thr?Ser?Val?Ser?Val?Ile?Asn?His
1 5 10 15
Thr?Pro?Pro?Gly?Ser?Tyr
20
<210>4
<211>15
<212>PRT
＜213〉from enterorrhagia Bacillus coil 0157: the aminoacid sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>4
Val?Pro?Gly?Val?Thr?Thr?Val?Ser?Met?Thr?Thr?Asp?Ser?Ser?Tyr
1 5 10 15
<210>5
<211>84
<212>DNA
＜213〉coding enterorrhagia Bacillus coil 0157: the nucleotide sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>5
gatatacgag?ggcttgatgt?ctatcaggcg?cgttttgacc?atcttcgtct?gattattgag 60
caaaataatt?tatatgtggc?cggg 84
<210>6
<211>81
<212>DNA
＜213〉coding enterorrhagia Bacillus coil 0157: the nucleotide sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>6
gtcactgtca?cagcagaagc?cttacgcttc?aggcagatac?agagagaatt?tcgtcaggca 60
ctgtctgaaa?ctgctcctgtg 81
<210>7
<211>66
<212>DNA
＜213〉coding enterorrhagia Bacillus coil 0157: the nucleotide sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>7
cttgaacata?tatctcaggg?gaccacatcg?gtgtctgtta?ttaaccacac?cccaccgggc 60
agttat 66
<210>8
<211>45
<212>DNA
＜213〉coding enterorrhagia Bacillus coil 0157: the nucleotide sequence of the B cell epitope peptide of H7 shiga toxin IIA1 subunit
<400>8
gtgcccggtg?tgacaacggt?ttccatgaca?acggacagca?gttat 45
Claims (4)
1. one kind from enterorrhagia Bacillus coil 0157: the B cell epitope peptide of H7 shiga toxin II A1 subunit is characterized in that the aminoacid sequence of described epitope peptide is: SEQ ID NO:1.
2. a coding claim 1 is described from enterorrhagia Bacillus coil 0157: the nucleotide sequence of the B cell epitope peptide of H7 shiga toxin II A1 subunit is characterized in that the nucleotides sequence of described epitope peptide is classified as: SEQ ID NO:5.
3. claim 1 is described from enterorrhagia Bacillus coil 0157: the application of the B cell epitope peptide of H7 shiga toxin IIA1 subunit in the preparation monoclonal antibody.
4. vaccine that prevents Enterohemorrhagic Escherichia coli (EHEC) infection, it is described from enterorrhagia Bacillus coil 0157 to it is characterized in that comprising claim 1: the B cell epitope peptide and the pharmaceutically acceptable carrier of H7 shiga toxin II A1 subunit.
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| CN101948546B (en) * | 2010-09-25 | 2012-11-07 | 中国人民解放军军事医学科学院微生物流行病研究所 | Fusion protein SAmB as well as coding gene and applications thereof |
| AU2016297920A1 (en) * | 2015-07-26 | 2018-01-18 | Molecular Templates, Inc. | Cell-targeting molecules comprising shiga toxin A subunit effectors and CD8+ T-cell epitopes |
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| CN1847397A (en) * | 2006-01-20 | 2006-10-18 | 中国人民解放军第三军医大学 | EHEC 0157 shiga-like toxin IIA resisting subunit monoclonal antibody 5F3 light and heavy chain variable region gene and its application |
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| 罗萍等.肠出血性大肠杆菌O157∶H7志贺毒素ⅡA1亚单位单克隆抗体的制备和生物学特性鉴定.《第三军医大学学报》.2008,第30卷(第8期),698-701. * |
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