CN100368546C - Food-poisoning fungus concatenated fusion mycinamicin and use thereof - Google Patents
Food-poisoning fungus concatenated fusion mycinamicin and use thereof Download PDFInfo
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- CN100368546C CN100368546C CNB2005100171472A CN200510017147A CN100368546C CN 100368546 C CN100368546 C CN 100368546C CN B2005100171472 A CNB2005100171472 A CN B2005100171472A CN 200510017147 A CN200510017147 A CN 200510017147A CN 100368546 C CN100368546 C CN 100368546C
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Abstract
The present invention relates to a poly recombinant toxin for food poisoning bacteria and a use thereof, which belongs to the technical field of biology. Clostridium botulinum toxin A gene Hc, staphylococcus aureus enterotoxin A gene SEA, staphylococcus aureus enterotoxin B gene SEB, and escherichia coli O157: H7 veroB subunit toxin genes VT<1B> and VT<2B> are connected in series via a catenative sequence linker to form Hc-VT<1B>-SEA-VT<2B>-SEB, a soluble poly recombinant toxin protein for food poisoning bacteria is coded and expressed by the Hc-VT<1B>-SEA-VT<2B>-SEB, and a use of the Hc-VT<1B>-SEA-VT<2B>-SEB is disclosed. The present invention has the advantages that serum antibodies of five bacteriotoxins are obtained, the five toxins have high specificity and high reaction sensitivity, and cross reactions with other similar toxins in a certain range are carried out simultaneously. The present invention is used for solving the problems of complicated and long food detection process and lack of universal poly recombinant toxin kits.
Description
Technical field
The invention belongs to biological technical field, specifically comprise multi-joint fusion bacteriotoxin and establishment method thereof, become the beginning of broad spectrum immunological detection method.
Background technology
Food poisoning bacteria is the problem that often runs in the food sanitation, and bacterium (toxin) property food poisoning is again one of modal form.Ordinary method to bacteriologic test in the food is will identify each bacterium (toxin) and type thereof when checking, advantage is that science is accurate, but this process is complicated and comparatively very long, and the toxigenic bacterium strain is determined whether after separation to producing the toxin of strain or what kind again.The method for quick that uses is difficult to satisfy the requirement that detects broad covered area again at present.In food aspect detection side's science of law of cause of disease or toxin, the one, seek accurate detection technique, be to seek Fast Detection Technique on the other hand, seek the detection technique of popularity besides.For in the food or the bacterium of other field or bacteriotoxin also do not have the detection method of broad sense, can judge whether contain suspicious food poisoning bacteria or toxin in the food within a short period of time simultaneously, and no matter which kind of bacterium or toxin; If feminine gender just shows this food safety within the specific limits, if the positive then need in limited range, further determine to detect.This has important economics meaning in foodstuffs industry, on food sanitation and safety sometimes even more meaningful than rigorous examination.This method of being set up on the bacteriotoxic basis of multi-joint fusion might become the beginning of broad spectrum immunological detection method.
Summary of the invention
The invention provides a kind of food poisoning fungus concatenated fusion mycinamicin and uses thereof, complicated and comparatively very long to solve present food inspection process, the problem of shortage popularity concatenated fusion mycinamicin test kit.The technical scheme that the present invention takes is:
5 toxin genes: clostridium botulinum toxin A gene Hc, staphylococcus aureus toxin A gene SEA, B gene SEB, intestinal bacteria O 157:H7veroB subunit toxin gene VT
1B, VT
2B, being together in series with catenation sequence linker forms Hc-VT
1B-SEA-VT
2B-SEB.
Hc-VT
1B-SEA-VT
2BThe nucleotide sequence of-SEB such as SEQ ID NO:1.
The nucleotide sequence of food poisoning fungus concatenated fusion mycinamicin, the solubility food poisoning proteic aminoacid sequence of fungus concatenated fusion mycinamicin such as the SEQ ID NO:2 of its coded expression.
A kind of antigen is expressed generation by food poisoning fungus concatenated fusion mycinamicin nucleotide sequence, and what this expression produced is the soluble proteins that genetic engineering bacterium is expressed.
A kind of antibody adopts this above-mentioned antigen, with adding isopyknic complete freund adjuvant immunizing rabbit for 2mg/ time for the first time, every 7 days more respectively with same amount albumen and incomplete freund adjuvant immunizing rabbit 4 times, survey antibody titer after blood sampling obtain serum.
The application of above-mentioned antibody in preparation food toxin detection kit.
Advantage of the present invention and beneficial effect are: with 5 toxin genes of Escherichia coli O 157: H7, streptococcus aureus and three kinds of bacterium of Clostridium botulinum or fragment with linker be together in series Hc-VT
1B-SEA-VT
2B-SEB, 2928bp obtains inclusion body and two kinds of expression products of solubility in pET22-b.Immunizing rabbit, obtain anti-5 kinds of bacteriotoxic serum antibodies, have very high specificity and reaction sensibility with five kinds of toxin, the toxin with the close type of certain limit (in belonging to together) has cross reaction simultaneously, has so just obtained the broad spectrum antibody of limited cross within the specific limits.For the series connection of polygene antigen reaches at food poisoning bacteria or toxin wide spectrum, the methodology basis has been established in the foundation of screening method fast.Complicated and comparatively very long in order to solve present food inspection process, the problem of shortage popularity concatenated fusion mycinamicin test kit
Description of drawings
The linker of Fig. 1, gene PCR of the present invention connects and amplification.
Fig. 2, SDS-PAGE electrophoresis result, M: albumen marker, 1, inductive HVSVS-pET-226 not, 2,37 ℃ of inclusion body protein supernatants, 3,37 ℃ of inclusion body proteins, 4,25 ℃ of inclusion body protein supernatants, 5,25 ℃ of inclusion body proteins, 6,4h/2PTG inductive HVSVS-pET-226 recombinant plasmid.
Embodiment
The poison by food preparation of nucleotide sequence of fungus concatenated fusion mycinamicin of embodiment 1 the present invention
Material and method
1.1 bacterial strain
Streptococcus aureus A B (26072,26075) Staphylococcus aureus A B (26072,26075), Clostridium botulinum A (62A) Clostridium botulinum A (62A), Escherichia coli O 157: H7E.coli O157:H7; Other strains: Pseudomonas aeruginosa Pseudomonasaerugionsa, streptococcus aureus C (C
1, C
2) Staphylococcus aureus C (C
1, C
2), streptococcus aureus E Staphylococcus aureus E, monocytosis Listeria monocytogenes Listeria monocytogene, small intestine colon yersinia enterocolitica (52207) Yersinia enterocolitica (52207), Fu Shi yersinia enterocolitica Yersinia frederiksenii, osculant yersinia enterocolitica Yersiniaintermedia, Ke Shi yersinia enterocolitica Yersinia kristensenii, pseudonodule yersinia enterocolitica Yersiniapseudotuberculosis, yersinia pestis Yersinia pestis, Vibrio parahemolyticus Vibrioparahaemolyticus, Arizona Salmonellas Salmonella arizonae, salmonella paratyphi A Salmonella paratyphiA, salmonella paratyphi C Salmonella paratyphi C, Salmonella choleraesuls Salmonella choleraesuis, Salmonella enteritidis Salmonellaenteritidis, salmonella typhi Salmonella typhimurium, Salmonella gallinarum Salmonella pullorum, salmonella dublin Salmonella Dublin, salmonella london Salmonella London, salmonella aberdeen Salmonella aberdeen, Newport Salmonellas Salmonella newport, Bacillus cereus Bacillus cereus, Bacillus subtilus Bacillus subtilis, anthrax bacillus (low virulent strain) Bacillus anthracis (low toxic strain), jejunum knee bacterium Campylobacter jejuni, bacillus ceylonensis A Shigella sonnei, clostridieum welchii Clostriaium welcii, Clostridium botulinum BClostridium botulinumB, Clostridium botulinum CClostridium botulinum, C..
1.2 molecular cloning
1.2.1 primer and catenation sequence linker:
Primer:
P15’-CCA TGG ACC TTT CCA AAT ACG TAG ATA ATC-3’
P25’-CGT GCC CGG ACC CAG TGG CCT TTC TCC CCA TC-3’
P35’-ACT GGG TCC GGG TCC GGG CAC GCC TGA TTG TGT AACTGG-3’
P45’-TGC CCG GAC CCG GAC CAC GAA AAA TAA CTT CGA TGAATC-3’
P55’-TTC GTG GTC CGG GTC CGG GCA GCG AGA AAA GCGAAG AAA-3’
P65’-GCC CGG ACC ACT TGT ATA TAA ATA TAT ATC AAT-3’
P75’-AAG TGG TCC GGG TCC GGG CGC GGA TTG TGC TAA AGGTAA-3’
P85’-TCG CCC GGA CCC GGA CCG TCA TTA TTA AAC TGC ACTTCA-3’
P95’-TGA CGG TCC GGG TCC GGG CGA GAG TCA ACC AGATCC TAA-3’
P105’-CTC GAG TTA TTC AAA TAC CCG AAC AGT AAT ACT-3’Linker：5’-ggt ccg ggt ccg ggc-3’
1.2.2 genome extracts
Extract the gene Hc that specification sheets extracts clostridium botulinum toxin A respectively by the Vitagene genome, the gene SEA of its nucleotide sequence such as SEQ ID NO:3, staphylococcus aureus toxin A, the gene SEB of its nucleotide sequence such as SEQ ID NO:4, Staphylococcus aureus enterotoxin B, its nucleotide sequence such as SEQ ID NO:5, intestinal bacteria O 157:H7veroB subunit toxin gene VT
1B, its nucleotide sequence such as SEQ ID NO:6 and intestinal bacteria O 157:H7veroB subunit toxin gene VT
2B, its nucleotide sequence such as SEQ ID NO:7.
1.2.3PCR amplifying target genes Hc (the heavy chain nontoxicity fragment of clostridium botulinum toxin A): 94 ℃ of 5min, 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 1min, circulation of every annealing descends 1 ℃, to 33 ℃, and 35 circulations, 4 ℃ of preservations, the PCR condition of other toxin gene: 94 ℃ of 5min, 94 ℃ of 1min, 43 ℃ (SEA), 48 ℃ (SEB), 57 ℃ of (VT
2B), 55 ℃ of (VT
1B), 50sec, 72 ℃ of 1min.Hc：1338bp，VT1B：2137bp，VT2B：210bp，SEB：414bp，SEA：699bp。
1.2.3 connect toxin gene Hc1332bp, VT
1B207bp, VT
2B210bp, SEA699bp, SEB 414bp, Linker.The recombination total length is 2928bp.See Fig. 1,
For the first time PCR:Hc, VT
1B, SEA, VT
2B, SEB, 5 gene fragments increase;
PCR:VT for the second time
1B-SEA, VT
2B-SEB, two genes that increase connect product:
PCR:VT for the third time
1B-SEA-VT
2B-SEB, four genes that increase connect product:
The 4th PCR:Hc-VT
1B-SEA-VT
2B-SEB, five genes that increase connect product.
This recombination Hc-VT
1B-SEA-VT
2BThe nucleotide sequence of-SEB such as SEQ ID NO:1.
Show that through order-checking recombinant toxin gene is respectively protogene or portion gene sequence.
The expression and purification of embodiment 2 recombinant toxins of the present invention
Hc-VT
1B-SEA-VT
2B-SEB gene, hereinafter to be referred as HVSVS, the pET-22b expression vector, in host bacterium E.coli DH5 α respectively at 37 ℃ or 25 ℃, 1～6h, the expression of perhaps spending the night, 1mmol/L IPTG induces.The soluble proteins of expressing at the pET-22b expression vector downcuts the target protein band behind the SDS-PAGE electrophoresis, reclaim the back as immunogen.See Fig. 2.
HVSVS 37 ℃, the expression-form of 4h in pET-22b are (4.69%) two kind of solubility (5.29%) and inclusion body, induce with 1mmol/L IPTG.Almost be solubility expression albumen (9.9%) entirely in the time of 25 ℃.The expressing protein molecular weight is 112.33kDa.Most of soluble proteins is positioned at endochylema, and small portion is positioned at the cell pericentral siphon.
This food poisoning proteic aminoacid sequence of fungus concatenated fusion mycinamicin such as SEQ ID NO:2.
The preparation of embodiment 3 anti-recombinant toxin antibody
To express bacterial sediment with TE (pH8.0) damping fluid and hang, will express after thalline smashes with ultrasonic wave, centrifugal, go precipitation; Supernatant is packed in the dialysis tubing, concentrate 2h with the PEG20000 dialysis, supernatant after concentrating downcuts the target protein band behind the SDS-PAGE electrophoresis, in homogenizer, make it pulp, with 2 times TE (pH8.0) damping fluid mixing, put 4 ℃ of 12h, during jolting 3～4 times, use the centrifugal 20min of 10000r/min then, supernatant is fusion toxin HVSVS expressing protein.Measure protein concentration with ultraviolet spectrophotometer.With the solubility expression albumen of purifying add for 2mg/ time isopyknic complete freund adjuvant with syringe mode of averaging mixing or fully after the emulsification immunizing rabbit take the subcutaneous multi-point injection method in back for the first time.The back immunizing rabbit was mixed 4 times with same amount albumen and incomplete freund adjuvant respectively in 7 days again in later every interval.Last immunity heart blood sampling in 7 days, 37 ℃ of 1h make blood coagulation, put 4 ℃ again and spend the night, and make blood clot retraction.The centrifugal 10min of 10000g separates acquisition serum.Obtain serum with taking a blood sample routinely behind agar diffusion or the ELISA method survey antibody titer.
Measure antibody titer with agar diffusion test and ELISA: agar diffusion 1.8% agar, serum dilutes 0 respectively
*, 2
*, 4
*, 8
*, 16
*, 32
*Etc. several extension rates, observe the situation of antigen-antibody precipitation band behind 24～48h; The ELISA method is diluted 5 kinds of several extent of dilution of natural toxin with the coating buffer of pH9.6, and every hole adds 100 μ L, measures its susceptibility then respectively.Do specific detection with toxin and 29 kinds of other common foodborne bacterial pathogenses in belonging to together.See Table 1.
Special (OD of table 1 antibody
490nm)
| Bacterial strain or toxin | O157 | VT1 | VT2 | Hc | C.b A | C.b B | C.b C | HVSVS |
| OD
490nmNegative control bacterial strain or toxin OD
490nm |
1.003 0.262 SEA 2.402 0.321 | 2.979 0.366 SEB 2.412 0.323 | 2.144 0.302 A 0.803 0.353 | 2.322 0.352 B 0.674 0.298 | 2.337 0.377 E 0.418 0.293 | 1.059 0.399 C 1 0.613 0.324 | 1.088 0.414 C 2 0.698 0.292 | 2.916 0.307 - - - |
Annotate: A, B, E, C
1, C
2Be streptococcus aureus, C.b
A, C.b
B, C.b
CBe Clostridium botulinum.
Agar diffusion test shows, VT
1BBe 64
*, the antibody titer of other toxin is 16
*The ELISA test-results shows, VT
1BBe 51200
*, SEA is 6400
*, VT
2BBe 12800
*, Hc is 6400
*, SEB is 6400
*The antibody titer that solubility expression albumen and inclusion body immunizing rabbit produce much at one.Do not have obvious visible ELISA reaction with toxin and other 26 kinds common foodborne bacterial pathogens cultures beyond relevant toxin belongs to, but certain cross reaction is arranged with toxin in these three kinds of Pseudomonas.Antibody is respectively Hc31.25ng/mL, VT with the ELISA reaction sensibility of corresponding toxin
1B3.75ng/mL, SEA125.00ng/mL, VT
2B7.50ng/mL, SEB62.50ng/mL.
The detection of embodiment 5 simulation foodstuff samples
5 kinds of toxigenic bacterium kind cultures are mixed with milk, be prepared into 5 dilution series, serum is diluted into 3000
*, measure simulation foodstuff samples, 30 parts of each dilution series analog samples with ELISA.
Detect for the ELISA of totally 150 duplicate samples of the analog sample of breast and positive findings all to have occurred, and contrast and have assorted bacterium interferential sample all negative.Different extent of dilution measurement results show that the detection sensitivity of ELISA can reach 4 * 10
-2Ng toxin/mL breast.See Table 2
Table 2 analog sample (breast) ELISA sensitivity testing
| Toxin | Dilution | Negative control | ||||
| 4(μg) | 4×10 -1 | 4×10 -2 | 4×10 -3 | 4×10 -4 | ||
| BoNTs SEA SEB VT | 2.02 2.22 2.13 2.73 | 2.00 2.10 2.10 2.42 | 1.60 1.50 1.60 2.00 | 1.40 1.40 1.43 2.00 | 0.70 0.65 0.70 1.60 | 0.15 0.20 0.18 0.80 |
Bacteriotoxin ELISA detection kit in embodiment 6 food
Reagent and material
1, polystyrene plastic microcomponent culture plate
2, coating buffer (pH9.6moL carbonate buffer solution)
3, washings (pH7.4,0.01moLPBS)
4, insulation liquid (contains BSA0.1g, 0.05%PBS-Tween-20/100mL)
5, substrate solution: 1. pH5.0, phosphoric acid salt-citrate buffer solution of 0.2moL; 2. use phosphoric acid salt-citrate buffer solution 100mL, matching while using adds O-Phenylene Diamine (OPD) 40mg, 30%H2O20.15mL
6, stop buffer (2moLH2SO4)
7, serum antibody (3000 times of dilutions)
8, enzyme mark goat anti-rabbit antibody (1: 400 times of dilution)
Working method
1. sample preparation
Milk food: fresh milk can be directly as sample, if dense thick with 2 times of PBS dilutions.
Meat-based food: supernatant with 3 times of PBS dilutions, is got as working sample in the broken back of digested tankage.
2. antigen coated: after doing 10 times of dilutions with 100 liquid, every hole adds 100 μ L, and not tame antigen is made blank, and 4 ℃ are spent the night.
3. washing: with washings washing 3 times, soaked into 3 minutes at every turn, drain.
4. the every hole of serum that will be incubated liquid dilution adds 100 μ L, 37 ℃ 1 hour.
5. wash equally 3 times.
6. add ELIAS secondary antibody 100 μ L/ holes, 37 ℃ 1 hour.
7. wash equally 3 times.
8. adding substrate, colour developing was put in the magazine 20 minutes in 100 μ L/ holes.
9. add stop buffer 50 μ L/ holes, static 5 minutes.
10.490nm survey the OD value.Differ promptly positive more than the twice with the feminine gender value.
Sequence table
＜110〉institute of animal husbandry and veterinary medicine of Jilin University
＜120〉food poisoning fungus concatenated fusion mycinamicin
<130>liuzs2005
<160>7
<170>PatentIn version 3.2
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atggaccttt ccaaatacgt agataatcaa agattattat ctacatttac tgaatatatt 60
aagaatatta ttaatacttc tatattgaat ttaagatatg aaagtaatca tttaatagac 120
ttatctaggt atgcatcaaa aataaatatt ggtagtaaag taaattttga tccaatagat 180
aaaaatcaaa ttcaattatt taatttagaa agtagtaaaa ttgaggtaat tttaaaaaat 240
gctattgtat ataatagtat gtatgaaaat tttagtacta gcttttggat aagaattcct 300
aagtatttta acagtataag tctaaataat gaatatacaa taataaattg tatggaaaat 360
aattcaggat ggaaagtatc acttaattat ggtgaaataa tctggacttt acaggatact 420
caggaaataa aacaaagagt agtttttaaa tacagtcaaa tgattaatat atcagattat 480
ataaacagat ggatttttgt aactatcact aataatagat taaataactc taaaatttat 540
ataaatggaa gattaataga tcaaaaacca atttcaaatt taggtaatat tcatgctagt 600
aataatataa tgtttaaatt agatggttgt agagatacac atagatatat ttggataaaa 660
tattttaatc tttttgataa ggaattaaat gaaaaagaaa tcaaagattt atatgataat 720
caatcaaatt caggtatttt aaaagacttt tggggtgatt atttacaata tgataaacca 780
tactatatgt taaatttata tgatccaaat aaatatgtcg atgtaaataa tgtaggtatt 840
agaggttata tgtatcttaa agggcctaga ggtagcgtaa tgactacaaa catttattta 900
aattcaagtt tgtatagggg gacaaaattt attataaaaa aatatgcttc tggaaataaa 960
gataatattg ttagaaataa tgatcgtgta tatattaatg tagtagttaa aaataaagaa 1020
tataggttag ctactaatgc atcacaggca ggcgtagaaa aaatactaag tgcattagaa 1080
atacctgatg taggaaatct aagtcaagta gtagtaatga agtcaaaaaa tgatcaagga 1140
ataacaaata aatgcaaaat gaatttacaa gataataatg ggaatgatat aggctttata 1200
ggatttcatc agtttaataa tatagctaaa ctagtagcaa gtaattggta taatagacaa 1260
atagaaagat ctagtaggac tttgggttgc tcatgggaat ttattcctgt agatgatgga 1320
tggggagaaa ggccactggg tccgggtccg ggcacgcctg attgtgtaac tggaaaggtg 1380
gagtatacaa aatataatga tgacgatacc tttacagtta aagtgggtga taaagaatta 1440
tttaccaaca gatggaatct tcagtctctt cttctcagtg cgcaaattac ggggatgact 1500
gtaaccatta aaactaatgc ctgtcataat ggagggggat tcagcgaagt tatttttcgt 1560
ggtccgggtc cgggcagcga gaaaagcgaa gaaataaatg aaaaagattt gcgaaaaaag 1620
tctgaattgc agggaacagc tttaggcaat cttaaacaaa tctattatta caatgaaaaa 1680
gctaaaactg aaaataaaga gagtcacgat caatttttac agcatactat attgtttaaa 1740
ggctttttta cagatcattc gtggtataac gatttattag tagattttga ttcaaaggat 1800
attgttgata aatataaagg gaaaaaagta gacttgtatg gtgcttatta tggttatcaa 1860
tgtgcgggtg gtacaccaaa caaaacagct tgtatgtatg gtggtgtaac gttacatgat 1920
aataatcgat tgaccgaaga gaaaaaagtg ccgatcaatt tatggctaga cggtaaacaa 1980
aatacagtac ctttggaaac ggttaaaacg aataagaaaa atgtaactgt tcaggagttg 2040
gatcttcaag caagacgtta tttacaggaa aaatataatt tatataactc tgatgttttt 2100
gatgggaagg ttcagagggg attaatcgtg tttcatactt ctacagaacc ttcggttaat 2160
tacgatttat ttggtgctca aggacagtat tcaaatacac tattaagaat atatagagat 2220
aataaaacga ttaactctga aaacatgcat attgatatat atttatatac aagtggtccg 2280
ggtccgggcg cggattgtgc taaaggtaaa attgagtttt ccaagtataa tgaggatgac 2340
acatttacag tgaaggttga cgggaaagaa tactggacca gtcgctggaa tctgcaaccg 2400
ttactgcaaa gtgctcagtt gacaggaatg actgtcacaa tcaaatccag tacctgtgaa 2460
tcaggctccg gatttgctga agtgcagttt aataatgacg gtccgggtcc gggcgagagt 2520
caaccagatc ctaaaccaga tgagttgcac aaatcgagta aattcactgg tttgatggaa 2580
aatatgaaag ttttgtatga tgataatcat gtatcagcaa taaacgttaa atctatagat 2640
caatttctat actttgactt aatatattct attaaggaca ctaagttagg gaattatgat 2700
aatgttcgag tcgaatttaa aaacaaagat ttagctgata aatacaaaga taaatacgta 2760
gatgtgtttg gagctaatta ttattatcaa tgttattttt ctaaaaaaac gaatgatatt 2820
aattcgcatc aaactgacaa acgaaaaact tgtatgtatg gtggtgtaac tgagcataat 2880
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Gln Arg Gly Leu Ile Val Phe His Thr Ser Thr Glu Pro Ser Val Asn
705 710 715 720
Tyr Asp Leu Phe Gly Ala Gln Gly Gln Tyr Ser Asn Thr Leu Leu Arg
725 730 735
Ile Tyr Arg Asp Asn Lys Thr Ile Asn Ser Glu Asn Met His Ile Asp
740 745 750
Ile Tyr Leu Tyr Thr Ser Gly Pro Gly Pro Gly Ala Asp Cys Ala Lys
755 760 765
Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp Asp Thr Phe Thr Val
770 775 780
Lys Val Asp Gly Lys Glu Tyr Trp Thr Ser Arg Trp Asn Leu Gln Pro
785 790 795 800
Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr Val Thr Ile Lys Ser
805 810 815
Ser Thr Cys Glu Ser Gly Ser Gly Phe Ala Glu Val Gln Phe Asn Asn
820 825 830
Asp Gly Pro Gly Pro Gly Glu Ser Gln Pro Asp Pro Lys Pro Asp Glu
835 840 845
Leu His Lys Ser Ser Lys Phe Thr Gly Leu Met Glu Asn Met Lys Val
850 855 860
Leu Tyr Asp Asp Asn His Val Ser Ala Ile Asn Val Lys Ser Ile Asp
865 870 875 880
Gln Phe Leu Tyr Phe Asp Leu Ile Tyr Ser Ile Lys Asp Thr Lys Leu
885 890 895
Gly Asn Tyr Asp Asn Val Arg Val Glu Phe Lys Asn Lys Asp Leu Ala
900 905 910
Asp Lys Tyr Lys Asp Lys Tyr Val Asp Val Phe Gly Ala Asn Tyr Tyr
915 920 925
Tyr Gln Cys Tyr Phe Ser Lys Lys Thr Asn Asp Ile Asn Ser His Gln
930 935 940
Thr Asp Lys Arg Lys Thr Cys Met Tyr Gly Gly Val Thr Glu His Asn
945 950 955 960
Gly Asn Gln Leu Asp Lys Tyr Arg Ser Ile Thr Val Arg Val Phe Glu
965 970 975
<210>3
<211>1338
<212>DNA
＜213〉clostridium botulinum toxin A
<400>3
atggaccttt ccaaatacgt agataatcaa agattattat ctacatttac tgaatatatt 60
aagaatatta ttaatacttc tatattgaat ttaagatatg aaagtaatca tttaatagac 120
ttatctaggt atgcatcaaa aataaatatt ggtagtaaag taaattttga tccaatagat 180
aaaaatcaaa ttcaattatt taatttagaa agtagtaaaa ttgaggtaat tttaaaaaat 240
gctattgtat ataatagtat gtatgaaaat tttagtacta gcttttggat aagaattcct 300
aagtatttta acagtataag tctaaataat gaatatacaa taataaattg tatggaaaat 360
aattcaggat ggaaagtatc acttaattat ggtgaaataa tctggacttt acaggatact 420
caggaaataa aacaaagagt agtttttaaa tacagtcaaa tgattaatat atcagattat 480
ataaacagat ggatttttgt aactatcact aataatagat taaataactc taaaatttat 540
ataaatggaa gattaataga tcaaaaacca atttcaaatt taggtaatat tcatgctagt 600
aataatataa tgtttaaatt agatggttgt agagatacac atagatatat ttggataaaa 660
tattttaatc tttttgataa ggaattaaat gaaaaagaaa tcaaagattt atatgataat 720
caatcaaatt caggtatttt aaaagacttt tggggtgatt atttacaata tgataaacca 780
tactatatgt taaatttata tgatccaaat aaatatgtcg atgtaaataa tgtaggtatt 840
agaggttata tgtatcttaa agggcctaga ggtagcgtaa tgactacaaa catttattta 900
aattcaagtt tgtatagggg gacaaaattt attataaaaa aatatgcttc tggaaataaa 960
gataatattg ttagaaataa tgatcgtgta tatattaatg tagtagttaa aaataaagaa 1020
tataggttag ctactaatgc atcacaggca ggcgtagaaa aaatactaag tgcattagaa 1080
atacctgatg taggaaatct aagtcaagta gtagtaatga agtcaaaaaa tgatcaagga 1140
ataacaaata aatgcaaaat gaatttacaa gataataatg ggaatgatat aggctttata 1200
ggatttcatc agtttaataa tatagctaaa ctagtagcaa gtaattggta taatagacaa 1260
atagaaagat ctagtaggac tttgggttgc tcatgggaat ttattcctgt agatgatgga 1320
tggggagaaa ggccactg 1338
<210>4
<211>699
<212>DNA
＜213〉staphylococcus aureus toxin A
<400>4
agcgagaaaa gcgaagaaat aaatgaaaaa gatttgcgaa aaaagtctga attgcaggga 60
acagctttag gcaatcttaa acaaatctat tattacaatg aaaaagctaa aactgaaaat 120
aaagagagtc acgatcaatt tttacagcat actatattgt ttaaaggctt ttttacagat 180
cattcgtggt ataacgattt attagtagat tttgattcaa aggatattgt tgataaatat 240
aaagggaaaa aagtagactt gtatggtgct tattatggtt atcaatgtgc gggtggtaca 300
ccaaacaaaa cagcttgtat gtatggtggt gtaacgttac atgataataa tcgattgacc 360
gaagagaaaa aagtgccgat caatttatgg ctagacggta aacaaaatac agtacctttg 420
gaaacggtta aaacgaataa gaaaaatgta actgttcagg agttggatct tcaagcaaga 480
cgttatttac aggaaaaata taatttatat aactctgatg tttttgatgg gaaggttcag 540
aggggattaa tcgtgtttca tacttctaca gaaccttcgg ttaattacga tttatttggt 600
gctcaaggac agtattcaaa tacactatta agaatatata gagataataa aacgattaac 660
tctgaaaaca tgcatattga tatatattta tatacaagt 699
<210>5
<211>414
<212>DNA
＜213〉Staphylococcus aureus enterotoxin B
<400>5
gagagtcaac cagatcctaa accagatgag ttgcacaaat cgagtaaatt cactggtttg 60
atggaaaata tgaaagtttt gtatgatgat aatcatgtat cagcaataaa cgttaaatct 120
atagatcaat ttctatactt tgacttaata tattctatta aggacactaa gttagggaat 180
tatgataatg ttcgagtcga atttaaaaac aaagatttag ctgataaata caaagataaa 240
tacgtagatg tgtttggagc taattattat tatcaatgtt atttttctaa aaaaacgaat 300
gatattaatt cgcatcaaac tgacaaacga aaaacttgta tgtatggtgg tgtaactgag 360
cataatggaa accaattaga taaatataga agtattactg ttcgggtatt tgaa 414
<210>6
<211>213
<212>DNA
＜213〉colon bacillus 0157: H7veroB
<400>6
atggatacgc ctgattgtgt aactggaaag gtggagtata caaaatataa tgatgacgat 60
acctttacag ttaaagtggg tgataaagaa ttatttacca acagatggaa tcttcagtct 120
cttcttctca gtgcgcaaat tacggggatg actgtaacca ttaaaactaa tgcctgtcat 180
aatggagggg gattcagcga agttattttt cgt 213
<210>7
<211>210
<212>DNA
＜213〉colon bacillus 0157: H7veroB
<400>7
gcggattgtg ctaaaggtaa aattgagttt tccaagtata atgaggatga cacatttaca 60
gtgaaggttg acgggaaaga atactggacc agtcgctgga atctgcaacc gttactgcaa 120
agtgctcagt tgacaggaat gactgtcaca atcaaatcca gtacctgtga atcaggctcc 180
ggatttgctg aagtgcagtt taataatgac 210
Claims (6)
1. the nucleotide sequence of the fungus concatenated fusion mycinamicin of poisoning by food is characterized in that: 5 toxin genes: clostridium botulinum toxin A gene Hc, staphylococcus aureus toxin A gene SEA, B gene SEB, intestinal bacteria O 157:H7 veroB subunit toxin gene VT
1B, VT
2B, being together in series with catenation sequence linker forms Hc-VT
1B-SEA-VT
2B-SEB, clostridium botulinum toxin A gene Hc wherein, its nucleotide sequence such as SEQ ID NO:3, staphylococcus aureus toxin A gene SEA, its nucleotide sequence such as SEQ ID NO:4, Staphylococcus aureus enterotoxin B gene SEB, its nucleotide sequence such as SEQ ID NO:5, intestinal bacteria O 157:H7veroB subunit toxin gene VT
1B, its nucleotide sequence such as SEQ ID NO:6 and Escherichia coli O 157: H7 veroB subunit toxin gene VT
2B, its nucleotide sequence such as SEQ ID NO:7, the nucleotide sequence of Linker: ggt ccg ggt ccg ggc.
2. the nucleotide sequence of food poisoning fungus concatenated fusion mycinamicin according to claim 1 is characterized in that: Hc-VT
1B-SEA-VT
2BThe nucleotide sequence of-SEB such as SEQ ID NO:1.
3. the nucleotide sequence of food poisoning fungus concatenated fusion mycinamicin according to claim 2, the solubility food poisoning proteic aminoacid sequence of fungus concatenated fusion mycinamicin such as the SEQ ID NO:2 of its coded expression.
4. an antigen is expressed generation by food poisoning fungus concatenated fusion mycinamicin nucleotide sequence as claimed in claim 2, and what this expression produced is the soluble proteins that genetic engineering bacterium is expressed.
5. antibody adopts as antigen as described in the claim 4, adds isopyknic complete Freund's adjuvant immunizing rabbit for the first time with 2mg/ time, more respectively with same amount albumen and incomplete Freund's adjuvant immunizing rabbit 4 times, surveys the acquisition serum of taking a blood sample behind the antibody titer every 7 days.
6. the application of antibody as claimed in claim 5 in preparation food toxin detection kit.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6080400A (en) * | 1995-03-24 | 2000-06-27 | Ophidian Pharmaceuticals, Inc. | Compositions for the prevention and treatment of verotoxin-induced disease |
| CN1458527A (en) * | 2002-08-21 | 2003-11-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chromatographic paper strip and method for quick detecting pathogen and toxin in food |
| CN1580263A (en) * | 2003-08-13 | 2005-02-16 | 中国科学院沈阳应用生态研究所 | Interleukin-2 and enterotoxin A fusion gene and its preparation |
| US6936423B1 (en) * | 1999-04-27 | 2005-08-30 | Binie V. Lipps | Anti-LTNF for in vitro assay of biological toxins |
-
2005
- 2005-09-21 CN CNB2005100171472A patent/CN100368546C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6080400A (en) * | 1995-03-24 | 2000-06-27 | Ophidian Pharmaceuticals, Inc. | Compositions for the prevention and treatment of verotoxin-induced disease |
| US6936423B1 (en) * | 1999-04-27 | 2005-08-30 | Binie V. Lipps | Anti-LTNF for in vitro assay of biological toxins |
| CN1458527A (en) * | 2002-08-21 | 2003-11-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chromatographic paper strip and method for quick detecting pathogen and toxin in food |
| CN1580263A (en) * | 2003-08-13 | 2005-02-16 | 中国科学院沈阳应用生态研究所 | Interleukin-2 and enterotoxin A fusion gene and its preparation |
Non-Patent Citations (1)
| Title |
|---|
| IL-2与金黄色葡萄球菌肠毒素A和B融合基因的克隆及表达. 杨立泉，吴文芳，时成波等.生物技术，第14卷第3期. 2004 * |
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