CN101215568B - Anthrax resisting polypeptide and its application and preparation method - Google Patents
Anthrax resisting polypeptide and its application and preparation method Download PDFInfo
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- CN101215568B CN101215568B CN2008100452126A CN200810045212A CN101215568B CN 101215568 B CN101215568 B CN 101215568B CN 2008100452126 A CN2008100452126 A CN 2008100452126A CN 200810045212 A CN200810045212 A CN 200810045212A CN 101215568 B CN101215568 B CN 101215568B
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Abstract
The invention provides an anti-anthrax polypeptide gene, a recombinant plasmid, a polypeptide, the application and a process for preparation which comprises following steps: operationally connecting a gene of an encoding antibody simulacrum with the gene of an encoding recombinant mutation bacillus anthracis proteantigen to obtain the gene which expresses a recombinant anti-anthrax polypeptide, inserting the gene of the encoding antibody simulacrum into the gene of the recombinant mutation bacillus anthracis proteantigen through the technique of double chain oligonucleotide point mutation to form the recombinant plasmid of the invention, transfecting the recombinant plasmid which is obtained into coli bacteria BL-21 project engineering bacteria to obtain engineering bacteria cells of the anti-anthrax polypeptide, obtaining the anti-anthrax polypeptide through extracting supernate which contains polypeptide from a great amount of increasing bacteria, centrifugal precipitation cells and saccharose with an addex-magnesiumand method and purifying the supernate with florisil column. The anti-anthrax polypeptide specifically can damage the biological activity of bacillus anthracis toxin and does not attack normal human cells.
Description
Technical field
The present invention relates to a kind of gene, recombinant plasmid, polypeptide and application thereof and preparation method of anthrax resisting polypeptide, described anthrax is the lethal infection disease due to anthrax bacillus or the anthrax toxin.
Background technology
Lethal infection disease due to anthrax bacillus or the anthrax toxin is the grave danger to human health always.In addition, in bio-terrorism attacked, anthrax bacillus or anthrax toxin also were the most appalling pathogenic agent and toxin always.To the lethal infection disease due to anthrax bacillus or the anthrax toxin, present means of prevention mainly is to adopt antibiotic, antitoxic serum, Antybody therapy and vaccine prevention injection.Because it is above-mentioned means are not very effective, human always at the new controlling way of positive searching.
Summary of the invention
One object of the present invention is to provide a kind of gene, recombinant plasmid, polypeptide of anthrax resisting polypeptide, and the biological activity of this peptide species specific destruction anthrax bacillus of energy or anthrax toxin but can not injure normal cell.A further object of the present invention provides the preparation method of above-mentioned anthrax resisting polypeptide.
The present inventor has made up a kind of simplification stand-in of antibody, the gene of encoding antibody stand-in is the described nucleotide sequence of SEQ ID NO.12 in the sequence table, the nucleotide sequence of above-mentioned encoding antibody stand-in is heavy chain CDR1 districts of anti-anthrax bacillus proteantigen-lethal gene complex body antibody (NCBI CAL58671) of producing with cynomolgus monkey respectively, and the gene in anti-anthrax bacillus proteantigen structural domain 1 antibody (NCBIABF69350) the light chain CDR3 district that heavy chain FR2 linkage section and home mouse produce is that the source structure forms.
Anthrax toxin with height bio-hazard is a kind of binary toxin, and its biocidal is extremely strong.Anthrax toxin is made of two kinds of compositions: 1) proteantigen (protective antigen, PA); 2) necrosin and edema factor (lethal factor, LF; Edema factor, EF).Experimentation on animals shows that proteantigen and necrosin are united use, can cause necrocytosis rapidly, does not then induce reaction but give animal respectively separately.
Because the basal component of anthrax resisting polypeptide of the present invention is PA, therefore must manage to remove PA to human body and the Normocellular toxicity of animal.Among the present invention, used two kinds of means to remove PA to Normocellular toxicity: 1) amino-acid residue of being responsible for associated receptor on the identification target cell among the PA to be suddenlyd change 2) connect above-mentioned antibody analog at the carboxyl terminal of PA.
The technical scheme that makes up anthrax resisting polypeptide of the present invention is as follows:
The gene (SEQ ID NO.2,4,6,8) of encoding mutant amino-acid residue is operably connected with proteantigen (PA) gene (seeing the SEQ ID NO.1 in the sequence table) of anthrax bacillus, thereby obtain the nucleotide sequence of coding recombination mutation anthrax bacillus proteantigen (PA) gene, the gene of coding recombination mutation anthrax bacillus proteantigen is the described nucleotide sequence of SEQ ID NO.10 in the sequence table.
With the carboxyl end groups of the genes of SEQ ID NO.12 of above-mentioned encoding antibody stand-in and the genes of SEQ ID NO.10 of coding recombination mutation anthrax bacillus proteantigen because of being connected, obtain the gene of express recombinant anthrax resisting polypeptide, this gene is the described nucleotide sequence of SEQ ID NO.14 in the sequence table.
Recombinant plasmid of the present invention, be with aforesaid SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 nucleotide sequence forms recombination mutation anthrax bacillus proteantigen gene (SEQ ID NO.10) in the gene of the proteantigen (SEQID NO.1) of double-stranded oligonucleotide point mutation technology insertion anthrax bacillus, the nucleotide sequence (SEQ ID NO.12) with aforesaid antibody analog forms because of (SEQ ID NO.10) through the carboxyl end groups of double-stranded oligonucleotide point mutation technology insertion recombination mutation anthrax bacillus proteantigen again.
Another aspect of the present invention provides the preparation method of anthrax resisting polypeptide of the present invention.The gene of the described encoding antibody stand-in of SEQ IDNO.12 in the sequence table is operably connected with the pET22b plasmid that has loaded coding recombination mutation anthrax bacillus proteantigen genes of SEQ ID NO.10 obtains the gene of coding reorganization anthrax resisting polypeptide, the gene that obtains imported in intestinal bacteria (E.coli) BL-21 (DE3) the engineering bacterium expression system express, promptly obtain anthrax resisting polypeptide of the present invention (called after PA-anti-PA through His-tag post (Ni Sepharose High Performance post) separation and purification, be called for short PA-aPA, below describe and to use this name).
Anthrax resisting polypeptide of the present invention can be used in the medicine of preparation treatment or prevention anthrax bacillus or anthrax toxin lethal infection.Can make the pharmaceutical composition that is suitable for clinical use by the polypeptide among the present invention is added pharmaceutically acceptable carrier or vehicle or optional additional components.Its mechanism is: anthrax resisting polypeptide monomer of the present invention can be formed a heterozygosis heptamer with wild-type anthrax bacillus proteantigen monomer and stride the fenestra road on target cell membrane, this heterozygosis heptamer is striden the fenestra road and partially or completely lost the biological activity that wild heptamer is striden the fenestra road.Wild-type anthrax bacillus necrosin and edema factor can't and pass heptamer and stride the fenestra road and enter cell with heptamer identification like this, thereby can't kill host cell; Simultaneously the antibody analog on the anthrax resisting polypeptide monomer of the present invention again can in and proteantigen-lethal gene complex body; This has just reached us and has prevented and treated the purpose of anthrax bacillus or anthrax toxin lethal infection.
Description of drawings
Fig. 1 contains the genes of SEQ ID NO.12 of encoding antibody stand-in and the structural representation of the recombinant plasmid pCHCA-PA1 of the recombination mutation anthrax bacillus proteantigen genes of SEQ ID NO.10 that encodes.
Fig. 2 is the structural representation of anthrax resisting polypeptide of the present invention (PA-aPA), among the figure, Mimetic is anti-anthrax bacillus proteantigen antibody analog, and S337K, G342S, I656R, N682S and D683K are the amino-acid residue that is suddenlyd change in the anthrax bacillus proteantigen.
The survival curve that Fig. 3 is protected through anthrax resisting polypeptide of the present invention by the mouse that wild-type anthrax bacillus proteantigen (PA)/necrosin (LF) mixture kills and wounds, X-coordinate is fate for survival, and ordinate zou is a mouse existence quantity.Curve 1 is an abdominal injection wild-type anthrax bacillus PA/LF mixture group, curve 2 is abdominal injection anthrax resisting polypeptide/LF mixture group, curve 3 is abdominal injection wild-type anthrax bacillus PA/LF mixture+anthrax resisting polypeptide group, and curve 4 is abdominal injection wild-type anthrax bacillus PA/LF mixture+antineoplastic dibasic polypeptide group.
Fig. 4 is that the mouse that wild-type anthrax bacillus proteantigen (PA)/necrosin (LF) mixture kills and wounds gives the survival curve that anthrax resisting polypeptide of the present invention is protected again after 6 hours, and X-coordinate is fate for survival, and ordinate zou is a mouse existence quantity.Curve 1 is a wild-type anthrax bacillus PA/LF mixture group, and curve 2 gives the anthrax resisting polypeptide group again for wild-type anthrax bacillus PA/LF mixture after 6 hours.
The survival curve that Fig. 5 is protected through anthrax resisting polypeptide of the present invention by the rat that wild-type anthrax bacillus proteantigen (PA)/necrosin (LF) mixture kills and wounds, X-coordinate is fate for survival, and ordinate zou is a mouse existence quantity.Curve 1 is a tail vein injection wild-type anthrax bacillus PA/LF mixture group, and curve 2 is wild-type anthrax bacillus PA/LF mixture+anthrax resisting polypeptide group.
Embodiment
The structure of the plasmid of embodiment 1 expression anthrax resisting polypeptide and the preparation of reorganization anthrax resisting polypeptide
Original plasmid is for having loaded the pET22b plasmid (7.6kb of wild-type anthrax bacillus proteantigen (PA) genes of SEQ ID NO.1, blank plasmid is a Novagan pET22b plasmid, the PA gene is loaded by Harvard Univ.Dr.J.Collier), through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategene company) after the aminoterminal of PA gene inserts the gene-CACCACCACCACCACCAC-of six Histidines, gene (SEQ ID NO.2,4,6,8) with the encoding mutant amino-acid residue is operably connected with wild-type anthrax bacillus proteantigen (PA) gene respectively, thereby obtains the nucleotide sequence SEQ ID NO.10 of coding recombination mutation anthrax bacillus proteantigen gene.After again the genes of SEQ ID NO.12 of encoding antibody stand-in being inserted into the carboxyl terminal G735 site of recombination mutation anthrax bacillus proteantigen gene, just prepared the recombinant plasmid pCHCA-PA1 (see figure 1) of anthrax resisting polypeptide.Above-mentioned recombinant plasmid is transfected in E.coli BL-21 (DE3) engineering bacteria prepares polypeptide, obtain the described anthrax resisting polypeptide of SEQ ID NO.15 in the sequence table.
The sudden change program is undertaken by Strategene QuickChange Site-Directed Mutagenesis Kit (catalog#200518) medicine-chest handbook:
1, prepares the point mutation reactant
5ul?10x?buffer
2ul (10ng) wild-type anthrax bacillus proteantigen plasmid
1.25ul (125ng) She Ji 5 '-3 ' oligonucleotide primer
1.25ul (125ng) She Ji 3 '-5 ' oligonucleotide primer
1ul?dNTP
Distilled water 50ul
1ul?pfu
(except that plasmid, primer and distilled water, being reagent that medicine-chest is equipped with)
2, carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, totally 20 circulations in 17 fens;
3, add (37 ℃, 1 hour) behind the Dpn1 restriction endonuclease 1ul digestion mother body D NA chain, get 1ul reactant and XL1-Blue competent cell 50ul ice and incubated 30 minutes, 42 ℃ of capable thermal shockings 45 seconds, were inserted in the ice 2 minutes again;
4, add NZY and train 220rpm behind the basic 0.5ml, 37 ℃ were shaken bacterium after 1 hour, got 50-100ul reactant bed board (LB training base adds 1% agar and adds the 50-100ug/ml penbritin, and 37 ℃ are spent the night);
5, choose bacterium after 18 hours, follow Qiagene, companies such as Gibco various commercial extract the plasmid medicine-chests extract plasmids all can, order-checking is determined to suddenly change successfully;
6, BL-21 (DE3) the engineering bacteria competent cell 50ul ice of plasmid 50ng and preparation was incubated 5 fens, 42 ℃, thermal shocking in 30 seconds, get 50-100ul reactant adding LB and train basic 0.5ml, 220rpm, 37 ℃ are shaken bacterium bed board after 1 hour (LB training base adds 1% agar and adds the 50ug/ml penbritin) and hatch picking colony after 18 hours for 37 ℃;
7, increase bacterium in a large number, 8-16 rises LB training base, 220rpm, 37 ℃, 3 hours, after adding 1M IPTG 1ml/ rises LB training base, temperature is reduced to 28 ℃, 220rpm growth 5 hours, the centrifugation thalline, 4 ℃, 4000g, 15 minutes, get 4 ℃, 20% sucrose, 20mM Tris, 1mM EDTA, 1mM PMSF 300ml suspension thalline, ice is incubated 8min, and 4 ℃, 8000g, 20 minutes centrifugal after, remove supernatant, add 4 ℃, 5mM MgSO
4The 1mM PMSF 200ml precipitation that suspends, ice is incubated 10min, 4 ℃, 10000g, 10 minutes centrifugal after, get the supernatant molecular weight 10 of packing into, 000 dialysis tubing is in 4 ℃, 20mMTris, 0.15M NaCl, after 10 liters of dialysed overnight of 5mM imidazole buffer (changing liquid twice), supernatant is splined on His-tag post (4 ℃), through the 1M imidazoles, 20mM Tris, 0.15M NaCl buffer solution elution, use 20mM Tris again, 0.15M NaCl can obtain the anthrax resisting polypeptide (see figure 2) of recombinating behind 1mM edta buffer liquid 5 liters of dialysis removing imidazoles.
The designed oligonucleotide sequence of S337K in the above-mentioned plasmid of suddenling change:
1、5’-3’
gtc?gca?att?gat?cat?AAA?cta?tct?cta?gca?ggg
2、3’-5’
ccc?tgc?tag?aga?tag?TTT?atg?atc?aat?tgc?gac
The designed oligonucleotide sequence of G342S in the above-mentioned plasmid of suddenling change:
1、5’-3’
ttt?aaa?aaa?ta?aat?TCT?aaa?tta?ccg?tta?tat
2、3’-5’
ata?taa?cgg?taa?ttt?AGA?att?ata?ttt?ttt?aaa
The designed oligonucleotide sequence of I656R in the above-mentioned plasmid of suddenling change:
1、5’-3’
ggg?ctt?aaa?gaa?gtt?AGA?aat?gac?aga?tat?gat
2、3’-5’
Atc?ata?tct?gtc?aat?TCT?aac?ttc?ttt?aag?ccc
Suddenly change N682S and the designed oligonucleotide sequence of D683K in the above-mentioned plasmid:
1、5’-3’
gat?ttt?aaa?aaa?tat?TCT?AAA?aaa?tta?ccg?tta
2、3’-5’
taa?cgg?taa?ttt?TTT?AGA?ata?ttt?ttt?aaa?atc
Prepare anti-Pa and the designed oligonucleotide sequence of PA-LF mixture antibody analog gene in the above-mentioned plasmid:
1、5’-3’
aaa?aaa?ggc?tat?gag?ata?gga?TCA?ACC?GCC?CTC?CAT?TGG?GTG?AGA?CAA?GCC?taa
tga?ctc?gag?cac?cac?cac
2、3’-5’
gtg?gtg?gtg?ctc?gag?tca?tta?GGC?TTG?TCT?CAC?CCA?ATG?GAG?GGC?GGT?TGA?tcc?tat
ctc?ata?gcc?ttt?ttt
3、5’-3’
ctc?cat?tgg?gtg?aga?caa?gcc?CCC?GGA?AAG?GGC?CTG?GAG?TGG?GTA?TCG?taa?tga?ctc
gag?cac?cac?cac
4、3’-5’
gtg?gtg?gtg?ctc?gag?tca?tta?CGA?TAC?CCA?CTC?CAG?GCC?CTT?TCC?GGG?ggc?ttg?tct
cac?cca?atg?gag
5、5’-3’
aag?ggc?ctg?gag?tgg?gta?tcg?CTG?AGA?TAT?GAC?GAA?TTT?CCT?TAT?ACG?taa?tga?ctc
gag?cac?cac?cac
6、3’-5’
gtg?gtg?gtg?ctc?gag?tca?tta?CGT?ATA?AGG?AAA?TTC?GTC?ATA?TCT?CAG?cga?tac?cca
ctc?cag?gcc?ctt
Embodiment 2: the provide protection that anthrax resisting polypeptide of the present invention kills and wounds mouse to wild-type anthrax bacillus PA/LF mixture
Being tried mouse is BALB/c immune deficiency nude mice, is divided into 4 test group, and each test group is 10.Wild-type anthrax bacillus PA/LF mixture, anthrax resisting polypeptide, antineoplastic dibasic polypeptide are all through intraperitoneal administration, and be administered once every day simultaneously, and administration is 6 times altogether, the no longer administration of mouse of dying in heaven.
Test group 1: abdominal injection wild-type anthrax bacillus PA/LF mixture group, injection volume was 20 μ g/20 μ g/ days.
Test group 2: abdominal injection anthrax resisting polypeptide/LF mixture group, injection volume were 20 μ g/20 μ g/ days.
Test group 3: abdominal injection wild-type anthrax bacillus PA/LF mixture+anthrax resisting polypeptide group, injection volume were 20 μ g/20 μ g+20 μ g/ days.
Test group 4: abdominal injection wild-type anthrax bacillus PA/LF mixture+antineoplastic dibasic polypeptide (seeing the Chinese patent application of application number 200710049653.9) group, injection volume was 20 μ g/20 μ g+20 μ g/ days.
The survival state of each test group mouse is seen Fig. 3, and as can be seen from Figure 3, the wild-type anthrax bacillus PA/LF mixture toxic action maximum in the test group 1 can cause mouse all dead (seeing the curve 1 among Fig. 3) in 7 days.The toxic action of anthrax resisting polypeptide in the test group 2/LF mixture greatly weakens, and does not cause animal dead (seeing the curve 2 among Fig. 3) in 20 days.The toxic action that has weakened wild-type anthrax bacillus PA/LF mixture of the anthrax resisting polypeptide success in the test group 3,90% mouse all can survive (seeing the curve 3 among Fig. 3) in 20 days.Antineoplastic dibasic polypeptide in the test group 4 fails to stop the toxic action of wild-type anthrax bacillus PA/LF mixture, mouse all dead (seeing the curve 4 among Fig. 3) in 12 days.
Embodiment 3: anthrax resisting polypeptide of the present invention postpones the provide protection that administration kills and wounds mouse to wild-type anthrax bacillus PA/LF mixture after 6 hours
Being tried mouse is BALB/c immune deficiency nude mice, is divided into 2 test group, and each test group is 5.Wild-type anthrax bacillus PA/LF mixture, anthrax resisting polypeptide are all through intraperitoneal administration, and anthrax resisting polypeptide was in wild-type anthrax bacillus PA/LF mixture administration administration again after 6 hours on 1st.Be administered once simultaneously every day afterwards.Administration is 6 times altogether, the no longer administration of mouse of dying in heaven.
Test group 1: abdominal injection wild-type anthrax bacillus PA/LF mixture group, injection volume was 20 μ g/20 μ g/ days.
Test group 2: abdominal injection wild-type anthrax bacillus PA/LF mixture+anthrax resisting polypeptide group, injection volume were 20 μ g/20 μ g+20 μ g/ days.
The survival state of each test group mouse is seen Fig. 4, and as can be seen from Figure 4, the wild-type anthrax bacillus PA/LF mixture toxic action maximum in the test group 1 can cause mouse all dead (seeing the curve 1 among Fig. 4) in 6 days.The toxic action that has weakened wild-type anthrax bacillus PA/LF mixture of the anthrax resisting polypeptide success in the test group 2, but abated effect is weaker than embodiment 2; 70% mouse can survive (seeing the curve 2 among Fig. 4) is arranged in 20 days approximately.
Embodiment 4: the provide protection that anthrax resisting polypeptide of the present invention kills and wounds rat to wild-type anthrax bacillus PA/LF mixture
Being tried rat is the common rat of SD, is divided into 2 test group, and each test group is 5.Wild-type anthrax bacillus PA/LF mixture, anthrax resisting polypeptide were administered once all through the tail intravenously administrable in per 6 hours, and administration is 4 times altogether, the no longer administration of rat of dying in heaven.
Test group 1: tail vein injection wild-type anthrax bacillus PA/LF mixture group, injection volume is 80 μ g/40 μ g/ time.
Test group 2: tail vein injection wild-type anthrax bacillus PA/LF mixture+anthrax resisting polypeptide group, injection volume are 80 μ g/40 μ g+80 μ g/ time.
The survival state of each rats in test groups is seen Fig. 5, and as can be seen from Figure 5, the wild-type anthrax bacillus PA/LF mixture toxic action maximum in the test group 1 can cause rat all dead (seeing the curve 1 among Fig. 5) in 24 hours.Anthrax resisting polypeptide in the test group 2 has successfully weakened the toxic action of wild-type anthrax bacillus PA/LF mixture, 100% rat all survive (seeing the curve 2 among Fig. 5) in 6 days.
SEQUENCE?LISTING
＜110〉Qiu Xiaoqing
＜120〉anthrax resisting polypeptide and application thereof and preparation method
<160>15
<170>PatentIn?Version?3.2
<210>1
<211>2211
<212>DNA
<213>Escherichia?coli
<220>
<221>misc_feature
<222>(1).....(2211)
＜223〉anthrax bacillus proteantigen gene
<400>1
atggaagtta?aacaggagaa?ccggttatta?aatgaatcag?aatcaagttc?ccaggggtta 60
ctaggatact?attttagtga?tttgaatttt?caagcaccca?tggtggttac?ttcttctact 120
acaggggatt?tatctattcc?tagttctgag?ttagaaaata?ttccatcgga?aaaccaatat 180
tttcaatctg?ctatttggtc?aggatttatc?aaagttaaga?agagtgatga?atatacattt 240
gctacttccg?ctgataatca?tgtaacaatg?tgggtagatg?accaagaagt?gattaataaa 300
gcttctaatt?ctaacaaaat?cagattagaa?aaaggaagat?tatatcaaat?aaaaattcaa 360
tatcaacgag?aaaatcctac?tgaaaaagga?ttggatttca?agttgtactg?gaccgattct 420
caaaataaaa?aagaagtgat?ttctagtgat?aacttacaac?tgccagaatt?aaaacaaaaa 480
tcttcgaact?caagaaaaaa?gcgaagtaca?agtgctggac?ctacggttcc?agaccgtgac 540
aatgatggaa?tccctgattc?attagaggta?gaaggatata?cggttgatgt?caaaaataaa 600
agaacttttc?tttcaccatg?gatttctaat?attcatgaaa?agaaaggatt?aaccaaatat 660
aaatcatctc?ctgaaaaatg?gagcacggct?tctgatccgt?acagtgattt?cgaaaaggtt 720
acaggacgga?ttgataagaa?tgtatcacca?gaggcaagac?acccccttgt?ggcagcttat 780
ccgattgtac?atgtagatat?ggagaatatt?attctctcaa?aaaatgagga?tcaatccaca 840
cagaatactg?atagtcaaac?gagaacaata?agtaaaaata?cttctacaag?taggacacat 900
actagtgaag?tacatggaaa?tgcagaagtg?catgcgtcgt?tctttgatat?tggtgggagt 960
gtatctgcag?gatttagtaa?ttcgaattca?agtacggtcg?caattgatca?ttcactatct 1020
ctagcagggg?aaagaacttg?ggctgaaaca?atgggtttaa?ataccgctga?tacagcaaga 1080
ttaaatgcca?atattagata?tgtaaatact?gggacggctc?caatctacaa?cgtgttacca 1140
acgacttcgt?tagtgttagg?aaaaaatcaa?acactcgcga?caattaaagc?taaggaaaac 1200
caattaagtc?aaatacttgc?acctaataat?tattatcctt?ctaaaaactt?ggcgccaatc 1260
gcattaaatg?cacaagacga?tttcagttct?actccaatta?caatgaatta?caatcaattt 1320
cttgagttag?aaaaaacgaa?acaattaaga?ttagatacgg?atcaagtata?tgggaatata 1380
gcaacataca?attttgaaaa?tggaagagtg?agggtggata?caggctcgaa?ctggagtgaa 1440
gtgttaccgc?aaattcaaga?aacaactgca?cgtatcattt?ttaatggaaa?agatttaaat 1500
ctggtagaaa?ggcggatagc?ggcggttaat?cctagtgatc?cattagaaac?gactaaaccg 1560
gatatgacat?taaaagaagc?ccttaaaata?gcatttggat?ttaacgaacc?gaatggaaac 1620
ttacaatatc?aagggaaaga?cataaccgaa?tttgatttta?atttcgatca?acaaacatct 1680
caaaatatca?agaatcagtt?agcggaatta?aacgtaacta?acatatatac?tgtattagat 1740
aaaatcaaat?taaatgcaaa?aatgaatatt?ttaataagag?ataaacgttt?tcattatgat 1800
agaaataaca?tagcagttgg?ggctgatgag?tcagtagtta?aggaggctca?tagagaagta 1860
attaattcgt?caacagaggg?attattgtta?aatattgata?aggatataag?aaaaatatta 1920
tcaggttata?ttgtagaaat?tgaagatact?gaagggctta?aagaagttat?aaatgacaga 1980
tatgatatgt?tgaatatttc?tagtttacgg?caagatggaa?aaacatttat?agattttaaa 2040
aaatataatg?ataaattacc?gttatatata?agtaatccca?attataaggt?aaatgtatat 2100
gctgttacta?aagaaaacac?tattattaat?cctagtgaga?atggggatac?tagtaccaac 2160
gggatcaaga?aaattttaat?cttttctaaa?aaaggctatg?agataggata?a 2211
<210>2
<211>3
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1).....(3)
＜223〉encoding mutant anthrax bacillus proteantigen gene G342S sequence
<400>2
tct
<210>3
<211>1
<212>PRT
＜213〉artificial sequence
＜223〉encoding mutant anthrax bacillus proteantigen G342S aminoacid sequence
<400>3
Ser
1
<210>4
<211>6
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1).....(6)
＜223〉encoding mutant anthrax bacillus proteantigen gene N682S-D683K sequence
<400>4
tctaaa
<210>5
<211>2
<212>PRT
＜213〉artificial sequence
＜223〉encoding mutant anthrax bacillus proteantigen N682S-D683K aminoacid sequence
<400>5
Ser?Lys
1
<210>6
<211>3
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1).....(3)
＜223〉encoding mutant anthrax bacillus proteantigen gene S337K sequence
<400>6
aaa
<210>7
<211>1
<212>PRT
＜213〉artificial sequence
＜223〉encoding mutant anthrax bacillus proteantigen S337K aminoacid sequence
<400>7
Lys
1
<210>8
<211>3
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1).....(3)
＜223〉encoding mutant anthrax bacillus proteantigen gene I656R sequence
<400>8
aga
<210>9
<211>1
<212>PRT
＜213〉artificial sequence
＜223〉encoding mutant anthrax bacillus proteantigen I656R aminoacid sequence
<400>9
Arg
1
<210>10
<211>2208
<212>DNA
＜213〉artificial sequence
<222>(1).....(2208)
＜223〉coding recombination mutation anthrax bacillus proteantigen gene
<400>10
atggaagtta?aacaggagaa?ccggttatta?aatgaatcag?aatcaagttc?ccaggggtta 60
ctaggatact?attttagtga?tttgaatttt?caagcaccca?tggtggttac?ttcttctact 120
acaggggatt?tatctattcc?tagttctgag?ttagaaaata?ttccatcgga?aaaccaatat 180
tttcaatctg?ctatttggtc?aggatttatc?aaagttaaga?agagtgatga?atatacattt 240
gctacttccg?ctgataatca?tgtaacaatg?tgggtagatg?accaagaagt?gattaataaa 300
gcttctaatt?ctaacaaaat?cagattagaa?aaaggaagat?tatatcaaat?aaaaattcaa 360
tatcaacgag?aaaatcctac?tgaaaaagga?ttggatttca?agttgtactg?gaccgattct 420
caaaataaaa?aagaagtgat?ttctagtgat?aacttacaac?tgccagaatt?aaaacaaaaa 480
tcttcgaact?caagaaaaaa?gcgaagtaca?agtgctggac?ctacggttcc?agaccgtgac 540
aatgatggaa?tccctgattc?attagaggta?gaaggatata?cggttgatgt?caaaaataaa 600
agaacttttc?tttcaccatg?gatttctaat?attcatgaaa?agaaaggatt?aaccaaatat 660
aaatcatctc?ctgaaaaatg?gagcacggct?tctgatccgt?acagtgattt?cgaaaaggtt 720
acaggacgga?ttgataagaa?tgtatcacca?gaggcaagac?acccccttgt?ggcagcttat 780
ccgattgtac?atgtagatat?ggagaatatt?attctctcaa?aaaatgagga?tcaatccaca 840
cagaatactg?atagtcaaac?gagaacaata?agtaaaaata?cttctacaag?taggacacat 900
actagtgaag?tacatggaaa?tgcagaagtg?catgcgtcgt?tctttgatat?tggtgggagt 960
gtatctgcag?gatttagtaa?ttcgaattca?agtacggtcg?caattgatca?taaactatct 1020
ctagcatctg?aaagaacttg?ggctgaaaca?atgggtttaa?ataccgctga?tacagcaaga 1080
ttaaatgcca?atattagata?tgtaaatact?gggacggctc?caatctacaa?cgtgttacca 1140
acgacttcgt?tagtgttagg?aaaaaatcaa?acactcgcga?caattaaagc?taaggaaaac 1200
caattaagtc?aaatacttgc?acctaataat?tattatcctt?ctaaaaactt?ggcgccaatc 1260
gcattaaatg?cacaagacga?tttcagttct?actccaatta?caatgaatta?caatcaattt 1320
cttgagttag?aaaaaacgaa?acaattaaga?ttagatacgg?atcaagtata?tgggaatata 1380
gcaacataca?attttgaaaa?tggaagagtg?agggtggata?caggctcgaa?ctggagtgaa 1440
gtgttaccgc?aaattcaaga?aacaactgca?cgtatcattt?ttaatggaaa?agatttaaat 1500
ctggtagaaa?ggcggatagc?ggcggttaat?cctagtgatc?cattagaaac?gactaaaccg 1560
gatatgacat?taaaagaagc?ccttaaaata?gcatttggat?ttaacgaacc?gaatggaaac 1620
ttacaatatc?aagggaaaga?cataaccgaa?tttgatttta?atttcgatca?acaaacatct 1680
caaaatatca?agaatcagtt?agcggaatta?aacgtaacta?acatatatac?tgtattagat 1740
aaaatcaaat?taaatgcaaa?aatgaatatt?ttaataagag?ataaacgttt?tcattatgat 1800
agaaataaca?tagcagttgg?ggctgatgag?tcagtagtta?aggaggctca?tagagaagta 1860
attaattcgt?caacagaggg?attattgtta?aatattgata?aggatataag?aaaaatatta 1920
tcaggttata?ttgtagaaat?tgaagatact?gaagggctta?aagaagttag?aaatgacaga 1980
tatgatatgt?tgaatatttc?tagtttacgg?caagatggaa?aaacatttat?agattttaaa 2040
aaatattcta?aaaaattacc?gttatatata?agtaatccca?attataaggt?aaatgtatat 2100
gctgttacta?aagaaaacac?tattattaat?cctagtgaga?atggggatac?tagtaccaac 2160
gggatcaaga?aaattttaat?cttttctaaa?aaaggctatg?agatagga 2208
<210>11
<211>734
<212>PRT
＜213〉artificial sequence
＜223〉coding recombination mutation anthrax bacillus proteantigen aminoacid sequence
<400>11
Glu?Val?Lys?Gln?Glu?Asn?Arg?Leu?Leu?Asn?Glu?Ser?Glu?Ser?Ser?Ser
1 5 10 15
Gln?Gly?Leu?Leu?Gly?Tyr?Tyr?Phe?Ser?Asp?Leu?Asn?Phe?Gln?Ala?Pro
20 25 30
Met?Val?Val?Thr?Ser?Ser?Thr?Thr?Gly?Asp?Leu?Ser?Ile?Pro?Ser?Ser
35 40 45
Glu?Leu?Glu?Asn?Ile?Pro?Ser?Glu?Asn?Gln?Tyr?Phe?Gln?Ser?Ala?Ile
50 55 60
Trp?Ser?Gly?Phe?Ile?Lys?Val?Lys?Lys?Ser?Asp?Glu?Tyr?Thr?Phe?Ala
65 70 75 80
Thr?Ser?Ala?Asp?Asn?His?Val?Thr?Met?Trp?Val?Asp?Asp?Gln?Glu?Val
85 90 95
Ile?Asn?Lys?Ala?Ser?Asn?Ser?Asn?Lys?Ile?Arg?Leu?Glu?Lys?Gly?Arg
100 105 110
Leu?Tyr?Gln?Ile?Lys?Ile?Gln?Tyr?Gln?Arg?Glu?Gln?Pro?Thr?Glu?Lys
115 120 125
Gly?Leu?Asp?Phe?Lys?Leu?Tyr?Trp?Thr?Asp?Ser?Gln?Asn?Lys?Lys?Glu
130 135 140
Val?Ile?Ser?Ser?Asp?Asn?Leu?Gln?Leu?Pro?Glu?Leu?Lys?Gln?Lys?Ser
145 150 155 160
Ser?Asn?Ser?Arg?Lys?Lys?Arg?Ser?Thr?Ser?Ala?Gly?Pro?Thr?Val?Pro
165 170 175
Asp?Lys?Asp?Asn?Asp?Gly?Ile?Pro?Asp?Ser?Leu?Glu?Val?Glu?Gly?Tyr
180 185 190
Thr?Val?Asp?Val?Lys?Asn?Lys?Arg?The?Phe?Leu?Ser?Pro?Trp?Ile?Ser
195 200 205
Asn?Ile?His?Glu?Lys?Lys?Gly?Leu?Thr?Lys?Tyr?Lys?Ser?Ser?Pro?Glu
210 215 220
Lys?Trp?Ser?Thr?Ala?Ser?Asp?Pro?Tyr?Ser?Asp?Phe?Glu?Lys?Val?Thr
225 230 235 240
Gly?Arg?Ile?Asp?Lys?Asn?Val?Ser?Pro?Glu?Ala?Arg?His?Pro?Leu?Val
245 250 255
Ala?Ala?Tyr?Pro?Ile?Val?His?Val?Asp?Met?Glu?Asn?Ile?Ile?Leu?Ser
260 265 270
Lys?Asn?Glu?Asp?Gln?Ser?Thr?Gln?Asn?Thr?Asp?Ser?Gln?Thr?Arg?Thr
275 280 285
Ile?Ser?Lys?Asn?Thr?Ser?Thr?Ser?Arg?Thr?His?Thr?Ser?Glu?Val?His
290 295 300
Gly?Asn?Ala?Glu?Val?His?Ala?Ser?Phe?Phe?Asp?Ile?Gly?Gly?Ser?Val
305 310 315 320
Ser?Ala?Gly?Phe?Ser?Asn?Ser?Asn?Ser?Ser?Thr?Val?Ala?Ile?Asp?His
325 330 335
Lys?Leu?Ser?Leu?Ala?Ser?Glu?Arg?Thr?Trp?Ala?Glu?Thr?Met?Gly?Leu
340 345 350
Asn?Thr?Ala?Asp?Thr?Ala?Arg?Leu?Asn?Ala?Asn?Ile?Arg?Tyr?Val?Asn
355 360 365
Thr?Gly?Thr?Ala?Pro?Ile?Tyr?Asn?Val?Leu?Pro?Thr?Thr?Ser?Leu?Val
370 375 380
Leu?Gly?Lys?Asn?Gln?Thr?Leu?Ala?Thr?Ile?Lys?Ala?Lys?Glu?Asn?Gln
385 390 395 400
Leu?Ser?Gln?Ile?Leu?Ala?Pro?Asn?Asn?Tyr?Tyr?Pro?Ser?Lys?Asn?Leu
405 410 415
Ala?Pro?Ile?Ala?Leu?Asn?Ala?Gln?Asp?Asp?Phe?Ser?Ser?Thr?Pro?Ile
420 425 430
Thr?Met?Asn?Tyr?Asn?Gln?Phe?Leu?Glu?Leu?Glu?Lys?Thr?Lys?Gln?Leu
435 440 445
Arg?Leu?Asp?Thr?Asp?Gln?Val?Tyr?Gly?Asn?Ile?Ala?Thr?Tyr?Asn?Phe
450 455 460
Glu?Asn?Gly?Arg?Val?Arg?Val?Asp?Thr?Gly?Ser?Asn?Trp?Ser?Glu?Val
465 470 475 480
Leu?Pro?Gln?Ile?Gln?Glu?Thr?Thr?Ala?Arg?Ile?Ile?Phe?Asn?Gly?Lys
485 490 495
Asp?Leu?Asn?Leu?Val?Glu?Arg?Arg?Ile?Ala?Ala?Val?Asn?Pro?Ser?Asp
500 505 510
Pro?Leu?Glu?Thr?Thr?Lys?Pro?Asp?Met?Thr?Leu?Lys?Glu?Ala?Leu?Lys
515 520 525
Ile?Ala?Phe?Gly?Phe?Asn?Glu?Pro?Asn?Gly?Asn?Leu?Gln?Tyr?Gln?Gly
530 535 540
Lys?Asp?Ile?Thr?Glu?Phe?Asp?Phe?Asn?Phe?Asp?Gln?Gln?Thr?Ser?Gln
545 550 555 560
Asn?Ile?Lys?Asn?Gln?Leu?Ala?Glu?Leu?Asn?Val?Thr?Asn?Ile?Tyr?Thr
565 570 575
Val?Leu?Asp?Lys?Ile?Lys?Leu?Asn?Ala?Lys?Met?Asn?Ile?Leu?Ile?Arg
580 585 590
Asp?Lys?Arg?Phe?His?Tyr?Asp?Arg?Asn?Asn?Ile?Ala?Val?Gly?Ala?Asp
595 600 605
Glu?Ser?Val?Val?Lys?Glu?Ala?His?Arg?Glu?Val?Ile?Asn?Ser?Ser?Thr
610 615 620
Glu?Gly?Leu?Leu?Leu?Asn?Ile?Asp?Lys?Asp?Ile?Arg?Lys?Ile?Leu?Ser
625 630 635 640
Gly?Tyr?Ile?Val?Glu?Ile?Glu?Asp?Thr?Glu?Gly?Leu?Lys?Glu?Val?Arg
645 650 655
Asn?Asp?Arg?Tyr?Asp?Met?Leu?Asn?Ile?Ser?Ser?Leu?Arg?Gln?Asp?Gly
660 665 670
Lys?Thr?Phe?Ile?Asp?Phe?Lys?Lys?Ser?Lys?Lys?Leu?Pro?Leu?Tyr?Ile
675 680 685
Ser?Asn?Pro?Asn?Tyr?Lys?Val?Asn?Val?Tyr?Ala?Val?Thr?Lys?Glu?Asn
690 695 700
Thr?Ile?Ile?Asn?Pro?Ser?Glu?Asn?Gly?Asp?Thr?Ser?Thr?Asn?Gly?Ile
705 710 715 720
Lys?Lys?Ile?Leu?Ile?Phe?Ser?Lys?Lys?Gly?Tyr?Glu?Ile?Gly
725 730
<210>12
<211>84
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1).....(84)
＜223〉encoding antibody stand-in gene order
<400>12
tcaaccgccc?tccattgggt?gagacaagcc?cccggaaagg?gcctggagtg?ggtatcgctg 60
agatatgacg?aatttcctta?tacg 84
<210>13
<211>28
<212>PRT
＜213〉artificial sequence
＜223〉encoding antibody stand-in aminoacid sequence
<400>5
Ser?Thr?Ala?Leu?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser
1 5 10 15
Pro?Arg?Tyr?Asp?Glu?Phe?Pro?Tyr?Thr
20 25
<210>14
<211>2292
<212>DNA
＜213〉artificial sequence
<222>(1).....(2292)
＜223〉coding anthrax resisting polypeptide gene
<400>14
atggaagtta?aacaggagaa?ccggttatta?aatgaatcag?aatcaagttc?ccaggggtta 60
ctaggatact?attttagtga?tttgaatttt?caagcaccca?tggtggttac?ttcttctact 120
acaggggatt?tatctattcc?tagttctgag?ttagaaaata?ttccatcgga?aaaccaatat 180
tttcaatctg?ctatttggtc?aggatttatc?aaagttaaga?agagtgatga?atatacattt 240
gctacttccg?ctgataatca?tgtaacaatg?tgggtagatg?accaagaagt?gattaataaa 300
gcttctaatt?ctaacaaaat?cagattagaa?aaaggaagat?tatatcaaat?aaaaattcaa 360
tatcaacgag?aaaatcctac?tgaaaaagga?ttggatttca?agttgtactg?gaccgattct 420
caaaataaaa?aagaagtgat?ttctagtgat?aacttacaac?tgccagaatt?aaaacaaaaa 480
tcttcgaact?caagaaaaaa?gcgaagtaca?agtgctggac?ctacggttcc?agaccgtgac 540
aatgatggaa?tccctgattc?attagaggta?gaaggatata?cggttgatgt?caaaaataaa 600
agaacttttc?tttcaccatg?gatttctaat?attcatgaaa?agaaaggatt?aaccaaatat 660
aaatcatctc?ctgaaaaatg?gagcacggct?tctgatccgt?acagtgattt?cgaaaaggtt 720
acaggacgga?ttgataagaa?tgtatcacca?gaggcaagac?acccccttgt?ggcagcttat 780
ccgattgtac?atgtagatat?ggagaatatt?attctctcaa?aaaatgagga?tcaatccaca 840
cagaatactg?atagtcaaac?gagaacaata?agtaaaaata?cttctacaag?taggacacat 900
actagtgaag?tacatggaaa?tgcagaagtg?catgcgtcgt?tctttgatat?tggtgggagt 960
gtatctgcag?gatttagtaa?ttcgaattca?agtacggtcg?caattgatca?taaactatct 1020
ctagcatctg?aaagaacttg?ggctgaaaca?atgggtttaa?ataccgctga?tacagcaaga 1080
ttaaatgcca?atattagata?tgtaaatact?gggacggctc?caatctacaa?cgtgttacca 1140
acgacttcgt?tagtgttagg?aaaaaatcaa?acactcgcga?caattaaagc?taaggaaaac 1200
caattaagtc?aaatacttgc?acctaataat?tattatcctt?ctaaaaactt?ggcgccaatc 1260
gcattaaatg?cacaagacga?tttcagttct?actccaatta?caatgaatta?caatcaattt 1320
cttgagttag?aaaaaacgaa?acaattaaga?ttagatacgg?atcaagtata?tgggaatata 1380
gcaacataca?attttgaaaa?tggaagagtg?agggtggata?caggctcgaa?ctggagtgaa 1440
gtgttaccgc?aaattcaaga?aacaactgca?cgtatcattt?ttaatggaaa?agatttaaat 1500
ctggtagaaa?ggcggatagc?ggcggttaat?cctagtgatc?cattagaaac?gactaaaccg 1560
gatatgacat?taaaagaagc?ccttaaaata?gcatttggat?ttaacgaacc?gaatggaaac 1620
ttacaatatc?aagggaaaga?cataaccgaa?tttgatttta?atttcgatca?acaaacatct 1680
caaaatatca?agaatcagtt?agcggaatta?aacgtaacta?acatatatac?tgtattagat 1740
aaaatcaaat?taaatgcaaa?aatgaatatt?ttaataagag?ataaacgttt?tcattatgat 1800
agaaataaca?tagcagttgg?ggctgatgag?tcagtagtta?aggaggctca?tagagaagta 1860
attaattcgt?caacagaggg?attattgtta?aatattgata?aggatataag?aaaaatatta 1920
tcaggttata?ttgtagaaat?tgaagatact?gaagggctta?aagaagttag?aaatgacaga 1980
tatgatatgt?tgaatatttc?tagtttacgg?caagatggaa?aaacatttat?agattttaaa 2040
aaatattcta?aaaaattacc?gttatatata?agtaatccca?attataaggt?aaatgtatat 2100
gctgttacta?aagaaaacac?tattattaat?cctagtgaga?atggggatac?tagtaccaac 2160
gggatcaaga?aaattttaat?cttttctaaa?aaaggctatg?agataggatc?aaccgccctc 2220
cattgggtga?gacaagcccc?cggaaagggc?ctggagtggg?tatcgctgag?atatgacgaa 2280
tttccttata?cg 2292
<210>15
<211>762
<212>PRT
＜213〉artificial sequence
＜223〉coding anthrax resisting polypeptide aminoacid sequence
<400>15
Glu?Val?Lys?Gln?Glu?Asn?Arg?Leu?Leu?Asn?Glu?Ser?Glu?Ser?Ser?Ser
1 5 10 15
Gln?Gly?Leu?Leu?Gly?Tyr?Tyr?Phe?Ser?Asp?Leu?Asn?Phe?Gln?Ala?Pro
20 25 30
Met?Val?Val?Thr?Ser?Ser?Thr?Thr?Gly?Asp?Leu?Ser?Ile?Pro?Ser?Ser
35 40 45
Glu?Leu?Glu?Asn?Ile?Pro?Ser?Glu?Asn?Gln?Tyr?Phe?Gln?Ser?Ala?Ile
50 55 60
Trp?Ser?Gly?Phe?Ile?Lys?Val?Lys?Lys?Ser?Asp?Glu?Tyr?Thr?Phe?Ala
65 70 75 80
Thr?Ser?Ala?Asp?Asn?His?Val?Thr?Met?Trp?Val?Asp?Asp?Gln?Glu?Val
85 90 95
Ile?Asn?Lys?Ala?Ser?Asn?Ser?Asn?Lys?Ile?Arg?Leu?Glu?Lys?Gly?Arg
100 105 110
Leu?Tyr?Gln?Ile?Lys?Ile?Gln?Tyr?Gln?Arg?Glu?Gln?Pro?Thr?Glu?Lys
115 120 125
Gly?Leu?Asp?Phe?Lys?Leu?Tyr?Trp?Thr?Asp?Ser?Gln?Asn?Lys?Lys?Glu
130 135 140
Val?Ile?Ser?Ser?Asp?Asn?Leu?Gln?Leu?Pro?Glu?Leu?Lys?Gln?Lys?Ser
145 150 155 160
Ser?Asn?Ser?Arg?Lys?Lys?Arg?Ser?Thr?Ser?Ala?Gly?Pro?Thr?Val?Pro
165 170 175
Asp?Lys?Asp?Asn?Asp?Gly?Ile?Pro?Asp?Ser?Leu?Glu?Val?Glu?Gly?Tyr
180 185 190
Thr?Val?Asp?Val?Lys?Asn?Lys?Arg?The?Phe?Leu?Ser?Pro?Trp?Ile?Ser
195 200 205
Asn?Ile?His?Glu?Lys?Lys?Gly?Leu?Thr?Lys?Tyr?Lys?Ser?Ser?Pro?Glu
210 215 220
Lys?Trp?Ser?Thr?Ala?Ser?Asp?Pro?Tyr?Ser?Asp?Phe?Glu?Lys?Val?Thr
225 230 235 240
Gly?Arg?Ile?Asp?Lys?Asn?Val?Ser?Pro?Glu?Ala?Arg?His?Pro?Leu?Val
245 250 255
Ala?Ala?Tyr?Pro?Ile?Val?His?Val?Asp?Met?Glu?Asn?Ile?Ile?Leu?Ser
260 265 270
Lys?Asn?Glu?Asp?Gln?Ser?Thr?Gln?Asn?Thr?Asp?Ser?Gln?Thr?Arg?Thr
275 280 285
Ile?Ser?Lys?Asn?Thr?Ser?Thr?Ser?Arg?Thr?His?Thr?Ser?Glu?Val?His
290 295 300
Gly?Asn?Ala?Glu?Val?His?Ala?Ser?Phe?Phe?Asp?Ile?Gly?Gly?Ser?Val
305 310 315 320
Ser?Ala?Gly?Phe?Ser?Asn?Ser?Asn?Ser?Ser?Thr?Val?Ala?Ile?Asp?His
325 330 335
Lys?Leu?Ser?Leu?Ala?Ser?Glu?Arg?Thr?Trp?Ala?Glu?Thr?Met?Gly?Leu
340 345 350
Asn?Thr?Ala?Asp?Thr?Ala?Arg?Leu?Asn?Ala?Asn?Ile?Arg?Tyr?Val?Asn
355 360 365
Thr?Gly?Thr?Ala?Pro?Ile?Tyr?Asn?Val?Leu?Pro?Thr?Thr?Ser?Leu?Val
370 375 380
Leu?Gly?Lys?Asn?Gln?Thr?Leu?Ala?Thr?Ile?Lys?Ala?Lys?Glu?Asn?Gln
385 390 395 400
Leu?Ser?Gln?Ile?Leu?Ala?Pro?Asn?Asn?Tyr?Tyr?Pro?Ser?Lys?Asn?Leu
405 410 415
Ala?Pro?Ile?Ala?Leu?Asn?Ala?Gln?Asp?Asp?Phe?Ser?Ser?Thr?Pro?Ile
420 425 430
Thr?Met?Asn?Tyr?Asn?Gln?Phe?Leu?Glu?Leu?Glu?Lys?Thr?Lys?Gln?Leu
435 440 445
Arg?Leu?Asp?Thr?Asp?Gln?Val?Tyr?Gly?Asn?Ile?Ala?Thr?Tyr?Asn?Phe
450 455 460
Glu?Asn?Gly?Arg?Val?Arg?Val?Asp?Thr?Gly?Ser?Asn?Trp?Ser?Glu?Val
465 470 475 480
Leu?Pro?Gln?Ile?Gln?Glu?Thr?Thr?Ala?Arg?Ile?Ile?Phe?Asn?Gly?Lys
485 490 495
Asp?Leu?Asn?Leu?Val?Glu?Arg?Arg?Ile?Ala?Ala?Val?Asn?Pro?Ser?Asp
500 505 510
Pro?Leu?Glu?Thr?Thr?Lys?Pro?Asp?Met?Thr?Leu?Lys?Glu?Ala?Leu?Lys
515 520 525
Ile?Ala?Phe?Gly?Phe?Asn?Glu?Pro?Asn?Gly?Asn?Leu?Gln?Tyr?Gln?Gly
530 535 540
Lys?Asp?Ile?Thr?Glu?Phe?Asp?Phe?Asn?Phe?Asp?Gln?Gln?Thr?Ser?Gln
545 550 555 560
Asn?Ile?Lys?Asn?Gln?Leu?Ala?Glu?Leu?Asn?Val?Thr?Asn?Ile?Tyr?Thr
565 570 575
Val?Leu?Asp?Lys?Ile?Lys?Leu?Asn?Ala?Lys?Met?Asn?Ile?Leu?Ile?Arg
580 585 590
Asp?Lys?Arg?Phe?His?Tyr?Asp?Arg?Asn?Asn?Ile?Ala?Val?Gly?Ala?Asp
595 600 605
Glu?Ser?Val?Val?Lys?Glu?Ala?His?Arg?Glu?Val?Ile?Asn?Ser?Ser?Thr
610 615 620
Glu?Gly?Leu?Leu?Leu?Asn?Ile?Asp?Lys?Asp?Ile?Arg?Lys?Ile?Leu?Ser
625 630 635 640
Gly?Tyr?Ile?Val?Glu?Ile?Glu?Asp?Thr?Glu?Gly?Leu?Lys?Glu?Val?Arg
645 650 655
Asn?Asp?Arg?Tyr?Asp?Met?Leu?Asn?Ile?Ser?Ser?Leu?Arg?Gln?Asp?Gly
660 665 670
Lys?Thr?Phe?Ile?Asp?Phe?Lys?Lys?Ser?Lys?Lys?Leu?Pro?Leu?Tyr?Ile
675 680 685
Ser?Asn?Pro?Asn?Tyr?Lys?Val?Asn?Val?Tyr?Ala?Val?Thr?Lys?Glu?Asn
690 695 700
Thr?Ile?Ile?Asn?Pro?Ser?Glu?Asn?Gly?Asp?Thr?Ser?Thr?Asn?Gly?Ile
705 710 715 720
Lys?Lys?Ile?Leu?Ile?Phe?Ser?Lys?Lys?Gly?Tyr?Glu?Ile?Gly?Ser?Thr
725 730 735
Ala?Leu?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
740 745 750
Ser?Pro?Arg?Tyr?Asp?Glu?Phe?Pro?Tyr?Thr
755 760
Claims (6)
1. the gene of the anthrax resisting polypeptide of encoding is characterized in that described gene is formed by connecting by the gene of coding recombination mutation anthrax bacillus proteantigen and the gene of encoding antibody stand-in, is the described nucleotide sequence of SEQ ID NO.14 in the sequence table.
2. the recombinant plasmid of an anthrax resisting polypeptide is characterized in that it contains the gene of the described coding anthrax resisting polypeptide of claim 1.
3. an anthrax resisting polypeptide is characterized in that it is formed by the described expression of recombinant plasmid of claim 2, is the described aminoacid sequence of SEQ ID NO.15 in the sequence table.
4. the application of the described anthrax resisting polypeptide of claim 3 in the medicine of preparation treatment or prevention anthrax bacillus or anthrax toxin lethal infection.
5. the preparation method of an anthrax resisting polypeptide is characterized in that may further comprise the steps:
The gene of the described encoding antibody stand-in of SEQ ID NO.12 in the sequence table is operably connected with the pET22b plasmid that has loaded the described coding recombination mutation of SEQ ID NO.10 anthrax bacillus proteantigen gene obtains the gene of coding reorganization anthrax resisting polypeptide, the gene that obtains imported in the engineering bacterium expression system express, promptly obtain anthrax resisting polypeptide through the His-tag column separating purification.
6. according to the preparation method of the described anthrax resisting polypeptide of claim 5, it is characterized in that the described coding recombination mutation of SEQ ID NO.10 anthrax bacillus proteantigen gene in the sequence table is formed by connecting by the proteantigen gene of the described anthrax bacillus of SEQ ID NO.1 in the gene of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, the described encoding mutant amino-acid residue of SEQ ID NO.8 in the sequence table and the sequence table.
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|---|---|---|---|
| CN2008100452126A CN101215568B (en) | 2008-01-18 | 2008-01-18 | Anthrax resisting polypeptide and its application and preparation method |
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|---|---|---|---|
| CN2008100452126A CN101215568B (en) | 2008-01-18 | 2008-01-18 | Anthrax resisting polypeptide and its application and preparation method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3341393B1 (en) * | 2015-08-27 | 2021-03-03 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Modified anthrax toxin protective antigen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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