CN1532283A - Novel anti-streptocouus pheumoniae polypeptide and its preparing method - Google Patents
Novel anti-streptocouus pheumoniae polypeptide and its preparing method Download PDFInfo
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- CN1532283A CN1532283A CNA031174922A CN03117492A CN1532283A CN 1532283 A CN1532283 A CN 1532283A CN A031174922 A CNA031174922 A CN A031174922A CN 03117492 A CN03117492 A CN 03117492A CN 1532283 A CN1532283 A CN 1532283A
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
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Abstract
The present invention discloses a kind of novel streptococcus pneumoniae resisting polypeptide and its preparation process. The recombinant streptococcus pneumoniae resisting polypeptide contains colicine capable of forming ion channel or its aquatic porous channel structure domain and streptococcus pneumoniae signal transducing polypeptide. The present invention also discloses the nucleotide sequence encoding the recombinant streptococcus pneumoniae resisting polypeptide and recombinant plasmid containing said nucleotide sequence. Compared with traditional antibiotics, the novel streptococcus pneumoniae resisting polypeptide has the advantage of no drug resistance the bacteria are induced to produce. The bacteria are hard to alter their phosphatide double molecular structure via mutation, and the recombinant polypeptide reaches the aim of killing bacteria via forming ion channel on the cell membrane of target bacteria directly.
Description
Technical field
The present invention relates to a kind of anti-streptococcus pneumoniae polypeptides of reorganization, the Nucleotide of this polypeptide of encoding and aminoacid sequence.
The invention still further relates to the preparation method of Ph-SP.
Background technology
Infectation of bacteria is human life and healthy main threat, since sulfanilamide (SN) and penicillin come out, the antibiotic of human invention successively mainly with by suppress bacterial cell wall synthetic, suppress or disturb the nucleic acid of bacterium and proteinic metabolism and route of synthesis to reach antibiotic purpose.Yet these antibiotic approach lure that easily bacterium undergos mutation and produce antibiotic resistance into.Therefore people are being devoted to the antibiotic of development of new always.Directly forming ionic channel and cause bacterium death on bacterial membrane is one of more promising antibiotic exploitation direction.Occurring in nature, bacteriotoxin quite a lot comes killing bacteria with this mechanism, its type specimen is exactly a kind of toxin protein of intestinal bacteria (Escherichia coli) excretory a---colicin (colicin), nineteen fifty-two Jacob finds some bacillus that its intestinal bacteria that can kill other strain system specifically and relevant strain are, as Shigella Sonnei (Jacob et al,) Sur labiosynthese d ' une colicine et son mode d ' action, Annals of thePasteur Institute.83:295-315 (1952)).Finkelstern in 1978 etc. have found that the colicin that can form ionic channel can form the voltage-dependent ionic channel on artificial bimolecular lipid membrane, thereby at the antibacterial mechanisms that has fundamentally disclosed this bacterioid toxin (Schein et al .Colicin K acts by forming voltagedependent channels in phospholipid bilayer membranes.Nature 276:159-163 (1973)).Qiu in 1996 and Finkelstein etc. have disclosed ionic channel that colicin Ia forms on artificial bimolecular lipid membrane open and stride film three-dimensional arrangement (Qiu et al. when closing, Major transmembrane movementassociated with colicin Ia channel gating.J.Gen.Physiology.107:313-328 (1996)), established theoretical basis on molecular level, designing and prepare novel antibiotic.
Recent two decades, people find that gradually bacterium also has the excretory signal conduction polypeptide with the external environment variation, regulating proteic gene in the after birth acceptor of preparation signal conduction polypeptide and the born of the same parents often is coupled at together, controlling growth and the division of bacterium in phase, but the research of this direction is made slow progress always, signal conduction polypeptide A grD such as staphylococcus aureus, until nineteen ninety-five is just made clear at last is an octapeptide (Ji et al., Celldensity control of staphylococcal virulence mediated by an octapeptide pheromone.Proc.Natl.Acad.Sci.USA 92:12055-12059 (1995)).Found that this octapeptide of the early stage of streptococcus aureus logarithmic phase or middle and advanced stage adding synthetic can stimulate or suppress growth (the Mayville et al. of streptococcus aureus in 1999, Structure activity analysis of synthetic autoinducingthiolactone peptides from staphylococcus aureus responsible for virulence.PNAS, 96:1218-1223 (1999)).
As mentioned above, colicin is a kind of ideal ionic channel antibiotic, shortcoming is to act on Gram-negative bacillus such as intestinal bacteria, if can utilize the distinctive signaling peptides of pathogenic bacterium as inductor, with colicin or its water-based pore passage structure or be induced to and form ionic channel near the specific bacterial film and kill and wound this pathogenic bacterium, should be a kind of ideal antibiotic exploitation direction.
Summary of the invention
One object of the present invention is to provide a kind of novel reorganization antimicrobial polypeptide, and it contains colicin and streptococcus pneumoniae signal conduction polypeptide, thereby can avoid bringing out drug-resistance of bacteria, improves sterilizing ability.
Another object of the present invention is to provide the nucleotide sequence of this Ph-SP of coding.
A further object of the present invention is to provide a recombinant plasmid, and it contains the nucleotide sequence of code book invention Ph-SP.
A further object of the present invention is to provide the monoamino-acid sequence, its Ph-SP of the present invention of encoding.
A further object of the present invention is to provide the pharmaceutical composition that contains Ph-SP of the present invention.
A further object of the present invention is to provide the application of the anti-streptococcus pneumoniae polypeptides of the present invention in the preparation antibacterials.
A further object of the present invention is to provide the preparation method of Ph-SP.
According to an aspect of the present invention, the gene that coding streptococcus pneumoniae signal is conducted polypeptide is operably connected with the colicin gene, thereby obtains the nucleotide sequence of the anti-streptococcus pneumoniae polypeptides of express recombinant.
In a preferred embodiment of the invention, adopt the signal of coding streptococcus pneumoniae to conduct the pheromone gene of the gene (SEQ ID NO.1) of polypeptide, its coding 17 amino acid whose peptides shown in SEQ ID NO:2 as antimicrobial polypeptide; The optional self energy of colicin forms colicin E1, Ia, Ib, A, B and N or its water-based pore passage structure territory of ionic channel, in a preferred embodiment of the invention, above-mentioned streptococcus pneumoniae signal conduction polypeptide gene is connected with the carboxyl terminal of colicin Ia (SEQ ID NO.3) and forms nucleotide sequence as SEQ ID NO.4.This nucleotide sequence coded aminoacid sequence as SEQ ID NO.5, this aminoacid sequence encoded polypeptide are anti-streptococcus pneumoniae polypeptides Ph-SP of the present invention.
In the Ph-SP that makes up, can induce colicin channel architecture territory to pass near bacterial outer membrane and the intermembrane space arrival target cell membrane as inductor with the signal of target bacteria after birth receptors bind conduction polypeptide, colicin water-based pore passage structure territory forms ionic channel on the target bacteria after birth then, make content leaks in the target bacteria born of the same parents and cause target bacteria death, thereby reach germ-resistant purpose.
According to a further aspect in the invention, the plasmid vector that comprises Nucleotide of the present invention as shown in Figure 1 is provided, and this plasmid vector is that the nucleotide sequence with aforesaid coding streptococcus pneumoniae signal conduction polypeptide inserts through double-stranded oligonucleotide point mutation technology and forms recombinant plasmid of the present invention on the 626th amino acids of colicin Ia gene.
Among the present invention, make up the original plasmid pSELECT of plasmid vector
TM-1 comes from Promega company, has wherein loaded colicin Ia and immunity protein gene, at streptococcus pneumoniae signal conduction gene design two pairs of primers, its sequence is respectively shown in SEQ ID NO.:6～SEQ ID NO:9.Utilize double-stranded oligonucleotide point mutation technology,, obtain recombinant plasmid, again the recombinant plasmid that obtains is transfected into the e. coli tg1 engineering bacteria and obtains host cell according to the medicine-chest operation of Strategene company through twice sudden change.
In accordance with a further aspect of the present invention, the pharmaceutical composition that contains Ph-SP of the present invention is provided, can makes the pharmaceutical composition that is suitable for clinical use by polypeptide of the present invention is added pharmaceutically acceptable carrier or vehicle or optional additional components.
According to another aspect of the invention, the preparation method of the anti-streptococcus pneumoniae polypeptides of the present invention is provided, the gene of coding streptococcus pneumoniae signal conduction polypeptide operationally is connected the gene that obtains the coding Ph-SP with the colicin gene, the gene that obtains imported in the expression system express, separate polypeptide expressed and obtain anti-streptococcus pneumoniae polypeptides of the present invention.
Anti-streptococcus pneumoniae polypeptides of the present invention is that compared to the advantage of traditional antibiotic it can not induce bacterium to produce traditional resistance.As everyone knows, bacterium can produce β-Nei Xiananmei by sudden change, reduce modes such as taking in, change the drug effect site change its cell wall structure, and the metabolism etc. that changes its protein and nucleic acid produces resistance to traditional antibiotic.But bacterium but is difficult to change by sudden change the structure of the phospholipid bilayer of its cytolemma, and this is distinctive feature of the present invention just, and recombinant polypeptide of the present invention reaches the sterilization purpose by directly form ionic channel on the after birth of target bacteria.
The expressed aminoacid sequence of the coded aminoacid sequence of nucleotide sequence SEQ ID NO.1 (SEQ ID NO:2) of coding streptococcus pneumoniae signal conduction polypeptide and Genebank AAC44440 sequence is identical among the present invention;
Brief description of drawings
Fig. 1 illustrates the structure of the plasmid pCHCSP that contains streptococcus pneumoniae signal conduction polypeptide and colicin Ia;
Fig. 2 illustrates the structure of anti-streptococcus pneumoniae polypeptides;
Fig. 3 is the anti-streptococcus pneumoniae polypeptides of the present invention (Ph-SP) extracorporeal disinfecting experimental result, and right figure is common streptococcus pneumoniae, and the Ph-SP of visible preparation is the same with penicillin, has effectively suppressed the growth of streptococcus pneumoniae.Left side figure is the penicillin resistant streptococcus pneumoniae, penicillin inhibition partly the pneumonia streptococcus bacteria growing, the Ph-SP fungistatic effect of preparation is relatively stronger at least 2 times than penicillin from the figure.
The embodiment of invention
Below in conjunction with accompanying drawing, describe the present invention in detail by description to preferred embodiment of the present invention.
[embodiment 1] expresses the structure and the Ph-SP preparation of the plasmid of anti-streptococcus pneumoniae polypeptides
Original plasmid is the pSELECT that has loaded colicin Ia and immunity protein gene
TM-1 commercial plasmid (plasmid size 8.3kb, Promega company) (UCSF, P.Gosh gifts).Through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategeue company) gene of the streptococcus pneumoniae signal peptide C omC that will encode is inserted on the I626 site of colicin Ia gene, has prepared the mutant plasmid pCHCEF (as shown in Figure 1) of anti-streptococcus pneumoniae engineered polypeptide.Mutant plasmid is transfected into E.coli TG1 engineering bacteria (AECOM, K.Jakes gifts) lining and prepares polypeptide.
The sudden change program is undertaken by Strategene QuikChange Site-Directed Mutagenesis Kit (Catalog#200518) medicine-chest handbook:
1. prepare the point mutation reactant:
5μl??10X?buffer
2 μ l (10ng) wild-type colicin plasmids
1.25 5 '-3 ' oligonucleotide primer (SEQ ID NO.6) of μ l (125ng) design
1.25 3 '-5 ' oligonucleotide primer (SEQ ID NO.7) of μ l (125ng) design
1μl?dNTP
Distilled water 50 μ l
1μl?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. with another primer (SEQ ID NO:8 and SEQ ID NO:9) is repeated aforesaid operations again one time;
4. add (37 ℃, 1 hour) behind the Dpnl restriction endonuclease 1 μ l digestion mother body D NA chain, get 1 μ l reactant and XL1-Blue competent cell 50 μ l ice and incubate, 42 ℃ of capable thermal shockings 45 seconds, were inserted in the ice 2 minutes again;
5. add NZY and train 220rpm behind the basic 0.5ml, 37 ℃ were shaken bacterium after 1 hour, got 50-100 μ l reactant bed board (LB training base adds 1% agar and adds 50 μ l/ml penbritins, and 37 ℃ are spent the night);
6. 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.
7. the TG1 engineering bacteria of plasmid 50ng and preparation is experienced polypeptide cell 50 μ l ice incubate 30 minutes 42 ℃, thermal shocking in 90 seconds, get 50-100 μ l reactant adding LB and train basic 0.5ml, 220rpm, 37 ℃ are shaken 37 ℃ on bacterium bed board after 1 hour (LB training base adds 1% agar and adds 50 μ g/ml penbritins), picking colony after 18 hours.
8. increase bacterium in a large number, 8-16 rises FB training base, 250rpm, 37 ℃, 6-8 hour; The centrifugation thalline, 4 ℃, 6000g, 20 minutes, get 4 ℃, 50mM borate buffer (2mM EDTA+2mM DTT) 50-80ml suspension thalline adds behind 0.2M PMSF 250 microlitres (4 ℃ of ultrasonications, 400W, 2 minutes), and the broken thalline of high speed centrifugation (4 ℃, 75000g, 1.5 hour), get supernatant and add the Vetstrep 5,000,000 deposit D NA of unit, and high speed centrifugation (4 ℃, 30000g, 10 minutes) after pack the dialysis tubing of molecular weight 15000 in 4 ℃, after 4 liters of dialysed overnight of 50mM borate buffer, and high speed centrifugation (4 ℃, 30000g, 10 minutes) back supernatant be splined on the CM ion exchange column, 4 ℃, 0.3M NaCl+50mM borate buffer wash-out can obtain Ph-SP Ph-SP, and its corresponding amino acid sequence is SEQ ID NO:5.
Designed oligonucleotide sequence in the above-mentioned preparation plasmid
1、
5’-3’(SEQ?ID?NO.6)
aaa?gcg?aat?aag?ttc?tgg?ggt?att?GAA?ATG?CGT?CTG?TCC?AAA?TTT?TTC?CGC?taa?ata
aaa?tat?aag?aca?ggc?tgt
3’-5’(SEQ?ID?NO.7)
aca?gcc?tgt?ctt?ata?ttt?tat?tta?GCG?GAA?AAA?TTT?GGA?CAG?ACG?CAT?TTC?aat?acc
cca?gaa?ctt?att?cgc?ttt
2、
5’-3’(SEQ?ID?NO.8)
atg?cgt?ctg?tcc?aaa?ttt?ttc?cgc?GAC?TTC?ATT?CTG?CAG?CGT?AAG?AAA?taa?ata?aaa
tat?aag?aca?ggc?tgt
3’-5’(SEQ?ID?NO.9)
aca?gcc?tgt?ctt?ata?ttt?tat?tta?TTT?CTT?ACG?CTG?CAG?AAT?GAA?GTC?gcg?gaa?aaa?ttt
gga?cag?acg?cat
[embodiment 2] Ph-SP is to the vitro inhibition effect of streptococcus pneumoniae
Bacterium is that USS strains A TCC 700671 (penicillin resistant streptococcus pneumoniae) and Chinese bacterial classification are preserved center bacterial strain 31201 (common streptococcus pneumoniaes), and as shown in Figure 3, left figure adds penicillin resistant streptococcus pneumoniae bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds 1% Tryptones, 1%NaCl, 0.5% yeast, 0.5% glucose, 1%HK
2PO
4Nutrient solution 10ml in, prepare 3 groups altogether, first group adds 0.3M NaCl+50mM borate buffer (the antimicrobial polypeptide amount of liquid that adds in amount and the experimental group is identical) in contrast, treat that bacteria growing is after two hours for second group, add 5 μ g/ml Benzylpenicillin sodiums, treat bacteria growing after two hours for the 3rd group, add the anti-streptococcus pneumoniae polypeptides of 5 μ g/ml (preserving liquid is 0.3M NaCl+50mM borate buffer).
Right figure adds common streptococcus pneumoniae bacterium liquid 5 microlitres (10
8CFU/ml level bacterium amount) adds 1% Tryptones, 1%NaCl, 0.5% yeast, 0.5% glucose, 1%HK
2PO
4Nutrient solution 10ml in, prepare 4 groups altogether, first group adds 0.3M NaCl+50mM borate buffer (the antimicrobial polypeptide amount of liquid that adds in amount and the experimental group is identical) in contrast, treat that bacterial growth is after two hours for second group, add 5 μ g/ml Benzylpenicillin sodiums, treat that bacterial growth adds 5 μ g/ml wild-type colicin Ia (preserving liquid is the 0.3MNaCl+50mM borate buffer) after two hours for the 3rd group, treat that bacterial growth adds the anti-streptococcus pneumoniae polypeptides of 5 μ g/ml (preserving liquid is 0.3M NaCl+50mM borate buffer) after two hours for the 4th group.
The above-mentioned liquid of respectively organizing places the 100ml Erlenmeyer flask, 200rpm, 37 ℃ of growths, the 100 μ l that per hour sample add in the 96 hole enzyme plates through spectrophotometer (A 595nm) colorimetric bacteria tested growth turbidity, and the growth curve of bacteria that draws comes the inhibitory effect of comparison antimicrobial polypeptide.
The molecular weight of anti-streptococcus pneumoniae polypeptides is 70,000, is 200 times of penicillin molecular weight 350, and as pressing with same medicine molecular amounts in the volume relatively, the fungicidal effectiveness of anti-streptococcus pneumoniae polypeptides is hundreds of times of penicillin.
[embodiment 3] anti-streptococcus pneumoniae polypeptides is tested penicillin resistant suis ATCC 700671 endogenous protectives
55 of mouse, body weight 20-25gm is divided into 4 groups: control group (n=10), penicillin group, Cephazolin group and anti-streptococcus pneumoniae polypeptides (Ph-SP) group, each medicine group is divided into 3 dosage groups again, every group of 5 mouse.(ATCC 700671, and the bacterium amount is 10 in abdominal injection lethal dose penicillin resistant suis for mouse
5CFU/ml) after 1 hour, inject the various medicines of diagram dosage once by the tail vein respectively, checked the dead mouse situation, and the results are shown in Table 1 in per 12 hours.
Table 1 Ph-SP is to penicillin resistant suis ATCC 700671 endogenous protective test-results
| Medicine | Dosage | Approach X dosage | Number of animals | Infect the dead distribution in back | Death toll | ||||||
| (mg/kg) | The M1/20g mouse is heavy | (only) | 24h | ?48h | ?72h | ?96h | ?120h | ?144h | ?168h | (only) | |
| ????Ph-SP | ????10 | ??IV× ????0.5 | ??5 | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 |
| ????5 | ??5 | ??0 | ??1 | ??0 | ??0 | ??0 | ??0 | ??0 | ??1 | ||
| ????2.5 | ??5 | ??0 | ??0 | ??1 | ??1 | ??0 | ??0 | ??0 | ??2 | ||
| Penicillin | ????10 | ??IV?X× ????0.5 | ??5 | ??3 | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 | ??3 |
| ????5 | ??5 | ??3 | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 | ??3 | ||
| ????2.5 | ??5 | ??5 | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 | ??5 | ||
| Cephazolin | ????10 | ??IV?X× ????0.5 | ??5 | ??2 | ??1 | ??0 | ??0 | ??0 | ??0 | ??0 | ??3 |
| ????5 | ??5 | ??3 | ??1 | ??0 | ??0 | ??0 | ??0 | ??0 | ??4 | ||
| ????2.5 | ??5 | ??2 | ??2 | ??1 | ??0 | ??0 | ??0 | ??0 | ??5 | ||
| Control group | ??IV?X× ????0.5 ??0.9％ ??NaCl | ??10 | ??9 | ??1 | ??0 | ??0 | ??0 | ??0 | ??0 | ??10 | |
The result shows, Ph-SP in animal body can anti-effectively streptococcus pneumoniae infection, and animal is had stronger provide protection.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
SEQUENCE?LISTING
＜110〉Yanghui Biotechnology Co Ltd, Chengdu
＜120〉a kind of novel anti streptococcus pneumoniae polypeptides and preparation method thereof
<130>1554
<160>9
<170>PatentIn?version?3.1
<210>1
<211>51
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(51)
＜223〉coding streptococcus pneumoniae signal conduction polypeptide
<400>1
gaa?atg?cgt?ctg?tcc?aaa?ttt?ttc?cgc?gac?ttc?att?ctg?cag?cgt?aag????48
Glu?Met?Arg?Leu?Ser?Lys?Phe?Phe?Arg?Asp?Phe?Ile?Leu?Gln?Arg?Lys
1???????????????5???????????????????10??????????????????15
aaa????????????????????????????????????????????????????????????????51
Lys
<210>2
<211>17
<212>PRT
＜213〉artificial sequence
<400>2
Glu?Met?Arg?Leu?Ser?Lys?Phe?Phe?Arg?Asp?Phe?Ile?Leu?Gln?Arg?Lys
1???????????????5???????????????????10??????????????????15
Lys
<210>3
<211>1878
<212>DNA
<213>Escherichia?coli
<220>
<221>misc?feature
<222>(1)..(1878)
＜223〉colicin gene
<400>3
atgtctgacc?ctgtacgtat?tacaaatccc?ggtgcagaat?cgctggggta?tgattcagat???60
ggccatgaaa?ttatggccgt?tgatatttat?gtaaaccctc?cacgtgtcga?tgtctttcat??120
ggtaccccgc?ctgcatggag?ttccttcggg?aacaaaacca?tctggggcgg?aaacgagtgg??180
gttgatgatt?ccccaacccg?aagtgatatc?gaaaaaaggg?acaaggaaat?cacagcgtac??240
aaaaacacgc?tcagcgcgca?gcagaaagag?aatgagaata?agcgtactga?agccggaaaa??300
cgcctctctg?cggcgattgc?tgcaagggaa?aaagatgaaa?acacactgaa?aacactccgt??360
gccggaaacg?cagatgccgc?tgatattaca?cgacaggagt?tcagactcct?gcaggcagag??420
ctgagagaat?acggattccg?tactgaaatc?gccggatatg?acgccctccg?gctgcataca??480
gagagccgga?tgctgtttgc?tgatgctgat?tctcttcgta?tatctccccg?ggaggccagg????540
tcgttaatcg?aacaggctga?aaaacggcag?aaggatgcgc?agaacgcaga?caagaaggcc????600
gctgatatgc?ttgctgaata?cgagcgcaga?aaaggtattc?tggacacccg?gttgtcagag????660
ctggaaaaaa?atggcggggc?agcccttgcc?gttcttgatg?cacaacaggc?ccgtctgctc????720
gggcagcaga?cacggaatga?cagggccatt?tcagaggccc?ggaataaact?cagttcagtg????780
acggaatcgc?ttaacacggc?ccgtaatgca?ttaaccagag?ctgaacaaca?gctgacgcaa????840
cagaaaaaca?cgcctgacgg?caaaacgata?gtttcccctg?aaaaattccc?ggggcgttca????900
tcaacaaatc?attctattgt?tgtgagcggt?gatccgagat?ttgccggtac?gataaaaatc????960
acaaccagcg?cagtcatcga?taaccgtgca?aacctgaatt?atcttctgag?ccattccggt???1020
ctggactata?aacgcaatat?tctgaatgac?cggaatccgg?tggtgacaga?ggatgtggaa???1080
ggtgacaaga?aaatttataa?tgctgaagtt?gctgaatggg?ataagttacg?gcaaagattg???1140
cttgatgcca?gaaataaaat?cacctctgct?gaatctgcgg?taaattcggc?gagaaataac???1200
ctcagtgcca?gaacaaatga?gcaaaagcat?gcaaatgacg?ctcttaatgc?cctgttgaag???1260
gaaaaagaga?atatacgtaa?ccagctttcc?ggcatcaatc?agaagatagc?ggaagagaaa???1320
agaaaacagg?atgaactgaa?ggcaacgaaa?gacgcaatta?atttcacaac?agagttcctg???1380
aaatcagttt?cagaaaaata?tggtgcaaaa?gctgagcagt?tagccagaga?gatggccggg???1440
caggctaaag?ggaagaaaat?acgtaatgtt?gaagaggcat?taaaaacgta?tgaaaagtac???1500
cgggctgaca?ttaacaaaaa?aattaatgca?aaagatcgtg?cagcgattgc?cgcagccctt???1560
gagtctgtga?agctgtctga?tatatcgtct?aatctgaaca?gattcagtcg?gggactggga???1620
tatgcaggaa?aatttacaag?tcttgctgac?tggatcactg?agtttggtaa?ggctgtccgg???1680
acagagaact?ggcgtcctct?ttttgttaaa?acagaaacca?tcatagcagg?caatgccgca???1740
acggctcttg?tggcactggt?cttcagtatt?cttaccggaa?gcgctttagg?cattatcggg???1800
tatggtttac?tgatggctgt?caccggtgcg?ctgattgatg?aatcgcttgt?ggaaaaagcg???1860
aataagttct?ggggtatt?????????????????????????????????????????????????1878
<210>4
<211>1932
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(4)..(1929)
＜223〉the anti-streptococcus pneumoniae polypeptides of encoding
<400>4
atg?tct?gac?cct?gta?cgt?att?aca?aat?ccc?ggt?gca?gaa?tcg?ctg?ggg???????48
Ser?Asp?Pro?Val?Arg?Ile?Thr?Asn?Pro?Gly?Ala?Glu?Ser?Leu?Gly
1???????????????5???????????????????10??????????????????15
tat?gat?tca?gat?ggc?cat?gaa?att?atg?gcc?gtt?gat?att?tat?gta?aac???????96
Tyr?Asp?Ser?Asp?Gly?His?Glu?Ile?Met?Ala?Val?Asp?Ile?Tyr?Val?Asn
20??????????????????25??????????????????30
cct?cca?cgt?gtc?gat?gtc?ttt?cat?ggt?acc?ccg?cct?gca?tgg?agt?tcc??????144
Pro?Pro?Arg?Val?Asp?Val?Phe?His?Gly?Thr?Pro?Pro?Ala?Trp?Ser?Ser
35??????????????????40??????????????????45
ttc?ggg?aac?aaa?acc?atc?tgg?ggc?gga?aac?gag?tgg?gtt?gat?gat?tcc??????192
Phe?Gly?Asn?Lys?Thr?Ile?Trp?Gly?Gly?Asn?Glu?Trp?Val?Asp?Asp?Ser
50??????????????????55??????????????????60
cca?acc?cga?agt?gat?atc?gaa?aaa?agg?gac?aag?gaa?atc?aca?gcg?tac??????240
Pro?Thr?Arg?Ser?Asp?Ile?Glu?Lys?Arg?Asp?Lys?Glu?Ile?Thr?Ala?Tyr
65??????????????????70??????????????????75
aaa?aac?acg?ctc?agc?gcg?cag?cag?aaa?gag?aat?gag?aat?aag?cgt?act??????288
Lys?Asn?Thr?Leu?Ser?Ala?Gln?Gln?Lys?Glu?Asn?Glu?Asn?Lys?Arg?Thr
80??????????????????85??????????????????90??????????????????95
gaa?gcc?gga?aaa?cgc?ctc?tct?gcg?gcg?att?gct?gca?agg?gaa?aaa?gat??????336
Glu?Ala?Gly?Lys?Arg?Leu?Ser?Ala?Ala?Ile?Ala?Ala?Arg?Glu?Lys?Asp
100?????????????????105?????????????????110
gaa?aac?aca?ctg?aaa?aca?ctc?cgt?gcc?gga?aac?gca?gat?gcc?gct?gat??????384
Glu?Asn?Thr?Leu?Lys?Thr?Leu?Arg?Ala?Gly?Asn?Ala?Asp?Ala?Ala?Asp
115?????????????????120?????????????????125
att?aca?cga?cag?gag?ttc?aga?ctc?ctg?cag?gca?gag?ctg?aga?gaa?tac??????432
Ile?Thr?Arg?Gln?Glu?Phe?Arg?Leu?Leu?Gln?Ala?Glu?Leu?Arg?Glu?Tyr
130?????????????????135?????????????????140
gga?ttc?cgt?act?gaa?arc?gcc?gga?tat?gac?gcc?crc?cgg?ctg?cat?aca??????480
Gly?Phe?Arg?Thr?Glu?Ile?Ala?Gly?Tyr?Asp?Ala?Leu?Arg?Leu?His?Thr
145?????????????????150?????????????????155
gag?agc?cgg?atg?ctg?ttt?gct?gat?gct?gat?tct?ctt?cgt?ata?tct?ccc??????528
Glu?Ser?Arg?Met?Leu?Phe?Ala?Asp?Ala?Asp?Ser?Leu?Arg?Ile?Ser?Pro
160?????????????????165?????????????????170?????????????????175
cgg?gag?gcc?agg?tcg?tta?atc?gaa?cag?gct?gaa?aaa?cgg?cag?aag?gat??????576
Arg?Glu?Ala?Arg?Ser?Leu?Ile?Glu?Gln?Ala?Glu?Lys?Arg?Gln?Lys?Asp
180?????????????????185?????????????????190
gcg?cag?aac?gca?gac?aag?aag?gcc?gct?gat?atg?ctt?gct?gaa?tac?gag??????624
Ala?Gln?Asn?Ala?Asp?Lys?Lys?Ala?Ala?Asp?Met?Leu?Ala?Glu?Tyr?Glu
195?????????????????200?????????????????205
cgc?aga?aaa?ggt?att?ctg?gac?acc?cgg?ttg?tca?gag?ctg?gaa?aaa?aat??????672
Arg?Arg?Lys?Gly?Ile?Leu?Asp?Thr?Arg?Leu?Ser?Glu?Leu?Glu?Lys?Asn
210?????????????????215?????????????????220
ggc?ggg?gca?gcc?ctt?gcc?gtt?ctt?gat?gca?caa?cag?gcc?cgt?ctg?ctc??????720
Gly?Gly?Ala?Ala?Leu?Ala?Val?Leu?Asp?Ala?Gln?Gln?Ala?Arg?Leu?Leu
225?????????????????230?????????????????235
ggg?cag?cag?aca?cgg?aat?gac?agg?gcc?att?tca?gag?gcc?cgg?aat?aaa??????768
Gly?Gln?Gln?Thr?Arg?Asn?Asp?Arg?Ala?Ile?Ser?Glu?Ala?Arg?Asn?Lys
240?????????????????245?????????????????250?????????????????255
ctc?agt?tca?gtg?acg?gaa?tcg?ctt?aac?acg?gcc?cgt?aat?gca?tta?acc??????816
Leu?Ser?Ser?Val?Thr?Glu?Ser?Leu?Asn?Thr?Ala?Arg?Asn?Ala?Leu?Thr
260?????????????????265?????????????????270
aga?gct?gaa?caa?cag?ctg?acg?caa?cag?aaa?aac?acg?cct?gac?ggc?aaa??????864
Arg?Ala?Glu?Gln?Gln?Leu?Thr?Gln?Gln?Lys?Asn?Thr?Pro?Asp?Gly?Lys
275?????????????????280?????????????????285
acg?ata?gtt?tcc?cct?gaa?aaa?ttc?ccg?ggg?cgt?tca?tca?aca?aat?cat??????912
Thr?Ile?Val?Ser?Pro?Glu?Lys?Phe?Pro?Gly?Arg?Ser?Ser?Thr?Asn?His
290?????????????????295?????????????????300
tct?att?gtt?gtg?agc?ggt?gat?ccg?aga?ttt?gcc?ggt?acg?ata?aaa?atc??????960
Ser?Ile?Val?Val?Ser?Gly?Asp?Pro?Arg?Phe?Ala?Gly?Thr?Ile?Lys?Ile
305?????????????????310?????????????????315
aca?acc?agc?gca?gtc?atc?gat?aac?cgt?gca?aac?ctg?aat?tat?ctt?ctg?????1008
Thr?Thr?Ser?Ala?Val?Ile?Asp?Asn?Arg?Ala?Asn?Leu?Asn?Tyr?Leu?Leu
320?????????????????325?????????????????330?????????????????335
agc?cat?tcc?ggt?ctg?gac?tat?aaa?cgc?aat?att?ctg?aat?gac?cgg?aat?????1056
Ser?His?Ser?Gly?Leu?Asp?Tyr?Lys?Arg?Asn?Ile?Leu?Asn?Asp?Arg?Asn
340?????????????????345?????????????????350
ccg?gtg?gtg?aca?gag?gat?gtg?gaa?ggt?gac?aag?aaa?att?tat?aat?gct??????1104
Pro?Val?Val?Thr?Glu?Asp?Val?Glu?Gly?Asp?Lys?Lys?Ile?Tyr?Asn?Ala
355?????????????????360?????????????????365
gaa?gtt?gct?gaa?tgg?gat?aag?tta?cgg?caa?aga?ttg?ctt?gat?gcc?aga??????1152
Glu?Val?Ala?Glu?Trp?Asp?Lys?Leu?Arg?Gln?Arg?Leu?Leu?Asp?Ala?Arg
370?????????????????375?????????????????380
aat?aaa?atc?acc?tct?gct?gaa?tct?gcg?gta?aat?tcg?gcg?aga?aat?aac??????1200
Asn?Lys?Ile?Thr?Ser?Ala?Glu?Ser?Ala?Val?Asn?Ser?Ala?Arg?Asn?Asn
385?????????????????390?????????????????395
ctc?agt?gcc?aga?aca?aat?gag?caa?aag?cat?gca?aat?gac?gct?ctt?aat??????1248
Leu?Ser?Ala?Arg?Thr?Asn?Glu?Gln?Lys?His?Ala?Asn?Asp?Ala?Leu?Asn
400?????????????????405?????????????????410?????????????????415
gcc?ctg?ttg?aag?gaa?aaa?gag?aat?ata?cgt?aac?cag?ctt?tcc?ggc?atc??????1296
Ala?Leu?Leu?Lys?Glu?Lys?Glu?Asn?Ile?Arg?Asn?Gln?Leu?Ser?Gly?Ile
420?????????????????425?????????????????430
aat?cag?aag?ata?gcg?gaa?gag?aaa?aga?aaa?cag?gat?gaa?ctg?aag?gca??????1344
Asn?Gln?Lys?Ile?Ala?Glu?Glu?Lys?Arg?Lys?Gln?Asp?Glu?Leu?Lys?Ala
435?????????????????440?????????????????445
acg?aaa?gac?gca?att?aat?ttc?aca?aca?gag?ttc?ctg?aaa?tca?gtt?tca??????1392
Thr?Lys?Asp?Ala?Ile?Asn?Phe?Thr?Thr?Glu?Phe?Leu?Lys?Ser?Val?Ser
450?????????????????455?????????????????460
gaa?aaa?tat?ggt?gca?aaa?gct?gag?cag?tta?gcc?aga?gag?atg?gcc?ggg??????1440
Glu?Lys?Tyr?Gly?Ala?Lys?Ala?Glu?Gln?Leu?Ala?Arg?Glu?Met?Ala?Gly
465?????????????????470?????????????????475
cag?gct?aaa?ggg?aag?aaa?ata?cgt?aat?gtt?gaa?gag?gca?tta?aaa?acg??????1488
Gln?Ala?Lys?Gly?Lys?Lys?Ile?Arg?Asn?Val?Glu?Glu?Ala?Leu?Lys?Thr
480?????????????????485?????????????????490?????????????????495
tat?gaa?aag?tac?cgg?gct?gac?att?aac?aaa?aaa?att?aat?gca?aaa?gat??????1536
Tyr?Glu?Lys?Tyr?Arg?Ala?Asp?Ile?Asn?Lys?Lys?Ile?Asn?Ala?Lys?Asp
500?????????????????505?????????????????510
cgt?gca?gcg?att?gcc?gca?gcc?ctt?gag?tct?gtg?aag?ctg?tct?gat?ata??????1584
Arg?Ala?Ala?Ile?Ala?Ala?Ala?Leu?Glu?Ser?Val?Lys?Leu?Ser?Asp?Ile
515?????????????????520?????????????????525
tcg?tct?aat?ctg?aac?aga?ttc?agt?cgg?gga?ctg?gga?tat?gca?gga?aaa??????1632
Ser?Ser?Asn?Leu?Asn?Arg?Phe?Ser?Arg?Gly?Leu?Gly?Tyr?Ala?Gly?Lys
530?????????????????535?????????????????540
ttt?aca?agt?ctt?gct?gac?tgg?atc?act?gag?ttt?ggt?aag?gct?gtc?cgg??????1680
Phe?Thr?Ser?Leu?Ala?Asp?Trp?Ile?Thr?Glu?Phe?Gly?Lys?Ala?Val?Arg
545?????????????????550?????????????????555
aca?gag?aac?tgg?cgt?cct?ctt?ttt?gtt?aaa?aca?gaa?acc?atc?ata?gca??????1728
Thr?Glu?Asn?Trp?Arg?Pro?Leu?Phe?Val?Lys?Thr?Glu?Thr?Ile?Ile?Ala
560?????????????????565?????????????????570?????????????????575
ggc?aat?gcc?gca?acg?gct?ctt?gtg?gca?ctg?gtc?ttc?agt?att?ctt?acc??????1776
Gly?Asn?Ala?Ala?Thr?Ala?Leu?Val?Ala?Leu?Val?Phe?Ser?Ile?Leu?Thr
580?????????????????585?????????????????590
gga?agc?gct?tta?ggc?att?atc?ggg?tat?ggt?tta?ctg?atg?gct?gtc?acc??????1824
Gly?Ser?Ala?Leu?Gly?Ile?Ile?Gly?Tyr?Gly?Leu?Leu?Met?Ala?Val?Thr
595?????????????????600?????????????????605
ggt?gcg?ctg?att?gat?gaa?tcg?ctt?gtg?gaa?aaa?gcg?aat?aag?ttc?tgg??????1872
Gly?Ala?Leu?Ile?Asp?Glu?Ser?Leu?Val?Glu?Lys?Ala?Asn?Lys?Phe?Trp
610?????????????????615?????????????????620
ggt?att?gaa?atg?cgt?ctg?tcc?aaa?ttt?ttc?cgc?gac?ttc?att?ctg?cag??????1920
Gly?Ile?Glu?Met?Arg?Leu?Ser?Lys?Phe?Phe?Arg?Asp?Phe?Ile?Leu?Gln
625?????????????????630?????????????????635
cgt?aag?aaa?taa??????????????????????????????????????????????????????1932
Arg?Lys?Lys
640
<210>5
<211>642
<212>PRT
＜213〉artificial sequence
<400>5
Ser?Asp?Pro?Val?Arg?Ile?Thr?Asn?Pro?Gly?Ala?Glu?Ser?Leu?Gly?Tyr
1???????????????5???????????????????10??????????????????15
Asp?Ser?Asp?Gly?His?Glu?Ile?Met?Ala?Val?Asp?Ile?Tyr?Val?Asn?Pro
20??????????????????25??????????????????30
Pro?Arg?Val?Asp?Val?Phe?His?Gly?Thr?Pro?Pro?Ala?Trp?Ser?Ser?Phe
35??????????????????40??????????????????45
Gly?Asn?Lys?Thr?Ile?Trp?Gly?Gly?Asn?Glu?Trp?Val?Asp?Asp?Ser?Pro
50??????????????????55??????????????????60
Thr?Arg?Ser?Asp?Ile?Glu?Lys?Arg?Asp?Lys?Glu?Ile?Thr?Ala?Tyr?Lys
65??????????????????70??????????????????75??????????????????80
Asn?Thr?Leu?Ser?Ala?Gln?Gln?Lys?Glu?Asn?Glu?Asn?Lys?Arg?Thr?Glu
85??????????????????90??????????????????95
Ala?Gly?Lys?Arg?Leu?Ser?Ala?Ala?Ile?Ala?Ala?Arg?Glu?Lys?Asp?Glu
100?????????????????105?????????????????110
Asn?Thr?Leu?Lys?Thr?Leu?Arg?Ala?Gly?Asn?Ala?Asp?Ala?Ala?Asp?Ile
115?????????????????120?????????????????125
Thr?Arg?Gln?Glu?Phe?Arg?Leu?Leu?Gln?Ala?Glu?Leu?Arg?Glu?Tyr?Gly
130?????????????????135?????????????????140
Phe?Arg?Thr?Glu?Ile?Ala?Gly?Tyr?Asp?Ala?Leu?Arg?Leu?His?Thr?Glu
145?????????????????150?????????????????155?????????????????160
Ser?Arg?Met?Leu?Phe?Ala?Asp?Ala?Asp?Ser?Leu?Arg?Ile?Ser?Pro?Arg
165?????????????????170?????????????????175
Glu?Ala?Arg?Ser?Leu?Ile?Glu?Gln?Ala?Glu?Lys?Arg?Gln?Lys?Asp?Ala
180?????????????????185?????????????????190
Gln?Asn?Ala?Asp?Lys?Lys?Ala?Ala?Asp?Met?Leu?Ala?Glu?Tyr?Glu?Arg
195?????????????????200?????????????????205
Arg?Lys?Gly?Ile?Leu?Asp?Thr?Arg?Leu?Ser?Glu?Leu?Glu?Lys?Asn?Gly
210?????????????????215?????????????????220
Gly?Ala?Ala?Leu?Ala?Val?Leu?Asp?Ala?Gln?Gln?Ala?Arg?Leu?Leu?Gly
225?????????????????230?????????????????235?????????????????240
Gln?Gln?Thr?Arg?Asn?Asp?Arg?Ala?Ile?Ser?Glu?Ala?Arg?Asn?Lys?Leu
245?????????????????250?????????????????255
Ser?Ser?Val?Thr?Glu?Ser?Leu?Asn?Thr?Ala?Arg?Asn?Ala?Leu?Thr?Arg
260?????????????????265?????????????????270
Ala?Glu?Gln?Gln?Leu?Thr?Gln?Gln?Lys?Asn?Thr?Pro?Asp?Gly?Lys?Thr
275?????????????????280?????????????????285
Ile?Val?Ser?Pro?Glu?Lys?Phe?Pro?Gly?Arg?Ser?Ser?Thr?Asn?His?Ser
290?????????????????295?????????????????300
Ile?Val?Val?Ser?Gly?Asp?Pro?Arg?Phe?Ala?Gly?Thr?Ile?Lys?Ile?Thr
305?????????????????310?????????????????315?????????????????320
Thr?Ser?Ala?Val?Ile?Asp?Asn?Arg?Ala?Asn?Leu?Asn?Tyr?Leu?Leu?Ser
325?????????????????330?????????????????335
His?Ser?Gly?Leu?Asp?Tyr?Lys?Arg?Asn?Ile?Leu?Asn?Asp?Arg?Asn?Pro
340?????????????????345?????????????????350
Val?Val?Thr?Glu?Asp?Val?Glu?Gly?Asp?Lys?Lys?Ile?Tyr?Asn?Ala?Glu
355?????????????????360?????????????????365
Val?Ala?Glu?Trp?Asp?Lys?Leu?Arg?Gln?Arg?Leu?Leu?Asp?Ala?Arg?Asn
370?????????????????375?????????????????380
Lys?Ile?Thr?Ser?Ala?Glu?Ser?Ala?Val?Asn?Ser?Ala?Arg?Asn?Asn?Leu
385?????????????????390?????????????????395?????????????????400
Ser?Ala?Arg?Thr?Asn?Glu?Gln?Lys?His?Ala?Asn?Asp?Ala?Leu?Asn?Ala
405?????????????????410?????????????????415
Leu?Leu?Lys?Glu?Lys?Glu?Asn?Ile?Arg?Asn?Gln?Leu?Ser?Gly?Ile?Asn
420?????????????????425?????????????????430
Gln?Lys?Ile?Ala?Glu?Glu?Lys?Arg?Lys?Gln?Asp?Glu?Leu?Lys?Ala?Thr
435?????????????????440?????????????????445
Lys?Asp?Ala?Ile?Asn?Phe?Thr?Thr?Glu?Phe?Leu?Lys?Ser?Val?Ser?Glu
450?????????????????455?????????????????460
Lys?Tyr?Gly?Ala?Lys?Ala?Glu?Gln?Leu?Ala?Arg?Glu?Met?Ala?Gly?Gln
465?????????????????470?????????????????475?????????????????480
Ala?Lys?Gly?Lys?Lys?Ile?Arg?Asn?Val?Glu?Glu?Ala?Leu?Lys?Thr?Tyr
485?????????????????490?????????????????495
Glu?Lys?Tyr?Arg?Ala?Asp?Ile?Asn?Lys?Lys?Ile?Asn?Ala?Lys?Asp?Arg
500?????????????????505?????????????????510
Ala?Ala?Ile?Ala?Ala?Ala?Leu?Glu?Ser?Val?Lys?Leu?Ser?Asp?Ile?Ser
515?????????????????520?????????????????525
Ser?Asn?Leu?Asn?Arg?Phe?Ser?Arg?Gly?Leu?Gly?Tyr?Ala?Gly?Lys?Phe
530?????????????????535?????????????????540
Thr?Ser?Leu?Ala?Asp?Trp?Ile?Thr?Glu?Phe?Gly?Lys?Ala?Val?Arg?Thr
545?????????????????550?????????????????555?????????????????560
Glu?Asn?Trp?Arg?Pro?Leu?Phe?Val?Lys?Thr?Glu?Thr?Ile?Ile?Ala?Gly
565?????????????????570?????????????????575
Asn?Ala?Ala?Thr?Ala?Leu?Val?Ala?Leu?Val?Phe?Ser?Ile?Leu?Thr?Gly
580?????????????????585?????????????????590
Ser?Ala?Leu?Gly?Ile?Ile?Gly?Tyr?Gly?Leu?Leu?Met?Ala?Val?Thr?Gly
595?????????????????600?????????????????605
Ala?Leu?Ile?Asp?Glu?Ser?Leu?Val?Glu?Lys?Ala?Asn?Lys?Phe?Trp?Gly
610?????????????????615?????????????????620
Ile?Glu?Met?Arg?Leu?Ser?Lys?Phe?Phe?Arg?Asp?Phe?Ile?Leu?Gln?Arg
625?????????????????630?????????????????635?????????????????640
Lys?Lys
<210>6
<211>75
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(75)
＜223〉primer
<400>6
aaagcgaata?agttctgggg?tattgaaatg?cgtctgtcca?aatttttccg?ctaaataaaa????60
tataagacag?gctgt?????????????????????????????????????????????????????75
<210>7
<211>75
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(75)
＜223〉primer
<400>7
acagcctgtc?ttatatttta?tttagcggaa?aaatttggac?agacgcattt?caatacccca????60
gaacttattc?gcttt?????????????????????????????????????????????????????75
<210>8
<211>72
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(72)
＜223〉primer
<400>8
atgcgtctgt?ccaaattttt?ccgcgacttc?attctgcagc?gtaagaaata?aataaaatat????60
aagacaggct?gt????????????????????????????????????????????????????????72
<210>9
<211>72
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(72)
＜223〉primer
<400>9
acagcctgtc?ttatatttta?tttatttctt?acgctgcaga?atgaagtcgc?ggaaaaattt????60
ggacagacgc?at????????????????????????????????????????????????????????72
Claims (12)
1. the Nucleotide of coding Ph-SP is characterized in that it contains
Coding can form the colicin of ionic channel or the gene in its water-based pore passage structure territory, and
The gene of coding streptococcus pneumoniae signal conduction polypeptide.
2. Nucleotide according to claim 1, the gene of wherein said coding streptococcus pneumoniae signal conduction polypeptide are connected in the C-terminal that described coding can form the gene in the colicin of ionic channel or its water-based pore passage structure territory.
3. Nucleotide according to claim 2, the wherein said colicin that forms ionic channel is selected from colicin E1, Ia, Ib, A, B and N.
4. Nucleotide according to claim 3, the wherein said colicin that forms ionic channel is colicin Ia.
5. Nucleotide according to claim 4 wherein contains the nucleotide sequence of SEQ ID NO.4.
6. a recombinant plasmid is characterized in that it contains
Coding can form the colicin of ionic channel or the gene in its water-based pore passage structure territory, and
The gene of coding streptococcus pneumoniae signal conduction polypeptide.
7. recombinant plasmid according to claim 6 wherein contains the nucleotide sequence of SEQ ID NO.4.
8. Ph-SP, it contains
Can form colicin or its water-based pore passage structure territory of ionic channel, and
Streptococcus pneumoniae signal conduction polypeptide.
9. anti-streptococcus pneumoniae polypeptides according to claim 8 wherein comprises the aminoacid sequence of SEQ ID NO.5.
10. the pharmaceutical composition that contains the described Ph-SP of claim 9.
11. the application of the described anti-streptococcus pneumoniae polypeptides of claim 9 in the preparation antibacterials.
12. the preparation method of Ph-SP may further comprise the steps:
The gene that the gene of coding streptococcus pneumoniae signal conduction polypeptide and coding can be formed the colicin of ionic channel or its water-based pore passage structure territory is operably connected and obtains to encode the gene of Ph-SP;
The gene that obtains imported in the expression system express;
Separate polypeptide expressed and obtain anti-streptococcus pneumoniae polypeptides of the present invention.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031174922A CN1532283A (en) | 2003-03-19 | 2003-03-19 | Novel anti-streptocouus pheumoniae polypeptide and its preparing method |
| PCT/CN2004/000047 WO2004083437A1 (en) | 2003-03-19 | 2004-01-14 | A new peptide against streptococcus pneumoniae and the method of producing it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031174922A CN1532283A (en) | 2003-03-19 | 2003-03-19 | Novel anti-streptocouus pheumoniae polypeptide and its preparing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1532283A true CN1532283A (en) | 2004-09-29 |
Family
ID=32997027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031174922A Withdrawn CN1532283A (en) | 2003-03-19 | 2003-03-19 | Novel anti-streptocouus pheumoniae polypeptide and its preparing method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1532283A (en) |
| WO (1) | WO2004083437A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007083175A1 (en) * | 2006-01-17 | 2007-07-26 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| CN108164586A (en) * | 2018-01-22 | 2018-06-15 | 西南医科大学 | Synthesis polypeptide and its application |
| WO2023050661A1 (en) * | 2021-09-28 | 2023-04-06 | 北京亦科信息菌素研究院有限公司 | Pheromonicin against sars-cov-2 and use thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1164612C (en) | 2001-09-11 | 2004-09-01 | 四川新泰克控股有限责任公司 | Artificial combined antibacterial engineering polypeptide and its preparation method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1164612C (en) * | 2001-09-11 | 2004-09-01 | 四川新泰克控股有限责任公司 | Artificial combined antibacterial engineering polypeptide and its preparation method |
| CN1396178A (en) * | 2002-06-17 | 2003-02-12 | 成都阳辉生物科技有限责任公司 | Engineered polypeptide resisting infection of coated virus (hepatitis B virus) and its preparing process |
-
2003
- 2003-03-19 CN CNA031174922A patent/CN1532283A/en not_active Withdrawn
-
2004
- 2004-01-14 WO PCT/CN2004/000047 patent/WO2004083437A1/en active Application Filing
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007083175A1 (en) * | 2006-01-17 | 2007-07-26 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| CN108164586A (en) * | 2018-01-22 | 2018-06-15 | 西南医科大学 | Synthesis polypeptide and its application |
| CN108164586B (en) * | 2018-01-22 | 2021-03-26 | 西南医科大学 | Synthetic polypeptides and uses thereof |
| WO2023050661A1 (en) * | 2021-09-28 | 2023-04-06 | 北京亦科信息菌素研究院有限公司 | Pheromonicin against sars-cov-2 and use thereof |
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| Publication number | Publication date |
|---|---|
| WO2004083437A1 (en) | 2004-09-30 |
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