CN1532282A - Novel antienterococci polypeptide and its preparing method - Google Patents

Abstract

The present invention discloses a kind of novel enterococcus resisting polypeptide and its preparation process. The recombinant enterococcus resisting polypeptide contains colicine capable of forming ion channel or its aquatic porous channel structure domain and enterococcus signal transducing polypeptide. The present invention also discloses the nucleotide sequence encoding the recombinant enterococcus resisting polypeptide and recombinant plasmid containing said nucleotide sequence. Compared with traditional antibiotics, the novel enterococcus resisting polypeptide of the present invention 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 of the present invention reaches the aim of killing bacteria via forming ion channel on the cell membrane of target bacteria directly.

Description

A kind of novel anti faecalis polypeptide and preparation method thereof

Technical field

The present invention relates to a kind of anti-faecalis polypeptide of reorganization, the Nucleotide of this polypeptide of encoding and aminoacid sequence.

The invention still further relates to the preparation method of Ph-EF.

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 ' unecolicine et son mode d ' action, Annals of the Pasteur 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 voltage dependent 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, Majortransmembrane movement associated 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., Cell density control ofstaphylococcal 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 autoinducing thiolactone peptides fromstaphylococcus 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 faecalis 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-EF 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-EF.

A further object of the present invention is to provide the monoamino-acid sequence, its Ph-EF of the present invention of encoding.

A further object of the present invention is to provide the pharmaceutical composition that contains Ph-EF of the present invention.

A further object of the present invention is to provide the application of the anti-faecalis polypeptide of the present invention in the preparation antibacterials.

A further object of the present invention is to provide the preparation method of Ph-EF.

According to an aspect of the present invention, the gene that coding faecalis signal is conducted polypeptide is operably connected with the colicin gene, thereby obtains the nucleotide sequence of the anti-faecalis polypeptide of express recombinant.

In a preferred embodiment of the invention, adopt the pheromone gene of the gene (SEQ ID NO.1) of the enterococcal signal conduction of coding polypeptide cCF10, seven peptides of its coding 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 faecalis 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-faecalis polypeptide Ph-EF of the present invention.

In the Ph-EF 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 faecalis 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, the original plasmid pSELECTTM-1 that makes up plasmid vector comes from Promega company, colicin Ia and immunity protein gene have wherein been loaded, at faecalis signal conduction gene design a pair of primer, its sequence is respectively shown in SEQ ID NO.:6 and SEQ ID NO:7.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.

In accordance with a further aspect of the present invention, the pharmaceutical composition that contains Ph-EF 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 present invention is provided the preparation method of anti-faecalis polypeptide, the gene of coding faecalis signal conduction polypeptide operationally is connected the gene that obtains the coding Ph-EF with the colicin gene, the gene that obtains imported in the expression system express, separate polypeptide expressed and obtain anti-faecalis polypeptide of the present invention.

Anti-faecalis polypeptide 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 faecalis signal conduction polypeptide and Genebank A30128 sequence is identical among the present invention.

Brief description of drawings

Fig. 1 illustrates the structure of the plasmid pCHCEF that contains faecalis signal conduction polypeptide cCF10 and colicin Ia;

Fig. 2 illustrates the structure of anti-faecalis polypeptide;

Fig. 3 is the anti-faecalis polypeptide of the present invention (Ph-EF) extracorporeal disinfecting experimental result, and wherein left side figure is common faecalis, and the Ph-EF of visible preparation is the same with penicillin, has effectively suppressed the faecalis growth.Right figure is a vancomycin-resistant enterococcus, and vancomycin has partly suppressed the faecalis growth, and the Ph-EF fungistatic effect of preparation is stronger at least one times than vancomycin.

Fig. 4 is the transmission electron microscope observing (amplify 15,000 times) of anti-faecalis polypeptide (Ph-EF) to vancomycin-resistant enterococcus (ATCC 700802 VRE) action effect.

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-EF preparation of the plasmid of anti-faecalis polypeptide

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) the faecalis signal polypeptide cCF10 gene of will encoding is inserted on the I626 site of colicin Ia gene, has prepared the mutant plasmid pCHCEF (as shown in Figure 1) of anti-faecalis 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. add (37 ℃, 1 hour) behind the Dpn1 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;

4. 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);

5.18 choose bacterium after hour, 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. 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.

7. 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-EF Ph-EF, and its corresponding amino acid sequence is SEQ ID NO:5.

Designed oligonucleotide sequence in the above-mentioned preparation plasmid

5’-3’(SEQ?ID?NO：6)

gcg?aat?aag?ttc?tgg?ggt?att?CTG?GTT?ACC?CTT?GTG?TTC?GTG?taa?ata?aaa?tat?aag?aca?ggc

3’-5’(SEQ?ID?NO：7)

gcc?tgt?ctt?ata?ttt?tat?tta?CAC?GAA?CAC?AAG?GGT?AAC?CAG?aat?acc?cca?gaa?ctt?att?cgc

[embodiment 2] Ph-EF is to enterococcal vitro inhibition effect

Bacterium is the USS bacterial strain, ATCC 29212 (common faecalis) and ATCC 700802 (vancomycin-resistant enterococcus), and as shown in Figure 3, left figure adds common faecalis 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, second group adds 3 μ g/ml Benzylpenicillin sodiums, the 3rd group adds the anti-faecalis polypeptide of 3 μ g/ml (preserving liquid is 0.3M NaCl+50mM borate buffer), treats that bacterial growth adds the anti-faecalis polypeptide of 3 μ g (preserving liquid is 0.3M NaCl+50mM borate buffer) after three hours for the 4th group.

Right figure adds vancomycin-resistant enterococcus 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 10 μ g/ml vancomycins, treat that bacterial growth adds the anti-faecalis polypeptide of 10 μ g/ml (preserving liquid is 0.3M NaCl+50mM borate buffer) after two hours for the 3rd 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.

As seen from Figure 3, the Ph-EF of preparation is the same with penicillin, has suppressed the faecalis growth effectively.Right figure is a vancomycin-resistant enterococcus, and vancomycin has partly suppressed the faecalis growth, and the Ph-EF fungistatic effect of preparation is stronger at least one times than vancomycin.

Anti-faecalis polypeptide molecular weight is 70,000, is 49 times of vancomycin molecular weight 1436, and as pressing with same medicine molecular amounts in the volume relatively, the fungicidal effectiveness of anti-faecalis polypeptide is the tens of and even hundreds of times of Norvancomycin.

[embodiment 3] anti-faecalis polypeptide is to the transmission electron microscope observing (dyeing of 1% phospho-wolframic acid is amplified 15,000 times) of vancomycin-resistant enterococcus (ATCC 700802) sterilization effect.

Anti-faecalis polypeptide to the transmission electron microscope observing of vancomycin-resistant enterococcus (ATCC 700802) sterilization effect (dyeing of 1% phospho-wolframic acid is amplified 15,000 times) as shown in Figure 4.Control, control group, bacterium adds in the aforementioned nutrient solution, and 37 ℃ of 200rpm growths still presented normal morphology after 2 hours; Ph-SA, the same control group of bacterial growth condition added anti-golden Portugal bacterium polypeptide (10 μ g/ml) growth after two hours, and the bacterium form is still normal; Vanco, the same control group of bacterial growth condition added vancomycin (20 μ g/ml) growth after 2 hours, and the bacterium form is still normal; Ph-EF, the same control group of bacterial growth condition, add anti-faecalis polypeptide (10 μ g/ml) growth half an hour after, the bacterium shrinkage, content leaks, after two hours, above-mentioned sick decrease more obvious.

Conclusion: anti-faecalis polypeptide can kill vancomycin-resistant enterococcus (ATCC 700802) effectively.

[embodiment 4] anti-faecalis polypeptide is tested vancomycin-resistant enterococcus ATCC 700802 endogenous protectives

55 of mouse, body weight 20-25gm is divided into 4 groups: control group (n=10), penicillin group, vancomycin group and anti-faecalis polypeptide (Ph-EF) group, each medicine group is divided into 3 dosage groups again, every group of 5 mouse.(ATCC 700802, and the bacterium amount is 10 at abdominal injection lethal dose vancomycin-resistant enterococcus 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-EF is to vancomycin-resistant enterococcus ATCC 700802 endogenous protective test-results

Medicine	Dosage	Approach * dosage	Number of animals	Infect the dead distribution in back							Death toll
													??(mg/kg)	The Ml/20g mouse is heavy	(only)	??24h	??48h	??72h	??96h	??120 ??h	??144 ??h	??168 ??h	Only
??Ph-E ??F	??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	??2	??0	??0	??0	??0	??0	??2
												Penicillin	??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	??1	??0	??0	??0	??0	??0	??0	??1
Vancomycin	??10	IV× 0.5	??5	??1	??0	??0	??0	??0	??0	??0													??1
											??5		??5	??0	??2	??1	??0	??0	??0	??0	??3
	??2.5		??5	??1	??2	??0	??1	??0	??0	??0												??4
											Control group		IV× 0.5 0.9％ NaCl	??10	??3	??3	??1	??0	??3	??0	??0		??10

The result shows that Ph-EF can infect by anti-effectively faecalis in animal body, 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 faecalis polypeptide and preparation method thereof

<130>1553

<160>7

<170>PatentIn?version?3.1

<210>1

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)..(21)

＜223〉coding faecalis cCF10 signal conduction polypeptide

<400>1

ctg?gtt?acc?ctt?gtg?ttc?gtg??????????????????????????21

Leu?Val?Thr?Leu?Val?Phe?Val

1???????????????5

<210>2

<211>7

<212>PRT

＜213〉artificial sequence

<400>2

Leu?Val?Thr?Leu?Val?Phe?Val

1???????????????5

<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>1902

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(4)..(1899)

＜223〉the anti-faecalis polypeptide of coding

<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?atc?gcc?gga?tat?gac?gcc?ctc?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?ctg?gtt?acc?ctt?gtg?ttc?gtg?taa?????????????????????????????1902

Gly?Ile?Leu?Val?Thr?Leu?Val?Phe?Val

625?????????????????630

<210>5

<211>632

<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?Leu?Val?Thr?Leu?Val?Phe?Val

625?????????????????630

<210>6

<211>63

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(63)

＜223〉primer

<400>6

gcgaataagt?tctggggtat?tctggttacc?cttgtgttcg?tgtaaataaa?atataagaca????60

ggc??????????????????????????????????????????????????????????????????63

<210>7

<211>63

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(63)

＜223〉primer

<400>7

gcctgtctta?tattttattt?acacgaacac?aagggtaacc?agaatacccc?agaacttatt????60

cgc??????????????????????????????????????????????????????????????????63

Claims (12)

1. the Nucleotide of coding Ph-EF 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 faecalis signal conduction polypeptide.

2. Nucleotide according to claim 1, the gene of wherein said coding faecalis 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 faecalis signal conduction polypeptide.

7. recombinant plasmid according to claim 6 wherein contains the nucleotide sequence of SEQ ID NO.4.

8. Ph-EF, it contains

Can form colicin or its water-based pore passage structure territory of ionic channel, and

Faecalis signal conduction polypeptide.

9. anti-faecalis polypeptide according to claim 8 wherein comprises the aminoacid sequence of SEQ ID NO.5.

10. the pharmaceutical composition that contains the described Ph-EF of claim 9.

11. the application of the described anti-faecalis polypeptide of claim 9 in the preparation antibacterials.

12. the preparation method of Ph-EF may further comprise the steps:

The gene that the gene of coding faecalis 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-EF;

The gene that obtains imported in the expression system express;

Separate polypeptide expressed and obtain anti-faecalis polypeptide of the present invention.

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