CN1317300C - Glycine-riched protein and active fragment and use thereof - Google Patents
Glycine-riched protein and active fragment and use thereof Download PDFInfo
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Abstract
The present invention discloses a glycine-rich protein, an active segment of the protein and applications of the protein. The glycine-rich protein provided by the present invention is selected from one of the following proteins: a protein with 1 to 6 amino acid residue sequences in a sequence table and a protein added with 1 to 20 amino acid residue at a carboxyl terminal and/or an amino terminal, wherein 1 to 6 amino acid residue of the protein added with 1 to 20 amino acid residue in the sequence table is deleted and/or inserted and/or replaced from 1 to 20 amino acid residue, and the protein added with 1 to 20 amino acid residue has antibacterial action. The active segment of the glycine-rich protein is a polypeptide with one of the following amino acid residue sequences: a polypeptide with 8 to 11 amino acid residue sequences in the sequence table and a polypeptide added with 1 to 5 amino acid residue at a carboxyl terminal and/or an amino terminal, wherein 8 to 11 amino acid residue of the polypeptide added with 1 to 5 amino acid residue in the sequence table is deleted and/or inserted and/or replaced from 1 to 5 amino acid residue, and the polypeptide added with 1 to 5 amino acid residue has antibacterial action. The glycine-rich protein of the present invention and the active segment of the protein can be used for antisepsis.
Description
Technical field
The present invention relates to be rich in glycine albumen and active fragments thereof and application, particularly this is rich in glycine albumen and the application of active fragments in antibacterium thereof.
Background technology
Over more than 20 year, the world of medicine does not find any new antibiotic family, but the development of resistance phenomenon is but very rapid, especially fearful is, the obstacle that tackles streptococcus aureus is just captured, and American physician has just been found bacterial strain that up-to-date antibiotic one vancomycin that can effectively suppress bacterium is had resistance.The resistance problem of pathogenic bacterium has day by day seriously threatened people's health.The microbiotic of seeking brand-new type is an effective way that solves the resistance problem.Antibacterial peptide is as the autarcetic bioactive molecule of biology, is used for resisting the infecting of germ of coming from the outside by various biologies, extensively exists in organic sphere.The people is that the natural antibacterial peptide of separating shows the anti-microbial activity height, has a broad antifungal spectrum, kind is many, and alternative scope is wide, the target bacterial strain is difficult for producing characteristics such as resistant mutation, thereby is considered to wide application prospect on medicine industry, foodstuffs industry and agricultural.
Antibacterial peptide is meant by genes encoding, usually below 10kDa, has the polypeptides matter of anti-microbial activity at synthetic relative molecular mass on the rrna, also is peptide antibiotics.Most of antibacterial peptide has thermostability, heats 10～15min down at 100 ℃ and still can keep its activity.Antibacterial peptide all has stronger resistance to bigger ionic strength and higher or lower pH value.The iso-electric point of most antibacterial peptides shows stronger positively charged ion feature greater than 7.Simultaneously, the part antibacterial peptide still possesses the ability of opposing trypsinase or pepsin hydrolysis.In addition, discover between the antibacterial peptide of different families on sequence, seldom to have homology, and have the conservative property of height between the different members of same family on the sequence, this means that its function is also very conservative.Antibacterial peptide is except having the effect of antibacterium or fungi, and some also has the function of protozoacide, virus or cancer cells.Antibacterial peptide extensively exists in organic sphere.Up to now, people are from bacterium, fungi, to batrachians, insect, higher plant, Mammals, found the nearly peptide antibiotics more than 700 kinds in human body.At present, existing multiple antibacterial peptide is carrying out preclinical study; In addition, antibacterial peptide transgenic animal, transgenic plant, antibacterial peptide is at food antiseptic, and fresh flower is fresh-keeping, makeup, the applied research of aspects such as seed pelleting and animal feedstuff additive is also in progress.
In view of antibacterial peptide has wide application prospect on medicine industry, foodstuffs industry and agricultural, seeking novel natural antibacterial peptide by all means from organic sphere just becomes one of focus in the present world wide.
Summary of the invention
The purpose of this invention is to provide a kind of glycine albumen and active fragments and their antibacterial application of being rich in.
The glycine albumen that is rich in provided by the present invention, name is called Glyrichin, is selected from least a in (clone from) following proteins family (Glyrichin family):
1) people Glyrichin (hGlyrichin) and mouse Glyrichin (mGlyrichin): all have the protein of the amino acid residue sequence of sequence 1 in the sequence table or the amino acid residue sequence of sequence in the sequence table 1 is added 1 to 20 amino-acid residue and has the protein of antibacterium effect through the disappearance of 1 to 20 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
2) chimpanzee (Pan troglodytes) Glyrichin: have the protein of the amino acid residue sequence of sequence 2 in the sequence table or the amino acid residue sequence of sequence in the sequence table 2 is added 1 to 20 amino-acid residue and has the protein of antibacterium effect through the disappearance of 1 to 20 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
3) dog (Canis familiaris) Glyrichin: have the protein of the amino acid residue sequence of sequence 3 in the sequence table or the amino acid residue sequence of sequence in the sequence table 3 is added 1 to 20 amino-acid residue and has the protein of antibacterium effect through the disappearance of 1 to 20 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
4) nematode (Caenorhabditis elegans) Glyrichin: have the protein of the amino acid residue sequence of sequence 4 in the sequence table or the amino acid residue sequence of sequence in the sequence table 4 is added 1 to 20 amino-acid residue and has the protein of antibacterium effect through the disappearance of 1 to 20 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
5) Arabidopsis Arabidopis thaliana (Arabidopsis thaliana) Glyrichin: have the protein of the amino acid residue sequence of sequence 5 in the sequence table or the amino acid residue sequence of sequence in the sequence table 5 is added 1 to 20 amino-acid residue and has the protein of antibacterium effect through the disappearance of 1 to 20 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
6) zebra fish (Danio rerio) Glyrichin: have the protein of the amino acid residue sequence of sequence 6 in the sequence table or the amino acid residue sequence of sequence in the sequence table 6 is added 1 to 20 amino-acid residue and has the protein of antibacterium effect through the disappearance of 1 to 20 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal.
The number of described disappearance and/or insertion and/or replacement and the amino-acid residue that adds at C-terminal and/or N-terminal is 1 to 10, is preferably 1 to 5, especially is preferably 1 to 3.
The above-mentioned proteic active fragments of glycine that is rich in is the polypeptide with one of following amino acid residue sequences:
1) has the polypeptide of the amino acid residue sequence of sequence 8 in the sequence table or the amino acid residue sequence of sequence in the sequence table 8 added 1 to 5 amino-acid residue and have the polypeptide of antibacterium effect through the disappearance of 1 to 5 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
2) has the polypeptide of the amino acid residue sequence of sequence 9 in the sequence table or the amino acid residue sequence of sequence in the sequence table 9 added 1 to 5 amino-acid residue and have the polypeptide of antibacterium effect through the disappearance of 1 to 5 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
3) has the polypeptide of the amino acid residue sequence of sequence 10 in the sequence table or the amino acid residue sequence of sequence in the sequence table 10 added 1 to 5 amino-acid residue and have the polypeptide of antibacterium effect through the disappearance of 1 to 5 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal;
4) has the polypeptide of the amino acid residue sequence of sequence 11 in the sequence table or the amino acid residue sequence of sequence in the sequence table 11 added 1 to 5 amino-acid residue and have the polypeptide of antibacterium effect through the disappearance of 1 to 5 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal.
Polypeptide with amino acid residue sequence of sequence 8 in the sequence table, name is called PEP1, derives from the people, mouse, chimpanzee and dog; Polypeptide with amino acid residue sequence of sequence 9 in the sequence table, name is called PEP2, derives from zebra fish; Polypeptide with amino acid residue sequence of sequence 10 in the sequence table, name is called PEP3, derives from nematode; Polypeptide with amino acid residue sequence of sequence 11 in the sequence table, name is called PEP4, derives from Arabidopis thaliana.
The number of described disappearance and/or insertion and/or replacement and the amino-acid residue that adds at C-terminal and/or N-terminal is for being preferably 1 to 3.
For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.
Described replacement mode is preferably replacement as shown in table 1.
Table 1.Glyrichin protein family can replace the amino acid tabulation
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala：Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Also can be rich in glycine albumen and active fragments is modified (not changing primary structure usually) to of the present invention.Modified forms comprises: the chemical species such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modify and also comprise glycosylation.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
The above-mentioned encoding gene that is rich in glycine albumen and active fragments thereof also belongs to protection scope of the present invention.
Described glycine albumen behaviour Glyrichin and the mouse Glyrichin of being rich in, its encoding gene has SEQ ID № in the sequence table: 7 dna sequence dna or with sequence table in SEQ ID №: 7 dna sequence dnas that limit have 90% above homology, and the dna sequence dna of the amino acid residue sequence of sequence 1 or the nucleotide sequence that the dna sequence dna that can limit with the sequence 7 in the sequence table under the rigorous condition of height is hybridized in the code sequence tabulation.
Sequence 7 in the sequence table is by 240 based compositions, and its open reading frame is from the 1st-240 bit base of 5 ' end, the people Glyrichin that coding has sequence 1 in the sequence table.
The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Contain the above-mentioned expression vector that is rich in the encoding gene of glycine albumen and active fragments thereof, clone and engineering bacteria also belong to protection scope of the present invention.
Of the present inventionly be rich in glycine albumen and active fragments can be used for antibacterium, specifically comprise the purposes of following several respects:
1) is used to prepare antibacterials;
2) be used to prepare and prevent and/or treat the biological goods of using of the different sorts with potential infectation of bacteria;
3) be used to produce the genetically modified organism of disease and insect resistance;
4) be used to prepare at the described derivative that is rich in glycine albumen and active fragments thereof or antagonist and part thereof, antibody;
5) be used to develop the medicine of regulation and control family member protein expression.
Use glycine protein family and the active fragments thereof of being rich in of the present invention to carry out antibioticly, have the anti-microbial activity height, has a broad antifungal spectrum, target bacterial strain are difficult for producing characteristics such as resistant mutation, have broad application prospects.
Description of drawings
Fig. 1 is the Northern Blot experimental result that is rich in glycine protein family human source gene
Fig. 2 is the restraining effect (A be S2 enlarged view, B be S5 enlarged view, C be AMP enlarged view, D be S12 enlarged view, E be L4 enlarged view) of expression product behind total length hGlyrichin and the 5 '-deletant gene transformation yeast to intestinal bacteria BL-21 thalline outgrowth
Fig. 3 A is pCM-19, pMG-19, and pMG-16, pMT-7 is to the influence of intestinal bacteria BL-21 growth
Fig. 3 B is pCM-19, pMG-19, and pMG-16, pMT-7 is to the influence of intestinal bacteria HB101 growth
Fig. 3 C is pCM-19, pMG-19, and pMG-16, pMT-7 is to the influence of bacillus coli DH 5 alpha growth
Fig. 4 is the influence of pCM-19 polypeptide to the growth of attenuation type plague bacillus
Fig. 5 is for being rich in 7 proteic multisequencing comparison results of glycine protein family
Fig. 6 is the secondary structure analysis result of hGlyrinchin
Fig. 7 is hGlyrinchin signal peptide analytical results figure
Fig. 8 is PEP1, PEP2, the test tube method test result of the anti-BL-21 bacterium of PEP3 and PEP4
Fig. 9 is PEP1, PEP2, the colony counting method test result of the anti-BL-21 bacterium of PEP3 and PEP4
Figure 10 is four kinds of artificial synthetic polypeptides and the interactional flow cytometry analysis result of BL-21 bacterium
Figure 11 is the hemolytic action test result of four kinds of artificial synthetic polypeptides
Embodiment
The contriver utilizes the inhibition difference to subtract the method for hybridization, and the mouse bone marrow difference expression gene of LTC (long term culture) being cultivated front and back has carried out a large amount of screenings, has obtained the EST clone of 131 differential expressions.Bioinformatic analysis confirms, these clones have represented gene and 7 brand-new genes of 26 kinds of known or part known functions, wherein 5 have complete open reading frame, article 3, register at GenBank, the mL52 in mouse source (mGlyrichin) gene is exactly one of them, this full length gene cDNA sequence 521bp, its GenBank
TMNumber of registration is AY028425.Relevant inhibition difference subtracts hybrid experiment and result referring to " experimental hematology magazine " 2002 the 10th phase 177-182 pages or leaves.
After obtaining mouse source mL52 gene, the contriver is according to mL52 gene design pair of degenerate primers, adopt the conventional PCR method amplification in laboratory people tire liver cDNA library, the sequence that acquisition people source has complete ORF is total to 240bp (sequence 7), and the aminoacid sequence of inferring according to the ORF sequence (sequence 1), called after Homo sapiens Glyrichin ((hGlyrichin)).Then, adopt Intnet-NCBI electronic PCR assignment of genes gene mapping method that this gene has been carried out chromosomal localization, confirm that it is positioned people 20q11.21 district.Adopt NCBI-BlastN to carry out the genome structure analysis, point out this gene to contain three exons and two introns.Further, in order to determine that this gene transcripts number and size carried out Northern Blot and analyzed.The result confirms that four cell strains selecting for use all have this expression of gene, and the single transcript (Fig. 1) about having only size for 600bp.Above-mentioned analysis and research result is verified is rich in the natural structure characteristic of glycine protein gene.
By the PCR design of primers, the 5 ' deletant gene from 5 ' end the 1st to the 60th bit base of pcr amplification total length hGlyrichin gene and 5 ' end deletion sequence 7.Total length hGlyrichin gene and 5 '-deletant gene that pcr amplification is obtained are inserted into respectively between the EcoRI and NotI of pPIC9K expression plasmid of yeast (Invitrogen), through transformed into escherichia coli BL-21 engineering bacteria and colony screening, have the plasmid of the hGlyrichin gene of total length and 5 ' disappearance after obtaining to make up respectively.The high purity plasmid that is obtained adopts electric commentaries on classics method to transform the GS115 yeast after the linearizing of SalI restriction endonuclease, and the MD plate screening through containing G418 (50ug/ml) obtains positive colony.Positive colony is inoculated into contains in the 5ml BMGY substratum, expression product is secreted in supernatant, makes active testing with supernatant.Active testing adopts the agar plate diffusion process, simultaneously with penbritin as active control.Through screening active ingredients, obtained the positive colony (S2, S12 and S5 clone) of expressing the positive colony (L4 clone) of total length hGlyrichin and expressing 5 '-disappearance hGlyrichin.L4, S2, S12 and S5 clone's fungistatic effect shows the positive colony (L4 that has transformed the hGlyrichin gene that no matter contains total length or 5 '-disappearance as shown in Figure 2, S2, S12 and S5) supernatant has the effect of obvious inhibition agar plate surface intestinal bacteria BL-21 growth.Above-mentioned experimental result confirms that yeast expression system can be expressed hGlyrinchin albumen, and expression product has anti-microbial activity; On behalf of the definite structure of this gene activity, the equal expression activity albumen of the gene of total length and 5 '-disappearance established firm foundations for further seeking.According to above-mentioned serial experiment result, personnel very easily utilize similar approach further to screen and obtain to have similar active minimum protein molecule in the industry.
After the yeast expressed hGlyrinchin albumen of above-mentioned experimental result explanation has anti-microbial effect, the contriver is according to the aminoacid sequence of hGlyrinchin gene, salvage the polypeptide of different lengths, directly prove the proteic anti-microbial effect of hGlyrinchin and find the core peptide section from protein level.Article four, the position of peptide section is respectively the 31-46 (pMG-16) of hGlyrichin aminoacid sequence, 42-60 (pCM-19), 53-59 (pMT-7), the individual amino acid of 61-79 (pMG-19).The purity of institute's section of synthesized peptide is 85-90%.Carrying out anti-microbial activity behind the above-mentioned peptide Duan Jingyong sterilization deionized water dissolving detects.The method that adopts is Kirby-Bauer disk diffusion (DavidT.Kingsbury et al.Microbiology, 2
NdEdition, ed by Harwal Publishing, 1990, page 37) method.The target bacteria that is adopted is e. coli bl21 (Fig. 3 A), intestinal bacteria HB101 (Fig. 3 B) and bacillus coli DH 5 alpha (Fig. 3 C) engineering bacteria.Fig. 3 A, label 1-5 is respectively pCM-1932,64,128,256 and 512 μ g among Fig. 3 B and Fig. 3 C, and label 6 is pMG-19 512 μ g, label 7 is pMG-16 512 μ g, label 8 is pMT-7 512 μ g, and label 9 is penbritin 512 μ g, and label 10 is a solvent control.Can find by result among the figure, above-mentioned four peptide species solution are under the identical condition of concentration, and are similar to the effect of three kinds of target bacterium, and the anti-microbial effect difference of different polypeptide solutions is bigger during with concentration, wherein pCM-19 and pMT-7 have strong anti-microbial effect, and pCM-19 is stronger than pMT-7.
Above-mentioned experimental result confirms that pCM-19 has definite anti-microbial effect, but just effective to engineering bacteria.In order to confirm whether it is effective equally to pathogenic bacterium, 100 μ g/200ul and 200 μ g/200ul pCM-19 peptide solutions are joined respectively contain 1 * 10
4In the attenuation plague bacillus liquid of CFU/200ul, 37 ℃ act on 1 hour, then all mixed solutions are coated on 1% agar plate surface and continue cultivation 3 days.The result of Fig. 4 shows that the attenuation plague bacillus after pCM-19 handles has almost completely lost energy for growth.Illustrate that pCM-19 has lethal effect equally to the attenuation plague bacillus.
Behind the anti-microbial effect that confirms hGlyrinchin and polypeptide fragment thereof, the contriver utilizes the information biology means that each member of family has been carried out the series of comparisons analysis.At first carried out the BlastP analysis, obtained totally 28 of the protein sequences of homology scoring value＞40, wherein 15 of different originated from fungus, nematode has 4 sequences.In order to verify the contact of above-mentioned homologous sequence on function, chosen 7 protein sequences such as representative, mouse, chimpanzee, Canis, nematode, Arabidopis thaliana respectively and further analyzed and researched, these 7 protein sequences have been contained from the highest homo sapiens that evolves to nematode and even the quite long space-time span of plant.Each member's of family sequence is all from GenBank, and their number of registration is respectively: CAC11117, AAK18748, XP_514807, XP_534406, CAB02486, AAH59661, NP_566324.
Use the DNAMAN analysis software to carry out above-mentioned each member's multisequencing compare of analysis, the result that obtains of institute as shown in Figure 5.Among the figure result reflect the common feature that is rich in each member of glycine protein family be exist a high conservative, about 65 amino acid lengths, the core texture territory of being rich in glycine, for determining that being rich in the glycine protein family provides strong evidence.In the upstream of this structural domain or the sequence homology in downstream lower, the first half of this conserved regions is tangible hydrophobic region.Up to the present, all members' of this family function is all unknown.
Further adopt multiple Intnet shareware that the aminoacid sequence feature that is rich in each member of glycine protein family is analyzed.Result such as table 2, these features are identical with the universals of present antibacterial peptide, especially meet the feature of cationic antibacterial peptide.
Table 2. is rich in the essential characteristic of glycine protein family protein sequence
| The species classification | The GeneBank sequence number | Length | Glycine % | Signal peptide | Score | Iso-electric point | Net charge | Molecular weight (dalton) |
| Homo sapiens (Homo Sapiens) | CAC11117 | 79 | 21.5 | 21～42 | 166 | 9.58 | 5 | 8182.83 |
| Mouse (Mus musculus) | AAK18748 | 79 | 21.5 | 21～42 | 166 | 9.58 | 5 | 8182.23 |
| Chimpanzee (Pan troglodytes) | XP_514807 | 211 | 16.1 | NO | 166 | 11.44 | 23 | 22654.21 |
| Dog (Canis familiaris) | XP_534406 | 124 | 22 | 1～19 | 166 | 10.51 | 9 | 13151.64 |
| Nematode (Caenorhabditis elegans) | CAB02486 | 82 | 22 | 20～42 | 84.3 | 10.45 | 6 | 8355.94 |
| Arabidopis thaliana (Arabidopsis thaliana) | NP_566324 | 74 | 18.9 | 1～26 | 50.8 | 9.83 | 6 | 7367.71 |
| Zebra fish (Danio rerio) | AAH59661 | 80 | 21.3 | 21～42 | 150 | 8.98 | 5 | 8381.03 |
Be rich in each member's secondary structure prediction of glycine protein family and adopted DNASTAR5.0 software, the result shows that each member's structure is very similar but is not quite similar, none is simple αLuo Xuanjiegou among 7 members, be one based between the β laminated structure or the antibacterial peptide family of αLuo Xuanjiegou arranged.Representative result is hGlyrinchin, as shown in Figure 6.With the Garnier-Robson model serves as that the main result who analyzes is: Homo sapiens, Mouse, Pantroglodytes, there is not the α spiral in 4 members such as Canis familiaris, be βZhe Die type peptide, Arabidopsis thaliana is based on βZhe Die, and only the aminoterminal in sequence has the possibility that forms the α spiral, and Danio rerio and Caenorhabditis elegans are α spiral-βZhe Die alternatively form structure.Hydrophilic, hydrophobic property analysis shows, all contains water repellent region, and majority is that two sections water repellent regions and hydrophilic region alternately exist.Each member's signal peptide of family is analyzed used software and is
Http:// www.cbs.dtu.dk/services/, except that chimpanzee Glyrinchin sequence no signal peptide, other member has signal peptide sequence.Between 20-45 and 1-20 amino acids, representative result (CAC11117) is as Fig. 7 respectively in the position of each member's signal peptide.The contriver also with each member in the family and existing antibacterial peptide storehouse (
Http:// aps.unmc.edu/ap/main.html) carried out systematic comparison, all prompt for antibacterial peptide.
The result of above-mentioned bioinformatic analysis confirms: be rich in (1) has height homologous conserved sequence between each member of glycine protein family, these structural similarity height have been pointed out similarity on the function; (2) each member's amino acid form and molecular weight is less relatively, contain signal peptide sequence, higher isoelectric point (pI), with positive electric charge, be rich in the feature that certain amino acid characteristics such as (glycine) meet most of antibacterial peptides; (3) compare with existing antibacterial peptide library database, all prompt for antibacterial peptide.To sum up, the natural existence of being rich in the glycine protein family of the strong prompting of bioinformatic analysis result.According to the information biology systems analysis as a result this conclusion of gained very high degree of recognition in the industry cycle should be arranged.
In view of be rich in each member of glycine protein family evolve the characteristics that go up high conservative with and the fact of Unknown Function, the contriver conducts a research to the function of this gene family, intends confirming to be rich in function of glycine protein family member and uses thereof.According to the information biology Comprehensive analysis results, between each member, selected the high conserved region domain amino acid sequence of 4 bigger genes of homology difference, synthetic corresponding 4 peptide sequences, these peptide sequences have been represented has identical (people, mouse, dog, chimpanzee) or the gene of 7 species of part identical sequence.Article four, the peptide section is synthetic by the biochemical (Shanghai) Co., Ltd. of gill, and sequence is respectively:
Derive from PEP1:Cys-Leu-Arg-Ile-Gly-Met-Arg-Gly-Arg-Glu-Leu-Met-Gly-Gly-Ile-Gly-Lys-Thr-Met-NH of people, mouse, chimpanzee and dog
2(sequence 8)
Derive from PEP2:Cys-Leu-Arg-Ile-Gly-Met-Arg-Gly-Arg-Glu-Leu-Met-Gly-Gly-Val-Gly-Lys-Thr-NH2 (sequence 9) of zebra fish
Derive from PEP3:Gly-Phe-Arg-Ala-Gly-Met-Arg-Gly-Lys-Asp-Leu-Leu-Leu-Gln-Thr-Gly-Lys-Thr-NH2 (sequence 10) of nematode
Derive from the PEP4:Ala-Ile-Arg-Val-Lys-Val-Pro-Gly-Leu-His-Lys-Val-Arg-Phe-Ile-Gly-Gln-Thr-NH2 (sequence 11) of Arabidopis thaliana
The purity of each peptide section is respectively: PEP1 88.7%, and PEP2 91.1%, PEP3 87.3% and PEP4 90.2%.Adopted test tube method and colony count method to measure the antibacterial effect of four peptide species subsequently.Fig. 8 and Fig. 9 result show that four kinds of peptide Duan Jun have the effect of anti-BL-21 bacteria growing, and wherein secondly antibacterial effect the best of PEP2 is PEP1 and PEP4, and that relatively poor is PEP3.The result of two kinds of different methods tests shows that the strong and weak trend of four kinds of peptide section anti-microbial effects is similar.
Same family different members anti-microbial effect mechanism may have similarity.The contriver adopts the pI fluorescein to follow the tracks of in antibacterial peptide and the bacterium interaction process the influence of bacterial film integrity, detects with peptide-bacterium interaction time prolongation, the variation of pI positive bacteria ratio by flow cytometer (FACS).The result shows among Figure 10, when bacterium respectively with different peptide solution 500ug/ml and pI25ug/ml 37 ℃ of actings in conjunction after 20 minutes, wherein the pI positive bacteria number after PEP1, PEP4 and the PEP2 solution effects reaches respectively about 35%, 70% and 94%, and pI positive bacteria ratio＜5% of solvent control and penbritin control group.PI positive bacteria ratio and control group are similar after the PEP3 solution-treated.Though, pI both can mix dead back bacterium also can mix the impaired bacterium of bacterial film, but both results' difference obviously shows, the antibiotic mechanism of the penbritin that antibacterial potency is determined is not the integrity that directly influences the bacterial film structure, and is tried three kinds of effects with obvious destruction bacterial film structural integrity in the peptide solution.In addition, after transforming and to obtain the amicillin resistance bacterium be that target bacteria carries out same experiment with pET-22b (+) empty carrier, the result who is obtained is almost completely consistent with a last experiment, the different of penbritin and the above-mentioned antibacterial peptide mechanism of action have further been confirmed, confirmed further that also antibacterial peptide brings into play the mechanism of germicidal action by the integrity of destroying the bacterial film structure, this mechanism of action conforms to the mechanism of action that has most of antibacterial peptide now.
The known portions antibacterial peptide has hemolytic action.In order to confirm to be rich in glycine protein family different members in the similarities and differences aspect the hemolytic action, the contriver continues to have carried out experimental study with four peptide sections representing 7 different plant species.The result of Figure 11 shows that four kinds of section of synthesized peptide all do not have hemolytic action to HRBC.This result has reflected one of common property between same family different members to a certain extent.
Above-mentioned result of study shows: the primary structure that (1) is rich in each member of glycine protein family has high similarity, the homology comparison scoring value of complete genome sequence reaches more than 40, and the scoring value relatively of the homology between 65 left and right sides amino acid guarding relatively between each member is higher.The similarity of different proteins primary structure and they the similarity height correlation on function in the industry cycle has quite high common recognition degree; (2) secondary structure between each member of family also has high similarity, and wherein 5 is simple β laminated structure, and 2 of the two mixed types of αLuo Xuanjiegou and β laminated structure do not have simple αLuo Xuanjiegou.These constitutional features explanations, being rich in the glycine protein family is an antibacterial peptide family based on the β laminated structure; (3) less, the pI value of each member's molecular weight of family more greatly, all with positive electric charge, be rich in certain seed amino acid (glycine/methionine(Met)), contain feature such as hydrophobic region and conform to the universals that existing antibacterial peptide is generally acknowledged; (4) each member and existing antibacterial peptide storehouse result relatively all prompts for antibacterial peptide in the family; (5) it is similar and all do not have a hemolytic action to come from peptide Duan Junyou anti-microbial effect, the mechanism of action in various degree of 7 species among the family member.The existence that the glycine protein family is rich in the strong prompting of these results, family member's composition number is at least more than 7.
The homologous sequence of experimental verification has only 7 among the present invention, the aminoacid sequence that compares scoring value＞40 with the hGlyrichin homology has 28, and personnel very easily adopt similar approach of the present invention to confirm biologic activity and and then the acquisition pharmaceutical protein or the polypeptide drug of other height homologous sequence in the industry.
By the recombinant DNA technology of routine, can utilize the encoding gene that is rich in glycine albumen or its active fragments of the present invention to express or produce reorganization be rich in glycine albumen or active fragments.As expressing by the following method or produce:
(1) transforms or the transduction host cell with containing the above-mentioned recombinant expression vector that is rich in the encoding gene of glycine albumen or its active fragments;
(2) cultivate described host cell;
(3) separation, protein purification from substratum or cell.
The carrier that sets out that is used to make up described recombinant expression vector can be bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus as adenovirus or other carrier.In a word, can use as long as can in host, duplicate with the plasmid and the carrier of stably express.
A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.Method well-known to those having ordinary skill in the art can be used to make up and contains Glyrichin DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.
Those of ordinary skills know how to select appropriate carriers, promotor, enhanser and host cell.Can carry out with routine techniques well known to those skilled in the art with described recombinant expression vector transformed host cell.The transformant that obtains can be cultivated with ordinary method, and that expresses genes encoding of the present invention is rich in glycine albumen or its active fragments.In aforesaid method be rich in glycine albumen or the extracellular can be expressed or be secreted into to its active fragments in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and albumen or its active fragments of purification of Recombinant of chemistry.When using Glyrichin albumen of the present invention or its active fragments, also can use other medicament simultaneously, as antibiotic such as penicillin.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
In the present invention, term " Glyrichin ", " Glyrichin family member ", " Glyrichin albumen " or " Glyrichin polypeptide " are used interchangeably, and all refer to have each member's aminoacid sequence of natural antibacterial peptide Glyrichin family (polypeptide of the albumen of sequence 1-6 or sequence 8-11 in the sequence table 1).They comprise the natural antibacterial peptide Glyrichin family member of containing or do not contain initial methionine, and the Glyrichin member protein that contains or do not contain signal peptide.
The variant form of different members comprises in the family: homologous sequence, conservative property varient, allelic variant, natural mutation, mutagenesis mutant.Variant form also comprises the fusion rotein that is rich in glycine albumen and active fragments thereof.Except the polypeptide of total length almost, the present invention has also comprised and has contained the proteic soluble fragments of glycine.Usually, this fragment have contain glycine protein polypeptide sequence at least about 7-25 continuous amino acid, usually at least about 35 continuous amino acids, more preferably at least about 45 continuous amino acids, best at least about 60 continuous amino acids.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
Below with the people source be rich in the glycine protein gene the clone, identify, bioinformatic analysis and anti-microbial activity test, Analysis on action mechanism and hemolytic action be embodiment, further how elaboration utilizes each member of glycine protein family of being rich in of the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, common molecular cloning: laboratory manual (New York:Cold Spring Harbor LaboratoryPress according to people such as normal condition such as Sambrook, 1989) condition described in, or the condition of advising according to manufacturer.
1, Ren Yuan is rich in the acquisition of the complete ORF sequence of glycine protein gene
Be rich in the glycine protein gene according to the mouse source and design following pair of degenerate primers amplification people tire liver cDNA library, obtain the people source and be rich in glycine albumen complete ORF sequence 240bp altogether, and the aminoacid sequence of inferring according to the ORF sequence:
5 ' primer Pa:5-CGATGCCGGTGGCCGTGGGTCCCT-3
3 ' primer Pb:5-TTAGCATCGTATGCCCATTCCA-3
The pcr amplification condition is: 94 ℃, 4 minutes, 1 circulation; 94 ℃, 40 seconds, 60 ℃, 50 seconds, 72 ℃, 1 minute, 30 circulations; 72 ℃, 7 minutes, 1 circulation.The PCR product is through Winzard PCR prepspurification kit purifying, recombinates in the pGEM-T carrier transformed into escherichia coli JM109 then, order-checking evaluation with the T4 ligase enzyme.The result shows the nucleotide sequence that people Glyrichin gene has sequence 7 in the sequence table, encoding sequence have sequence 1 in the table amino acid residue sequence be rich in glycine albumen people Glyrichin.
2, Northern blot identifies
Cultivator 4 kinds of tumor cell line HepG2, HeLa, Jurket and HEK293 use Winzard plusRNA purification kit (available from Promega company) to extract total RNA then.Respectively get the total RNA of 20 micrograms, on 1.2% denaturing formaldehyde sepharose, separate, and forward the Hybond-N+ nylon membrane to.With the complete ORF of HGlyrichin is probe, with the Prime-a-gene test kit mark of Promega company.Results of hybridization shows that people Glyrichin gene all has expression in the tumor cell line of 4 kinds of different tissue sources of being tested as shown in Figure 1, and pointing out it may be a kind of natural antibacterial peptide of wide expression.Only there is a transcript in announcement simultaneously, and size is about 600bp.
The detection of embodiment 2, hGlyrichin yeast expression and expression product fungicidal activity thereof
By PCR design of primers (total length upstream primer: 5 '-AGGAATTCATGCCGGTGGCCGTGGGTCCCTAC-3 '; 5 ' deletant upstream primer: 5 '-AGGAATTCATGGGCTTCGTGATGGGTTGC-3 '; Total length downstream primer: 5 '-AAGGAAAAAAGCGGCCGCTTAGCATCGGATGCCCATCCCA ATG-3 '), with the pET-22b plasmid that contains the hGlyrichin total length is template, the 5 ' deletant gene from 5 ' end the 1st to the 60th base of difference pcr amplification total length hGlyrichin gene and 5 ' end deletion sequence 7 under the guiding of total length upstream primer and total length downstream primer, 5 ' deletant upstream primer and total length downstream primer.Its PCR reaction system (except that primer) and reaction conditions are with the step 3 of embodiment 1.
Total length hGlyrichin gene and 5 '-deletant gene that pcr amplification is obtained are inserted into respectively between the EcoRI and NotI of pPIC9K expression plasmid of yeast (Invitrogen), through transformed into escherichia coli BL-21 engineering bacteria and colony screening, have the plasmid of the hGlyrichin gene of total length and 5 ' disappearance after obtaining to make up respectively.The high purity plasmid that is obtained adopts electric commentaries on classics method to transform the GS115 yeast after the linearizing of SalI restriction endonuclease, and the MD plate screening through containing G418 (50ug/ml) obtains positive colony.Positive colony is inoculated into contains in the 5ml BMGY substratum, 30 ℃ of shaking tables are cultured to OD
600During=2.0-6.0, use BMMY (contain final concentration be 1% methyl alcohol) substratum to be diluted to OD then
600=1.0, continue to cultivate.Per 24 hours later on add methyl alcohol is 0.5% to final concentration, and different time is got 1ml and joined in the Eppendorff centrifuge tube behind the inducing culture, and centrifugal 3000rpm stays supernatant to make active testing.Active testing adopts the agar plate diffusion process, promptly evenly be coated with test bacterium (intestinal bacteria BL-21) at the LB planar surface that contains 1% agar, place the filter paper of the about 6mm of diameter again in each dice central authorities, the different clone of 15ul supernatant is added drop-wise on the scraps of paper, equivalent adds after 20 minutes, adds altogether 3 times.37 ℃ of cultivations were observed the size of antibacterial ring in 4 hours to obtain positive colony.Be that the penbritin of 100mg/ml is as active control simultaneously with 10ul concentration.Through screening active ingredients, obtained the positive colony (S2, S12 and S5 clone) of expressing the positive colony (L4 clone) of total length hGlyrichin and expressing 5 '-disappearance hGlyrichin.L4, S2, S12 and S5 clone's fungistatic effect as shown in Figure 2, show the positive colony (S2 that has transformed the hGlyrichin gene that no matter contains total length or 5 '-disappearance, S5, S12 represents 5 '-and disappearance hGlyrichin expresses positive colony, on behalf of the hGlyrichin full-length gene, L4 express positive colony) supernatant has the effect that obvious inhibition agar plate surface intestinal bacteria BL-21 grows.Above-mentioned experimental result confirms that yeast expression system can be expressed hGlyrinchin albumen, and expression product has anti-microbial activity; On behalf of the definite structure of this gene activity, the equal expression activity albumen of the gene of total length and 5 '-disappearance established firm foundations for further seeking.According to above-mentioned serial experiment result, personnel very easily utilize similar approach further to screen and obtain to have similar active minimum protein molecule in the industry.
The anti-microbial effect analysis of embodiment 3, synthetic different lengths hGlyrichin protein polypeptide
1, the colibacillary effect of anti-Gram-negative
According to the aminoacid sequence of hGlyrichin gene, by the biochemical company limited of gill salvage the polypeptide of different lengths, the position of four peptide sections is respectively the 31-46 (H of hGlyrichin aminoacid sequence
3N
+-Met-Ala-Ala-Gly-Ala-Leu-Phe-Gly-Thr-Phe-Ser-Cys-Leu-Arg-Ile-Gly-COO
-, pMG-16), and 42-60 (sequence 8, pCM-19), 53-59 (pMT-7, H
3N
+-Met-Gly-Gly-Ile-Gly-Lys-Thr-COO
-), 61-79 (pMG-19, H
3N
+-Met-Gln-Ser-Gly-Gly-Thr-Phe-Gly-Thr-Phe-Met-Ala-Ile-Gly-Met-Gly-Ile-Arg-Cys-COO
-) individual amino acid.The purity of institute's section of synthesized peptide is 85-90%.Be dissolved into 20mg/ml with aseptic deionized water.The method that antibacterial experiment adopts is Kirby-Bauer diskdiffusion (David T.Kingsbury et al.Microbiology, 2
NdEdition, ed by HarwalPublishing, 1990, page 37) method.To contain the glass dish that 1% agar LB substratum is tiled in 15cm diameter behind the autoclaving, after the agar condensation with OD
600=4.0 30 μ l bacterium liquid (e. coli bl21s, HB101 or DH5 α) be diluted to 2ml through LB, be uniformly coated on LB agar solid culture primary surface respectively, in the grid of each delimitation, place the sterilization filter paper of a diameter 8mm, 15 μ l specimen are dripped on the filter paper.With plate be placed in 37 ℃ of incubators cultivate 4-10 hour after, take out the plate observations, with penbritin positive control and solvent (aseptic deionized water) negative control group relatively, with the big or small result of determination of inhibition zone.Result such as Fig. 3 A shown in Fig. 3 B and Fig. 3 C, show that above-mentioned four peptide species solution are under the identical condition of concentration, effect to three kinds of target bacterium is similar, the anti-microbial effect difference of different polypeptide solutions is bigger during with concentration, and wherein pCM-19 and pMT-7 have strong anti-microbial effect, and pCM-19 is stronger than pMT-7.Fig. 3 A, label 1-5 is respectively pCM-19 32,64,128,256 and 512 μ g among Fig. 3 B and Fig. 3 C, and label 6 is pMG-19 512 μ g, label 7 is pMG-16 512 μ g, label 8 is pMT-7 512 μ g, and label 9 is penbritin 512 μ g, and label 10 is a solvent control.
2, the effect of anti-Gram-negative plague bacillus
Containing 1 * 10
4The pCM-19 polypeptide solution that adds 200 μ l aseptic deionized waters (negative control), 100 μ g/200 μ l and 200 μ g/200 μ l in the every test tube of CFU/200 μ l attenuation plague bacillus (epidemiological study institute of Military Medical Science Institute) respectively, 37 ℃ act on 1 hour, whole mixed solutions are uniformly coated on 1% agar plate surface, continue to cultivate 3 days.The result shows that the contrast flat board has the growth of a large amount of plague bacilluses as shown in Figure 4, and has lost energy for growth fully with attenuation plague bacillus after 100 μ g or the 200 μ g pCM-19 polypeptide effects.Among Fig. 4, A is the aseptic deionized water contrast; B is 100 μ g effect groups; C is 200 μ g effect groups.
Embodiment 4, derive from the checking of four peptide species anti-microbial effects of different family members
According to the bioinformatic analysis result, the high conserved region of people's (identical), zebra fish, nematode and 4 kinds of genes of Arabidopis thaliana, 18-19 aminoacid sequence after being right after hydrophobic region in being rich in the glycine protein family, have been selected with mouse, dog, chimpanzee conserved regions aminoacid sequence 100%, entrust the biochemical company limited of gill (Shanghai) synthetic corresponding 4 peptide section PEP1, PEP2, PEP3 and PEP4, sequence is respectively:
Derive from PEP1:Cys-Leu-Arg-Ile-Gly-Met-Arg-Gly-Arg-Glu-Leu-Met-Gly-Gly-Ile-Gly-Lys-Thr-Met-NH of people, mouse, chimpanzee and dog
2(sequence 8)
Derive from PEP2:Cys-Leu-Arg-Ile-Gly-Met-Arg-Gly-Arg-Glu-Leu-Met-Gly-Gly-Val-Gly-Lys-Thr-NH2 (sequence 9) of zebra fish
Derive from PEP3:Gly-Phe-Arg-Ala-Gly-Met-Arg-Gly-Lys-Asp-Leu-Leu-Leu-Gln-Thr-Gly-Lys-Thr-NH2 (sequence 10) of nematode
Derive from the PEP4:Ala-Ile-Arg-Val-Lys-Val-Pro-Gly-Leu-His-Lys-Val-Arg-Phe-Ile-Gly-Gln-Thr-NH2 (sequence 11) of Arabidopis thaliana
The purity of each peptide section is respectively: PEP1 88.7%, and PEP2 91.1%, PEP3 87.3% and PEP4 90.2%.
Use water as solvent with 4 peptide sections be dissolved into respectively 20ug/ul be placed on-20 ℃ standby.Adopt " test tube method " and " colony count method " to measure the anti-microbial effect of 4 kinds of peptides respectively.
Test tube method: get OD
600=0.3 o'clock E.coli BL-21 bacterium 5 μ l, add LB liquid nutrient medium to 20 μ l, be water 10 μ l, penbritin 10 μ l (100 μ g) with four kinds of peptide liquid, 10 μ l (100 μ g), control group respectively, 37 ℃ hatch 1 hour after, the mixed solution of hatching in each pipe is joined in the different test tubes that contain 6ml LB liquid nutrient medium, 37 ℃, cultivate under the 175rpm condition, between 0-9 after the inoculation hour, per hour OD is surveyed in sampling
600Value dynamic observes the influence of four kinds of peptides to bacterial growth.Every kind of peptide, solvent control and penbritin contrast are 3 samples.Draw according to experimental result.Data are the equal numerical value plus-minus standard deviation (Fig. 8) of 3 samples.The result shows that four kinds of peptide Duan Jun have the effect of anti-BL-21 bacteria growing, and wherein secondly antibacterial effect the best of PEP2 is PEP1 and PEP4, and that relatively poor is PEP3.
Colony count method: get OD
600=0.3 o'clock E.coli BL-21 bacterium liquid 5 μ l add behind LB nutrient solution to the 20 μ l respectively and four kinds of peptide liquid, 10 μ l (100 μ g), 37 ℃ hatch 1 hour after, get be equivalent to 200 bacterium colonies hatch bacterium liquid, add the LB liquid nutrient medium to 1.0ml, evenly be applied to the LB flat board, be inverted to cultivate after 12-18 hour for 37 ℃ and count colony number.Every peptide is established 3 parallel incubation tubes.Display data is the equal numerical value plus-minus standard deviation of 3 samples.Control group is the solvent of dissolving peptide, every group of 3 samples.The result as shown in Figure 9, four kinds of peptide Duan Jun have the effect of anti-BL-21 bacteria growing, wherein secondly antibacterial effect the best of PEP2 is PEP1 and PEP4, that relatively poor is PEP3.
The result of two kinds of different methods tests shows that the strong and weak trend of four kinds of peptide section anti-microbial effects is similar.
Get OD respectively
600=0.5 o'clock the e. coli bl21 bacterium liquid that does not contain pET-22b (+) and contain the E.coli BL-21 of pET-22b (+) (with pET-22b (+) transformed into escherichia coli BL21, each 100 μ l of e. coli bl21-pET22b (+)/BL21) bacterium liquid that contains pET-22b (+) that screening obtains, respectively with four peptide species PEP1, PEP2, PEP3 and PEP4 solution, every peptide species solution 100 μ l (200 μ g), add again PI (propidium iodide) to final concentration be 25 μ g/ml, the final concentration of peptide is 500 μ g/ml in the system at this moment, at 37 ℃, 160rpm acts on 20 minutes down.Control group replaces peptide solution with solvent or equivalent penbritin solution.Whole samples of hatching after 20 minutes are washed unnecessary PI dye liquor off with physiological saline, and carry out flow cytometry analysis with 500 μ l physiological saline after resuspended.Test result the percentage ratio that transfect cell accounts for analysis of cells with the pI positive and is represented.The result as shown in figure 10, show when bacterium respectively with different peptide solution 500ug/ml and pI 25ug/ml 37 ℃ of actings in conjunction after 20 minutes, wherein the pI positive bacteria number after PEP1, PEP4 and the PEP2 solution effects reaches respectively about 35%, 70% and 94%, and pI positive bacteria ratio＜5% of solvent control (physiological saline) and penbritin control group.PI positive bacteria ratio and control group are similar after the PEP3 solution-treated.Though, pI both can mix dead back bacterium also can mix the impaired bacterium of bacterial film, but both results' difference obviously shows, the antibiotic mechanism of the penbritin that antibacterial potency is determined is not the integrity that directly influences the bacterial film structure, and is tried three kinds of effects with obvious destruction bacterial film structural integrity in the peptide solution.In addition, after unloaded the conversion and to obtain the amicillin resistance bacterium be that target bacteria carries out same experiment with pET-22b (+), the result who is obtained is almost completely consistent with a last experiment, the different of penbritin and the above-mentioned antibacterial peptide mechanism of action have further been confirmed, confirmed further that also antibacterial peptide brings into play the mechanism of germicidal action by the integrity of destroying the bacterial film structure, this mechanism of action conforms to the mechanism of action that has most of antibacterial peptide now.
Embodiment 6, derive from the hemolytic action test of four peptide species of different family members
Fresh anti-freezing normal people whole blood is after physiological saline is washed 3 times, with resuspended 8% (v/v) of physiological saline, get the resuspended liquid of 100 μ l and add 90ul physiological saline, 100 μ l 0.1%TritonX-100 mixings with the different peptide solutions of 100 μ l, 100 μ l physiological saline, 10 μ l deionized waters respectively, hatch centrifugal 1000g after 1 hour for 37 ℃, 5min gets supernatant and surveys 570nm place light absorption value.Every kind of peptide solution is established 125ug/ml, three concentration of 250ug/ml and 500ug/ml.Each test group is established 3 parallel sample.The gained experimental result is drawn.Data are represented with the mean plus-minus standard deviation of 3 samples.The result shows that four kinds of section of synthesized peptide all do not have hemolytic action to HRBC as shown in figure 11.This result has reflected one of common property between same family different members to a certain extent.Among the figure, water meter shows that 10 μ l deionized waters add 90ul physiological saline control group.
Sequence table
<160>11
<210>1
<211>79
<212>PRT
＜213〉Genus Homo people (Homo sapiens) and mouse (Mus musculus)
<400>1
Met Pro Val Ala Val Gly Pro Tyr Gly Gln Ser Gln Pro Ser Cys Phe
1 5 10 15
Asp Arg Val Lys Met Gly Phe Val Met Gly Cys Ala Val Gly Met Ala
20 25 30
Ala Gly Ala Leu Phe Gly Thr Phe Ser Cys Leu Arg Ile Gly Met Arg
35 40 45
Gly Arg Glu Leu Met Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly
50 55 60
Gly Thr Phe Gly Thr Phe Met Ala Ile Gly Met Gly Ile Arg Cys
65 70 75
<210>2
<211>211
<212>PRT
＜213〉chimpanzee (Pan troglogytes)
<400>2
Met Cys His Leu Arg Glu Arg Val Arg Gly Ser Gly Gln Gln Gly Gly
1 5 10 15
Gln Glu Gly Ser Gly Ile Phe Ser Cys Val Ala Ala Arg Arg Gly Gln
20 25 30
Val Arg Ala Ser Arg Arg Ala Gly Pro Glu Arg Thr Ala Gln Ala Gln
35 40 45
Pro Pro Ser Gly Arg Arg Gly Leu Arg Pro Trp Ser Arg Cys His Arg
50 55 60
His Cys Arg Pro Pro Pro Ser Gly Ser Glu Gln His Gly Pro Ala Gly
65 70 75 80
Arg Ala His Leu Pro Lys Pro Leu Gln Ser Trp Ala Pro Gly Ser Arg
85 90 95
Lys Cys Cys Pro Ala Leu Arg Lys Arg Phe Ile Gly Pro Arg Ala Pro
100 105 110
Ser Pro Ser Phe Ser Val Arg Asp Val Glu Leu Ser Asp Arg Gly Arg
115 120 125
Glu Arg Gly Glu Met Pro Val Ala Val Gly Pro Tyr Gly Gln Ser Gln
130 135 140
Pro Ser Cys Phe Asp Arg Val Lys Met Gly Phe Val Met Gly Cys Ala
145 150 155 160
Val Gly Met Ala Ala Gly Ala Leu Phe Gly Thr Phe Ser Cys Leu Arg
165 170 175
Ile Gly Met Arg Gly Arg Glu Leu Met Gly Gly Ile Gly Lys Thr Met
180 185 190
Met Gln Ser Gly Gly Thr Phe Gly Thr Phe Met Ala Ile Gly Met Gly
195 200 205
Ile Arg Cys
210
<210>3
<211>124
<212>PRT
＜213〉dog (Canis familiaris)
<400>3
Met Glu Leu Gly Tyr Ala Leu Ile Phe Leu Pro Gly Leu Pro Arg Thr
1 5 10 15
Leu Arg Phe Ala Pro His Pro Ala Ser Ser Gly Pro Arg Pro Gly Trp
20 25 30
Arg Leu Gly Pro Tyr Ser Pro Asn Leu Arg Thr Pro Gln Met Pro Val
35 40 45
Ala Val Gly Pro Tyr Gly Gln Ser Gln Pro Ser Cys Phe Asp Arg Val
50 55 60
Lys Met Gly Phe Val Met Gly Cys Ala Val Gly Met Ala Ala Gly Ala
65 70 75 80
Leu Phe Gly Thr Phe Ser Cys Leu Arg Ile Gly Met Arg Gly Arg Glu
85 90 95
Leu Met Gly Gly Ile Gly Lys Thr Met Met Gln Ser Gly Gly Thr Phe
100 105 110
Gly Thr Phe Met Ala Ile Gly Met Gly Ile Arg Cys
115 120
<210>4
<211>82
<212>PRT
＜213〉nematode (Caenorhabditis elegans)
<400>4
Met Pro Val Pro Ser Gly Tyr Ala Ala His Pro Gln Gly Ser Gln Pro
1 5 10 15
Ser Cys Phe Thr Lys Ile Arg Met Gly Leu Met Met Gly Ala Met Ile
20 25 30
Gly Gly Ala Thr Gly Ile Leu Leu Gly Gly Phe Met Gly Phe Arg Ala
35 40 45
Gly Met Arg Gly Lys Asp Leu Leu Leu Gln Thr Gly Lys Thr Val Ala
50 55 60
Gln Ser Gly Gly Ser Phe Gly Val Phe Met Gly Val Ala Gln Gly Leu
65 70 75 80
Arg Cys
<210>5
<211>74
<212>PRT
＜213〉Arabidopis thaliana (Arabidopsis thaliana)
<400>5
Met Ala Lys Asn Ser Cys Leu Ala Lys Ile Thr Ala Gly Val Ala Val
1 5 10 15
Gly Gly Ala Leu Gly Gly Ala Val Gly Ala Val Tyr Gly Thr Tyr Glu
20 25 30
Ala Ile Arg Val Lys Val Pro Gly Leu His Lys Val Arg Phe Ile Gly
35 40 45
Gln Thr Thr Leu Ser Ser Ala Ala Ile Phe Gly Leu Phe Leu Gly Ala
50 55 60
Gly Ser Leu Ile His Cys Gly Lys Gly Tyr
65 70
<210>6
<211>80
<212>PRT
＜213〉zebra fish (Danio rerio)
<400>6
Met Pro Val Ser Val Gly Ser Tyr Gly Gln Gln Ala Gln Pro Ser Cys
1 5 10 15
Phe Asp Arg Val Lys Met Gly Phe Met Met Gly Phe Ala Val Gly Met
20 25 30
Ala Ala Gly Ala Met Phe Gly Thr Phe Ser Cys Leu Arg Ile Gly Met
35 40 45
Arg Gly Arg Glu Leu Met Gly Gly Val Gly Lys Thr Met Met Gln Ser
50 55 60
Gly Gly Thr Phe Gly Thr Phe Met Ala Ile Gly Met Gly Ile Arg Cys
65 70 75 80
<210>7
<211>240
<212>DNA
＜213〉Genus Homo people (Homo sapiens)
<400>7
atgccggtgg ccgtgggtcc ctacggacag tcccagccaa gctgcttcga ccgtgtcaaa 60
atgggcttcg tgatgggttg cgccgtgggc atggcggccg gggcgctctt cggcaccttt 120
tcctgtctca ggatcggaat gcggggtcga gagctgatgg gcggcattgg gaaaaccatg 180
atgcagagtg gcggcacctt tggcacattc atggccattg ggatgggcat ccgatgctaa 240
<210>8
<211>19
<212>PRT
＜213〉Genus Homo people (Homo sapiens), mouse (Mus musculus), chimpanzee (Pan troglodytes), dog (Canis familiaris)
<400>8
Cys Leu Arg Ile Gly Met Arg Gly Arg Glu Leu Met Gly Gly Ile Gly
5 10 15
Lys Thr Met
19
<2l0>9
<211>18
<212>PRT
＜213〉zebra fish (Danio rerio)
<400>9
Cys Leu Arg Ile Gly Met Arg Gly Arg Glu Leu Met Gly Gly Val Gly
5 10 15
Lys Thr
18
<210>10
<211>18
<212>PRT
＜213〉nematode (Caenorhabditis elegans)
<400>10
Gly Phe Arg Ala Gly Met Arg Gly Lys Asp Leu Leu Leu Gln Thr Gly
5 10 15
Lys Thr
18
<210>11
<211>18
<212>PRT
＜213〉Arabidopis thaliana (Arabidopsis thaliana)
<400>11
Ala Ile Arg Val Lys Val Pro Gly Leu His Lys Val Arg Phe Ile Gly
5 10 15
Gln Thr
18
Claims (7)
1, a kind of proteic active fragments of glycine that is rich in, be polypeptide, or the amino acid residue sequence of sequence in the sequence table 9 is added 1 to 5 amino-acid residue and has the polypeptide of antibacterium effect through the disappearance of 1 to 5 amino-acid residue and/or insertion and/or replacement and at C-terminal and/or N-terminal with amino acid residue sequence of sequence 9 in the sequence table.
2, the described encoding gene that is rich in the proteic active fragments of glycine of claim 1.
3, contain the described expression vector that is rich in the encoding gene of the proteic active fragments of glycine of claim 2.
4, contain the described clone that is rich in the encoding gene of the proteic active fragments of glycine of claim 2.
5, contain the described engineering bacteria that is rich in the encoding gene of the proteic active fragments of glycine of claim 2.
6, the described proteic active fragments of glycine that is rich in of claim 1 is in the application of preparation in the anti-bacterial drug.
7, the described application of encoding gene in the preparation anti-bacterial drug of being rich in the proteic active fragments of glycine of claim 2.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341604A (en) * | 2001-09-11 | 2002-03-27 | 四川大学华西医院 | Artificially combined antibacterial engineering polypeptide and preparation method thereof |
| CN1367180A (en) * | 2001-01-22 | 2002-09-04 | 中国科学院上海生物化学研究所 | Novel natural antibacterial peptide, its code sequence and application |
| CN1382721A (en) * | 2001-04-26 | 2002-12-04 | 上海博德基因开发有限公司 | Polypeptide-Gly-enriched protein-56.98 and polynucleotide for coding it |
-
2005
- 2005-06-09 CN CNB2005100769081A patent/CN1317300C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1367180A (en) * | 2001-01-22 | 2002-09-04 | 中国科学院上海生物化学研究所 | Novel natural antibacterial peptide, its code sequence and application |
| CN1382721A (en) * | 2001-04-26 | 2002-12-04 | 上海博德基因开发有限公司 | Polypeptide-Gly-enriched protein-56.98 and polynucleotide for coding it |
| CN1341604A (en) * | 2001-09-11 | 2002-03-27 | 四川大学华西医院 | Artificially combined antibacterial engineering polypeptide and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Novel antibacterial insect immune peptides and proteins Glinski,Zd.et al,university of veterinary medicine,Vol.42 No.1 1998 * |
| 植物细胞壁中富含甘氨酸的蛋白质 胡延章，四川师范大学学报(自然科学版)，第23卷第4期 2000 * |
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| CN1699416A (en) | 2005-11-23 |
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