WO2005056591A1 - Proteines riches en glycine, leurs sequences codantes et leurs applications - Google Patents

Proteines riches en glycine, leurs sequences codantes et leurs applications Download PDF

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WO2005056591A1
WO2005056591A1 PCT/CN2004/001435 CN2004001435W WO2005056591A1 WO 2005056591 A1 WO2005056591 A1 WO 2005056591A1 CN 2004001435 W CN2004001435 W CN 2004001435W WO 2005056591 A1 WO2005056591 A1 WO 2005056591A1
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sequence
amino acid
acid residues
terminus
protein
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PCT/CN2004/001435
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French (fr)
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Shifu Zhao
Yong Zhang
Lili Yin
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Beijing Institute Of Radiation Medicine
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Priority to EP04802449A priority Critical patent/EP1693381A4/en
Priority to JP2006543349A priority patent/JP2008504003A/ja
Priority to US10/582,420 priority patent/US20070054356A1/en
Publication of WO2005056591A1 publication Critical patent/WO2005056591A1/zh

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
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    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
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    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a glycine-rich protein and its coding gene and application, and particularly to human and mouse glycine-rich protein and its coding gene, and their application in antibacterial.
  • the artificially isolated natural antibacterial peptides show high antibacterial activity, wide antibacterial spectrum, many types, wide range of options, and target strains are not easy to produce resistance mutations, so they are considered to have the following characteristics in the pharmaceutical industry, food industry, and agriculture Broad application prospects.
  • Antibacterial peptides refer to polypeptides that are encoded by genes and have a relative molecular mass of less than 10 kDa, which are synthesized on the ribosome. They have antibacterial activity and are also called polypeptide antibiotics. Most antibacterial peptides have thermal stability, and they can still maintain their activity when heated at 100 ⁇ for 10-15 minutes. Antibacterial peptides are more resistant to larger ionic strength and higher or lower pH values. Most antibacterial peptides have an isoelectric point greater than 7 and exhibit strong cationic characteristics. At the same time, some antibacterial peptides also have the ability to resist hydrolysis by trypsin or pepsin.
  • antimicrobial peptides are used in food preservation, fresh flowers preservation, cosmetics, seed coatings, and animal feed additives. Also underway.
  • antibacterial peptides in the pharmaceutical industry, food industry and agriculture, finding new types of natural antibacterial peptides from the biological world through various channels has become the world's hot invention disclosure.
  • the object of the present invention is to provide a glycine-rich protein and its coding gene and their antibacterial use.
  • the glycine-rich protein provided by the present invention is selected from (cloned from) at least one of the following protein families (Glyrichin family)-
  • K Glyrichin hGlyrichin
  • mouse Glyrichin proteins that have the sequence of amino acid residues of sequence 1 in the sequence list or the sequence of the amino acid residues of sequence 1 in the sequence list by 1 to 20 amino acid residues Deletions, insertions and / or substitutions, and proteins with 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and having antibacterial effects
  • Glyrichin a protein having the sequence of amino acid residues of sequence 4 in the sequence listing or a sequence of amino acid residues of sequence 4 in the sequence listing by deletion, insertion, and / or deletion of 1 to 20 amino acid residues Substitute and add 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and have antibacterial effect;
  • Glyrichin a protein having the sequence ⁇ ! J in the table ⁇ (J 5 amino acid residue sequence or the sequence of the amino acid residue sequence 5 in the sequence table by 1
  • Nematode ⁇ Caenorhabditis elegans Glyrichin: a protein having the amino acid residue sequence of the sequence. Lj. 6 in the sequence ⁇ lj table or the sequence of the amino acid residue of sequence 6 in the sequence table by deletion of 1 to 20 amino acid residues, Insertions and / or substitutions and proteins with 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and having antibacterial effects;
  • nematode i Caenorhabditis elegans' Glyrichin with sequence in sequence listing 7 amino acid residue sequence of the protein or the amino acid residue sequence of sequence 7 in the sequence listing after the deletion, insertion and / or substitution of 1 to 20 amino acid residues and the addition of 1 to 20 at the carboxy-terminus and / or amino-terminus Amino acid residues and proteins with antibacterial effects;
  • Schizosaccharomyces pomhe Glyrichin a protein having the sequence of amino acid residues of sequence 8 in the sequence listing or a sequence of amino acid residues of sequence 8 in the sequence listing having 1 to 20 amino acid residues deleted, inserted, and / or Substitute and add 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and have antibacterial effect;
  • Sacchromyces cerevisiae Glyrichin a protein having the sequence of the amino acid residue of the sequence 9 in the table ⁇ ! J or the sequence of the amino acid residue of the sequence 9 in the sequence table is deleted by 1 to 20 amino acid residues , Insertions and / or substitutions and proteins having 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and having antibacterial effects;
  • Arabidopsis thai i ana Glyrichin a protein having the sequence of the amino acid residue of sequence 10 in the sequence listing or the sequence of the amino acid residue of sequence 10 in the sequence listing is deleted, inserted, and / Or a protein having an antibacterial effect by adding 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus;
  • Plasmodium falciparum 3D7) Glyrichin a protein having the sequence of amino acid residues of sequence 11 in the sequence listing or a sequence of amino acid residues of sequence 11 in the sequence listing having 1 to 20 amino acid residues deleted, inserted, and / or Substitute and add 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and have antibacterial effect;
  • Plasmodium yoelii yoelii ' Glyrichin: a protein having the amino acid residue sequence of sequence 12 in the sequence ⁇ ij table or the sequence of the amino acid residue sequence of sequence 12 in the sequence table is deleted by 1 to 20 amino acid residues, Insertion and / or substitution and addition of 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and having an antibacterial effect;
  • Magr porthe grisea Glyrichin a protein having the sequence of the amino acid residue of sequence 13 in the sequence listing or the sequence of the amino acid residue of sequence 13 in the sequence listing is deleted, inserted, and / or 1 to 20 amino acid residues Substitute and add 1 to 20 amino acid residues at the carboxy-terminus and / or amino-terminus and have antibacterial effect;
  • Neurospora crassa Glyrichin a protein having the sequence of amino acid residues of sequence 14 in the sequence listing or a sequence of amino acid residues of sequence 14 in the sequence listing having deletions, insertions, and / or substitutions of 1 to 20 amino acid residues And at the carboxy terminus and / or amino terminus A protein with 1 to 20 amino acid residues added at the end and having an antibacterial effect.
  • the glycine-rich protein is preferably human Glyrichin and mouse Glyrichin.
  • the number of the deletions, insertions and / or substitutions and amino acid residues added at the carboxy-terminus and / or amino-terminus is preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3 .
  • substitution method is preferably a substitution as shown in Table 1.
  • Table 1. Glyrichin protein family substitutable amino acid list Initial residues Representative substitutions Preferred substitutions
  • the glycine-rich protein of the present invention can be modified (usually without changing the primary structure), such as acetylation, carboxylation, glycosylation, and phosphorylation.
  • the above-mentioned glycine-rich protein encoding gene also belongs to the protection scope of the present invention.
  • the human Glyrichin (hGlyrichin) coding gene may have the DNA sequence of SEQ ID No : 2 in the Sequence Listing or have more than 90% homology with the DNA sequence defined by SEQ ID Na : 2 in the Sequence Listing, and the coding sequence The DNA sequence of the amino acid residue sequence of Sequence 1 in the list or the nucleotide sequence that can hybridize with the DNA sequence defined by Sequence 2 in the Sequence Listing under high stringency conditions;
  • the high stringency condition is that the hybridization refers to washing the membrane with a solution containing 0.1 X SSPE (or 0.1 X SSC) and 0.1% SDS at 65 ° C.
  • the expression vector, the cell line and the engineering bacteria containing the above-mentioned glycine-rich protein-encoding gene belong to the protection scope of the present invention.
  • the glycine-rich protein described in the present invention can be used for antibacterial, and specifically includes the following uses:
  • Figure 1 shows the evolutionary tree analysis of 12 sequences in the homology comparison of Glyrichin family members.
  • Figure 2 shows the conserved Glyrich domain.
  • Figure 3 shows the sequence identity of Glyrich domain among different members.
  • Figure 4 shows the structural characteristics of the hGlyrichin family
  • Figure 5A is a Northern blot analysis of hGlyrichin transcript size and expression profile using hGlyrichin gene as a probe
  • Figure 5B is the result of human Glyrichin in vitro transcription and translation test
  • Figure 6A is a semi-logarithmic growth curve of E. coli BL21 transformed with a pET-22b (+) empty vector under 0.5 mM IPTG-induced and non-induced conditions
  • Figure 6B is a semi-logarithmic growth curve of E. coli BL21 transformed with pET-22b (+)-UBF and pET-22b (+)-PTP under 0.5mM IPTG-induced and non-induced conditions
  • Figure 6C is a semi-logarithmic growth curve of E. coli BL21 transformed with pET-22b-hGlyrichin positive clones 1 and 8 under 0.5 mM IPTG-induced and non-induced conditions
  • Figure 6D shows E. coli BL21 transformed with pET-22b-hGlyrichin-positive clone 1.
  • Figure 8 shows the inhibitory effect of the expression products of full-length hGlyrichin and 5'-deletion genes on the growth of E. coli BL-21 cells
  • Figure 9 shows the effects of full-length hGlyrichin, 5 'and 3' deletion genes on BL-21 growth after transformation
  • the inventors used the method of suppressive subtractive hybridization to screen a large number of differentially expressed genes of mouse bone marrow stromal cells before and after LTC (long term culture) culture, and obtained 131 differentially expressed EST clones. Bioinformatics analysis confirmed that these clones represent 26 genes with known or partially known functions and 7 new genes, 5 of which have complete open reading frames.
  • the mouse-derived mGlyrichin gene (GenBank number AY028425) is One of them, the glycine-rich protein mGlyrichir, whose coding sequence has the amino acid residue sequence of Sequence 1 in the table
  • the inventors designed a pair of primers based on murine Glyrichin gene, using human fetal liver mRNA as a template, and obtaining a complete GRF of human Glyrichin by RT-PCR amplification, a total of 240bp, and speculated based on the ORF Amino acid sequence.
  • the inventors used bioinformatics to analyze the sequence in detail, and performed homology comparison between members of the Glyrichin family-multiple sequence alignment.
  • Figure 1 shows the evolutionary process of the Glyrichin gene from lower to higher levels and the distance between the members.
  • Figure 2 shows the conserved Glyrich domains among members of the Glyrichin family.
  • Figure 3 shows the conserved amino acids are shown in reverse characters.
  • Figure 3 The sequence identity of the glycine-rich domain (Glyrich domain) between different members of the Glyrichin family is shown in Figure 3. It can be seen how close the relationship between each two members is.
  • Electronic PCR The results show that the hGlyrichin gene is located in the human chromosome 20qll.
  • 21 region is composed of three exons, and encodes a small molecular protein of 79 amino acids.
  • a comparison of BlastP homology at the amino acid level shows that the gene is very conservative in evolution. It exists in fungi (such as Neurospora, yeast, etc.), plants (such as Arabidopsis, etc.), Plasmodium, nematodes, Drosophila, Anopheles , Zebrafish, mouse to human and other species, which make up the Glyrichin homology protein family; it shows high homology among different species.
  • human Glyrichin (hGlyrichin) is composed of 79 amino acid residues, with a molecular weight of 8.8 kDa, an isoelectric point of 9. 36, and a pH of 7 with 4. 79 positive charges and a glycine content of 21. 52. %.
  • the inventor made predictions of the secondary structure of hGlyrichin. The structural characteristics of hGlyrichin are shown in Figure 4, which is the result of SignalP analysis.
  • the 13 Glyrichin protein family members of the present invention are distributed in all species that have been sequenced from fungi to humans. Those skilled in the art can easily infer from this that it may also exist in other species whose genomes have not yet been sequenced.
  • the structural common feature is the existence of a Glycine-rich region with a length between 59-68 amino acids. This region basically covers the entire sequence of human and mouse Glyrichin.
  • Glyrichin in other species also has other sequences (such as signal peptides) at the amino or carboxyl terminus of this conserved region, but is not conserved (Figure 2, Figure 3). This shows that the glycine conserved region determines the most basic biological activity of members of the Glyrichin protein family. It also shows that the organism gradually eliminated redundant sequences during the evolution process, making the molecule shorter and more compact.
  • the recombinant glycine-rich protein can be expressed or produced using the gene encoding the glycine-rich protein of the present invention by conventional recombinant DNA technology.
  • the following methods can be used for expression or production:
  • the starting vector used to construct the recombinant expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus or other vectors well known in the art. In short, as long as it can replicate and stabilize expression plasmids and vectors in the host, it can be used.
  • expression vectors usually contain origins of replication, promoters, marker genes and translation control elements. Methods known to those skilled in the art can be used to construct expression vectors containing Glyrichin-encoded DNA sequences and appropriate transcription / translation control signals.
  • Transformation of a host cell with the recombinant expression vector can be performed using conventional techniques well known to those skilled in the art.
  • the obtained transformants can be cultured by a conventional method to express a glycine-rich protein encoded by the gene of the present invention.
  • the glycine-rich protein in the above method can be expressed intracellularly, or on a cell membrane, or secreted extracellularly.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Using the invention
  • Glyrichin protein other agents, such as antibiotics such as penicillin, can also be used at the same time.
  • antibiotics such as penicillin
  • Glyrichin Glyrichin family member
  • Glyrichin protein Glyrichin polypeptide
  • Glyrichin polypeptide amino acid sequences of members of the Glyrichin family with natural antibacterial peptides (sequence 1 in Sequence Listing 1, Proteins or polypeptides of sequences 3-14). They include members of the Glyrichin family of natural antimicrobial peptides with or without a starting methionine, and Glyrichin member proteins with or without a signal peptide.
  • Glyrich domain refers to the most conserved polypeptide in sequences 1 to 13 in the sequence listing. Its amino acid length is between 59-68, the isoelectric point is greater than 7, and it has a positive charge at pH 7.0. The Glycine content is between A peptide with the highest content of 20 amino acids and containing at least one hydrophobic region. Their sequence and structural characteristics are defined in Figure 3, Figure 4 and Table 2.
  • Glyrichin family refers to a polypeptide having a Glyrich domain and having amino acid homology of 30% or more with each other in this domain segment, including the 13 polypeptides mentioned in the present invention and other unreported polypeptides. From the perspective of evolutionary biology, they come from the same ancestor.
  • the invention also provides a fusion protein comprising a Glyrichin polypeptide or a fragment thereof.
  • the invention also includes soluble fragments of Glyrichin polypeptides.
  • this fragment has a number of consecutive amino acid sequences in a Glyrichin polypeptide sequence. It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modification includes: the chemical form of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation modifications during the synthesis and processing or further processing steps of the polypeptide. This modification can be achieved by exposing the polypeptide to an enzyme that undergoes glycosylation, such as mammalian glycosylase Or deglycosylation). Modified forms also include sequences with phosphorylated amino acid residues (eg, serine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize their solubility.
  • human and mouse Glyrichin is a human and mouse
  • the widely expressed natural immune molecule with antibacterial activity is a naturally occurring antibacterial peptide, which is expected to develop into the pharmaceutical industry and all the different fields that need to prevent bacterial infections. This is also the basic function and activity of each member of the Glyrichin family.
  • examples of the cloning, expression, purification, and antibacterial activity test of a human-derived Glyrichin (human Glyrichin, hGlyrichin) gene are taken as examples to further illustrate the present invention.
  • mice-derived Glyrichin gene the following pair of degenerate primers was designed and human fetal liver RNA was used as a template for RT-PCR reaction:
  • the PCR reaction system is a conventional molecular biology system.
  • the PCR amplification conditions are: 94 ° C, 4 minutes, 1 cycle; 94 ° C, 40 seconds, 60 ° C, 50 seconds, 72 ° C, 1 minute, 30 cycles;. C, 7 minutes, 1 cycle.
  • the PCR product was purified by the Winzard PCR preps purification kit (purchased from Promega), and recombined into pGEM-T vector with T 4 ligase to obtain the recombinant plasmid pGEM-T / hGlyrichin, which was then transformed into E. coli JM109 and identified by sequencing.
  • the results show that the human Glyrichin gene has the nucleotide sequence of Sequence 2 in the sequence listing, Glycine-rich protein coding sequence having the amino acid residue sequence of sequence 1 in the table
  • PCR reaction system The conventional (refer to the refined molecular biology guide) PCR reaction system is used.
  • the PCR amplification conditions are: 94 ° C, 4 minutes, 1 cycle; 94 ° C, 40 seconds, 60 ° C, 50 seconds, 72 ° C, 1 minute, 30 cycles; 72 ° C, 7 minutes, 1 cycle.
  • pT7 vector purchased from Promega
  • T 4 DNA ligase to construct the pT7-hGlyrichin plasmid.
  • the constructed plasmid is subjected to transcription and translation reactions in the following systems:
  • Rabbit reticulocyte lysate 12. 5 ⁇ 1
  • Reaction buffer ⁇ . ⁇ ⁇ ⁇
  • the materials used in the reaction system were from the TNT kit of Promega, and S35-Met was purchased from Amersham Bioscience.
  • 1 is the molecular weight standard
  • 2 is the translation product of the unrelated gene UBF (GenBankUBF-fl AF294842; Chinese Journal of Applied Physiology, 20: 66, 2004)
  • 3 is the hGlyrichin gene translation product
  • 4 is the positive control translation provided by the kit product.
  • Example 2 Growth inhibition test of endogenously induced hGlyrichin (mGlyrichin) on E. coli BL21
  • PGEM-T / hGlyrichin was used as a template, primers 1: 5, -GGAATTCCATATGCCGGT GGCCGTGGGTC-3 'and bow
  • Product 2 5,-CCGCTCGAGTTAGCATCGGATGCCCATC-3, and the hGlyrichin gene was amplified by conventional PCR.
  • the PCR reaction system except primers) and reaction conditions were the same as those in step 3 of Example 1.
  • the obtained hGlyrichin gene amplification product was digested with Nde I and Xho I, and T 4 DNA ligase was used to ligate the Nde I and Xho I sites of the expression vector pET-22b (+) digested with Nde I and Xho I. Between spots, E. coli BL21 was transformed, and positive clones 1 and 8 (containing the plasmid pET-22b-hGlyrichin) were identified by enzyme digestion.
  • control plasmids pET-22b (+)-UBF and pET-22b (+)-PTP will use UBF gene (GenBankUBF- fl AF294842; Chinese Journal of Applied Physiology, 20: 66, 2004) and PTP gene (parathyroid gland The 34-peptide gene sequence of the hormone) (GenBank Let 000315) was cloned into the multiple cloning sites of pET-22b (+) to obtain control plasmids pET- 22b (+) -UBF and pET- 22b (+) -PTPo
  • FIG. 6A shows that the bacterial growth curve of the single-turn empty vector pET-22b (+) with or without IPTG basically matched the growth curve of the bacteria
  • 6B shows that in the case of single transfer of two known genes with no antibacterial activity UBF and PTP, with and without the addition of IPTG, the growth of bacteria is also not affected
  • Figures 6C and 6D show that when transferred into Glyrichin After that, the growth curve of bacteria was significantly different with and without the addition of IPTG. In the former case, the growth of bacteria was significantly inhibited.
  • Example 3 Purification of free hGlyrichin expression product and detection of bactericidal activity ⁇ Mouse Glyrichin cDNA i 1Ji Ji Ji Gong No. 5, ⁇ CGGGATCCCGATGCCGGTGGCC
  • the cells were induced to express in vitro with 0.5 mM IPTG for 5 hours, and the cells were collected; resuspended in PBS and repeatedly freeze-thaw lysed; centrifuged at 4 V, 12000 rpm for 20 minutes, and the supernatant was taken for SDS-PAGE electrophoresis. Results As shown in Figure 7, the GST-Glyrichin fusion protein is expressed in the supernatant with a molecular weight of 34KD.
  • lane 1 is the protein molecular weight standard
  • lane 2 is the GST-hGlyrichin fusion protein-induced expression product
  • lane 3 is the uninduced GST_hGlyrichin fusion protein expression product
  • lane 4 is a GST protein-induced expression product
  • lane 5 is an uninduced GST protein expression product.
  • the GST-Glyrichin fusion protein induced by expression was purified by Sepharose 4B GST purification column (Pharmcia Inc.) and cut with enterokinase (Roche) to obtain free hGlyrichin protein.
  • the GST-Glyrichin fusion protein was detected by a 96-well plate method And free hGlyrichin protein for antibacterial activity (E. coli DH5 ⁇ and Bacillus subtilis DB430), and the minimum inhibitory concentration (MIC) was determined.
  • the concentration of the diluted bacterial solution was 10 4 -10 5 CFU / ml, and 80 microliters of the bacterial solution per well was inoculated into a 96-well plate, and the peptide was in a certain ratio.
  • the full-length hGlyrichin gene and 5'-deletion gene obtained by PCR were inserted between EcoRI and Notl of pPIC9K yeast expression plasmid (Invitrogen), and transformed into E. coli BL-21 engineered bacteria and cloned. After construction, Plasmids with full-length and 5 'deleted hGlyrichin genes, respectively.
  • the obtained high-purity plasmid was linearized with Sail endonuclease, and then GS115 yeast was transformed by electroporation. Positive clones were obtained by screening on MD plates containing G418 (50ug / ml).
  • coli BL-21 is evenly coated on the surface of the LB plate containing 1% agar, and then a filter paper sheet with a diameter of about 2 mm is placed in the center of each small square, and 15 ul of different clones are cloned. Supernatant was added dropwise to a piece of paper, and 20 minutes later, it was added in equal amounts for 3 times. Incubate at 37 ° C for 4 hours to observe the size of the bacteriostatic ring to obtain positive clones. At the same time, 10 ⁇ l ampicillin was used as an active control at a concentration of 100 mg / ml.
  • the inventor designed a series of PCR primers using genetic recombination technology:
  • Primer 1 5 -GGAATTCCATATGCCGGTGGCCGTGGGTC-3 '
  • Primer 2 5 -GGAATTCCATATGGGCTTCGTGATGGGTTGC-3 ''
  • Primer 3 5 '— CCGGCTC GAG TTA GAA TGT GCC AAA GGT-3' Primer 4: 5, -CCGCTCGAGTTAGCATCGGATGCCCATC-3 '
  • PCR amplification of the full-length hGlyrichin gene (primer 1 and primer 4 combination), 5 'deletion gene 2 from the 5' end of the 5 'deletion base gene 1 to 60 (the combination of primer 2 and primer 4) and 3 The 3 'deletion gene from base 211-240 of the 5' end of the 2 'end deletion sequence 2 (primer 1 and primer 3 combination).
  • PCR reaction system except primers
  • reaction The conditions are the same as Step 3 in Example 1.
  • HGlyrichin gene fragments of different lengths obtained by PCR amplification were separately ligated with pET-22b (+) plasmid according to the method described in Example 2, transformed into E. coli BL-21 bacteria, picked and cloned.
  • FIG. 9 shows that E. coli BL-21 transformed with an empty vector has no effect on growth with or without IPTG; and after transforming a plasmid with a full-length or partially deleted target gene, no IPTG is added. Induction, bacterial growth was not affected, and the growth of transformants was significantly inhibited within 5 hours after IPTG induction.
  • the X-axis is the hours after IPTG induction (in parentheses), and the Y-axis is the OD600 light absorption value.
  • Glyrichin family for the first time, obtained its conserved sequence, and proved that hGlyrichin has an antibacterial effect. Glyrichin can be used in the medical field and all the different fields where antibiotics need to be applied, and has broad application prospects.

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Description

富含甘氨酸蛋白及其编码基因与应用 技术领域
本发明涉及富含甘氨酸蛋白及其编码基因与应用， 特别涉及人和小鼠 富含甘氨酸蛋白及其编码基因， 及它们在在抗细菌中的应用。
背景技术
20多年来， 医药界没有发现任何新的抗菌素家族， 但是抗药性现象的 发展却极为迅速， 人们甚至可以用肉眼观察到细菌的基因突变。 更为可怕 的是， 对付金黄色葡萄球菌的障碍刚刚攻克， 美国医生就发现了对可有效 抑制细菌的最新抗菌素一万古霉素具有抗性的菌株。 致病菌的抗药性问题 已经日益严重地威胁着人们的健康。 寻找全新类型的抗生素是解决抗药性 问题的一条有效途径。 抗菌肽作为生物天然免疫的活性分子， 被各种生物 用来抵御来自外界病菌的侵染， 在生物界中广泛存在。 人为分离出来的天 然抗菌肽表现出抗菌活性高， 抗菌谱广， 种类多， 可供选择的范围广， 靶 菌株不易产生抗性突变等特点， 因而在医药工业、 食品工业和农业上被认 为有着广阔的应用前景。
抗菌肽是指由基因编码，在核糖体上合成的相对分子质量通常在 lOkDa 以下， 具有抗菌活性的多肽类物质， 也叫多肽抗生素。 大部分抗菌肽具有 热稳定性， 在 100Ό下加热 10〜15min仍能保持其活性。 抗菌肽对较大的 离子强度和较高或较低的 pH值均具有较强的抗性。 多数抗菌肽的等电点 大于 7， 表现出较强的阳离子特征。 同时， 部分抗菌肽尚具备抵抗胰蛋白 酶或胃蛋白酶水解的能力。 此外， 研究发现不同家族的抗菌肽之间在序列 上极少存在同源性， 而同一家族的不同成员之间序列上却存在高度的保守 性，这意味着其功能也非常保守。抗菌肽除了具有抗细菌或真菌的作用外， 有些还具有抗原虫、 病毒或癌细胞的功能。 抗菌肽在生物界中广泛存在。 迄今为止， 人们已经从细菌、 真菌， 到两栖类、 昆虫、 高等植物、 哺乳动 物、 直至人类体内发现了多达 700种以上的多肽抗生素。 目前， 已有多种 抗菌肽正在进行临床前的可行性研究； 此外， 抗菌肽转基因动物、 转基因 植物以及抗菌肽在食品防腐， 鲜花保鲜， 化妆品， 种子包衣和动物饲料添 加剂等方面的应用研究也正在进行之中。 鉴于抗菌肽在医药工业、 食品工业和农业上有着广阔的应用前景， 通 过各种途径从生物界中寻找新型的天然抗菌肽已成为目前世界范围内热 发明公开
本发明的目的是提供一种富含甘氨酸蛋白及其编码基因以及它们的 抗菌用途。
本发明所提供的富含甘氨酸蛋白， 名称为 Glyrichin, 选自 （克隆自） 下述蛋白质家族 （Glyrichin家族） 中的至少一种-
1 ) K Glyrichin (hGlyrichin) 禾口小鼠 Glyrichin (mGlyrichin) ： 均具有序列表中序列 1的氨基酸残基序列的蛋白质或将序列表中序列 1的 氨基酸残基序列经过 1至 20个氨基酸残基的缺失、插入和 /或取代以及在 羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋 白质；
2 )斑马鱼 i Danio rerio) Glyrichin: 具有序列表中序列 3的氨基酸 残基序列的蛋白质或将序列表中序列 3的氨基酸残基序列经过 1至 20个氨 基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
3 )按蚊 { Anopheles gambiae) Glyrichin: 具有序列表中序列 4的氨 基酸残基序列的蛋白质或将序列表中序列 4的氨基酸残基序列经过 1至 20 个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质;
) 果虫 C Drosophila melanogas) Glyrichin: 具有序歹 !j表中序歹 (J 5 的氨基酸残基序列的蛋白质或将序列表中序列 5的氨基酸残基序列经过 1
'至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
5 ) 线虫 { Caenorhabditis elegans) Glyrichin: 具有序歹 lj表中序歹 lj . 6的氨基酸残基序列的蛋白质或将序列表中序列 6的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
6 ) 线虫 i Caenorhabditis elegans') Glyrichin: 具有序列表中序列 7的氨基酸残基序列的蛋白质或将序列表中序列 7的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
7 ) 芽殖酵母 ( Schizosaccharomyces pomhe Glyrichin : 具有序列 表中序列 8的氨基酸残基序列的蛋白质或将序列表中序列 8的氨基酸残基 序列经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
8 )酉良酒酵母 ( Sacchromyces cerevisiae) Glyrichin: 具有序歹 !j表中 序列 9的氨基酸残基序列的蛋白质或将序列表中序列 9的氨基酸残基序列 经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或 氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
9 ) 拟南芥 iArabidopsis thai i ana Glyrichin: 具有序列表中序列 10的氨基酸残基序列的蛋白质或将序列表中序列 10的氨基酸残基序列经 过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨 基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
10 ) 疟原虫 Plasmodium falciparum 3D7) Glyrichin: 具有序列表中序 列 11的氨基酸残基序列的蛋白质或将序列表中序列 11的氨基酸残基序列 经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或 氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
11 ) 痕原虫 ( Plasmodium yoelii yoelii') Glyrichin: 具有序歹 ij表中 序列 12的氨基酸残基序列的蛋白质或将序列表中序列 12的氨基酸残基序 列经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 / 或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
12 )稻痕病菌 Magr porthe grisea Glyrichin: 具有序列表中序列 13的氨基酸残基序列的蛋白质或将序列表中序列 13的氨基酸残基序列经 过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨 基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
13 ) 脉孢菌 Neurospora crassa Glyrichin: 具有序列表中序列 14 的氨基酸残基序列的蛋白质或将序列表中序列 14的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质。
所述富含甘氨酸蛋白优选为人 Glyrichin和小鼠 Glyrichin。
其中， 所述缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 的氨基酸残基的数目优选为 1至 10个， 更有选为 1至 5个， 最优选为 1 至 3个。
所述取代方式优选为如表 1所示的取代。 表 1. Glyrichin蛋白家族可取代氨基酸列表 最初的残基 代表性的取代 优选的取代
Ala(A) Val； Leu; lie Val
Arg (R) Lys ;Gln; Asn Lys
Asn (N) Gln;His;Lys;Arg Gin
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro; Ala Ala
His(H) Asn ; Gin; Lys ; Arg Arg
Ile(I) Leu; Val; Met; Ala: Phe Leu
Leu (L) Ile;Val;Met;Ala;Phe lie
Lys(K) Arg; Gin; Asn Arg
Met (M) Leu; Phe; lie Leu
Phe (F) Leu； Val； lie； 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) He ; Leu; Met ; Phe; Ala Leu 本发明的富含甘氨酸蛋白可进行修饰（通常不改变一级结构） ， 如乙 酰化， 羧基化， 糖基化和磷酸化。
上述富含甘氨酸蛋白的编码基因也属于本发明的保护范围。
其中， 所述人 Glyrichin (hGlyrichin) 的编码基因可具有序列表中 SEQ ID No : 2的 DNA序列或与序列表中 SEQ ID Na : 2限定的 DNA序列具 有 90 %以上同源性，且编码序列表中序列 1的氨基酸残基序列的 DNA序列 或在高严谨条件下可与序列表中的序列 2限定的 DNA序列杂交的核苷酸序 列；
所述高严谨条件为杂交系指用含 0. 1 X SSPE (或 0. 1 X SSC)、0. 1% SDS 的溶液在 65°C下洗膜。
含有上述富含甘氨酸蛋白的编码基因的表达载体， 细胞系及工程菌均 属于本发明的保护范围。
本发明所述的富含甘氨酸蛋白可用于抗细菌， 具体包括以下几方面的 用途：
1 ) 用于制备预防和 /或治疗人或畜细菌性感染疾病的药物。
2 ) 用于制备预防和 /或治疗具有潜在细菌感染的不同种类生物用制 口
3 ) 用于生产抗病虫害的转基因生物。
4) 用于制备针对所述富含甘氨酸蛋白的衍生物或拮抗剂及其配体、 抗体。
附图说明
图 1为 Glyrichin家族成员同源性比较中的 12条序列进化树分析图 图 2为保守的 Glyrich结构域
图 3为 Glyrich结构域在不同成员间的序列一致性
图 4为 hGlyrichin家族的结构特点图
图 5A为以 hGlyrichin基因为探针检测 hGlyrichin转录本大小和表 达谱的 Northern blot分析
图 5B为人 Glyrichin体外转录和翻译试验的结果
图 6A为转化了 pET- 22b (+)空载体的大肠杆菌 BL21在 0. 5mM IPTG诱 导和不诱导条件下的半对数生长曲线 图 6B为转化了 pET- 22b ( + ) - UBF和 pET- 22b ( + ) - PTP的大肠杆菌 BL21在 0. 5mM IPTG诱导和不诱导条件下的半对数生长曲线
图 6C为转化了 pET- 22b- hGlyrichin阳性克隆 1和 8的大肠杆菌 BL21 在 0. 5mM IPTG诱导和不诱导条件下的半对数生长曲线
图 6D为转化了 pET- 22b-hGlyrichin阳性克隆 1的大肠杆菌 BL21在
0. 5mM IPTG诱导和不诱导条件下的两次重复实验结果的半对数生长曲线 图 7为 hGlyrichin原核表达产物的 PAGE电泳结果
图 8为全长 hGlyrichin和 5' -缺失体基因转化酵母后表达产物对大 肠杆菌 BL-21菌体生长的抑制作用
图 9为 hGlyrichin全长、 5' 和 3' 缺失体基因对转化后 BL- 21菌生 长的影响
实施发明的最佳方式
发明人利用抑制性差减杂交的方法， 对 LTC ( long term culture) 培 养前、 后的小鼠骨髓基质细胞差异表达基因进行了大量筛选， 获得了 131 个差异表达的 EST克隆。 生物信息学分析证实， 这些克隆代表了 26种已 知或部分已知功能的基因和 7条全新的基因， 其中 5条具有完整的开放阅 读框架， 鼠源的 mGlyrichin基因 （GenBank号为 AY028425 ) 就是其中之 一， 编码序列具有表中序列 1 的氨基酸残基序列的富含甘氨酸蛋白 mGlyrichir
在得到鼠源 mGlyrichin后， 发明人根据鼠源 Glyrichin基因设计了 一对引物， 以人胎肝 mRNA为模板， 通过 RT-PCR扩增获得人源 Glyrichin 完整的 0RF序列共 240bp， 以及根据 0RF序列推测的氨基酸序列。 发明人 利用生物信息学手段对序列进行了详细分析， 进行了 Glyrichin家族成员 间的同源性比较一多序列对齐。
为研究 Glyrichin家族各成员的亲缘关系， 对图 1所示的 12条序列 进行了进化树分析， 图 1显示出 Glyrichin基因从低等到高等的进化历程 及各成员间亲缘关系的远近。 Glyrichin家族成员间保守的甘氨酸富含区 (Glyrich domain) 如图 2所示 （图中， 保守的氨基酸用反显字表示） 。 富含甘氨酸结构域 （Glyrich结构域） 在 Glyrichin家族不同成员间的序 列一致性如图 3所示，可以看出每两个成员间的亲缘关系的远近。电子 PCR 结果显示， hGlyrichin基因定位于人染色体 20qll. 21区域， 由三个外显 子组成， 编码 79个氨基酸的小分子蛋白。 氨基酸水平 BlastP同源性比较 显示该基因在进化上十分保守， 存在于从真菌 （如脉孢菌、 酵母菌等） 、 植物 （如拟南芥等） 、 疟原虫、 线虫、 果蝇、 按蚊、 斑马鱼、 小鼠到人等 物种中， 组成 Glyrichin同源性蛋白家族； 在不同种属间显示出很高的同 源性， 已知人源 Glyrichin与鼠源相比 100%同源， 与斑马鱼 Glyrinchin 相比具有 90%同源性， 与果蝇的同源性有 62%, 与酵母的同源性也有 46%; 家族内不同成员间的亲缘关系以进化树表示 （图 1 ) 。
其中，具有序列 1、序列 3-14的氨基酸残基序列的 Glyrichin蛋白家 族成员的序列特征如表 2所示。 表 2.具有序列 1、 序列 3-14的氨基酸残基序列的 Glyrichin蛋白家
族成员的序列特征 多肽长度 等 电 电荷 甘氨酸含 物种属名
(aa) 点 数（ρΗ7· 0 ) 量 (%)
Homo sapiens 79 9. 36 4. 79 21. 52
Mus musculus 79 9. 36 4. 79 21. 52
Danio rerio 80 9. 81 4. 82 21. 2
Anopheles gambiae 128 9. 73 10. 08 14. 1
Drosophila melanogaster 79 9. 66 4. 82 20. 3
Caenorhabdi tis elegans 145 10. 79 12. 01 14. 48
Schizosa ccharomyc es 107 10. 04 4. 05 13. 08 pombe
Saccharomyces 112 9. 74 5. 04 13. 27 cerevisiae
Plasmodium falciparum 168 9. 66 5. 15 15. 94 3D7
Plasmodium Yoelii Yoelii 167 9. 16 4. 15 15. 28
Magnaporthe grisea 107 11. 75 8. 24 13. 08
Arabidopsis thaliana 74 9. 84 6. 18 14. 86 其中， 人 Glyrichin (hGlyrichin) 由 79个氨基酸残基组成， 分子量 为 8. 8kDa， 等电点 9. 36， 在 pH值为 7的条件下带 4. 79个正电荷， 甘氨 酸含量为 21. 52 %。发明人进行了 hGlyrichin二级结构预测， hGlyrichin 的结构特点如图 4， 为 SignalP分析结果。 Glyrichin家族其他成员的等 电点均大于 7， 在 pH=7时都带正电荷， 且都存在强疏水区域。上述结果表 明 Glyrichin家族具有已知抗菌肽典型的结构特征。
本发明的 13个 Glyrichin蛋白家族成员分布在从真菌到人的所有已 完成基因组测序的物种中。 本领域的普通技术人员可以轻易地据此推断出 它也可能存在于其他基因组尚未完成测序的物种中。 结构上共同的特点是 存在一个甘氨酸 （Glycine) 富含区， 长度在 59-68个氨基酸之间。 该区 域基本涵盖了人源和鼠源 Glyrichin 全部的序列。 存在于其他物种中的 Glyrichin除了共同的甘氨酸富含区外， 还在此保守区的氨基端或羧基端 存在其他序列 （如信号肽等） ， 但不具有保守性 （图 2、 图 3 ) 。 这说明 甘氨酸保守区决定了 Glyrichin蛋白家族成员最基本的生物活性； 也从另 一方面说明生物在进化过程中逐渐淘汰了冗余序列， 使分子变得更短小、 结构更紧凑。
通过常规的重组 DNA技术， 可利用本发明的富含甘氨酸蛋白的编码基 因表达或生产重组的富含甘氨酸蛋白。 如可按以下方法进行表达或生产：
( 1 ) 用含有上述富含甘氨酸蛋白编码基因的重组表达载体转化或转 导宿主细胞；
(2) 培养所述宿主细胞；
( 3 ) 从培养基或细胞中分离、 纯化蛋白质。
用于构建所述重组表达载体的出发载体可为本领域熟知的细菌 质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病 毒或其它载体。 总之， 只要能在宿主体内复制和稳定表达质粒和载体 都可以用。
表达载体的一个重要特征是通常含有复制起点、 启动子、 标记基因和 翻译控制元件。 本领域的技术人员熟知的方法能用于构建含 Glyrichin编 码 DNA序列和合适的转录 /翻译控制信号的表达载体。
本领域普通技术人员都清楚如何选择适当的载体、 启动子、 增强子和 宿主细胞。 用所述重组表达载体转化宿主细胞可用本领域技术人员熟知的 常规技术进行。 获得的转化子可用常规方法培养， 表达本发明的基因编码 的富含甘氨酸蛋白。 在上述方法中的富含甘氨酸蛋白可在细胞内、 或在细 胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、 化学的和其 它特性通过各种分离方法分离和纯化重组的蛋白。 在使用本发明
Glyrichin蛋白时， 还可同时使用其它药剂， 如青霉素等抗菌素。
在本发明中，术语" Glyrichin "、 "Glyrichin家族成员 "、 "Glyrichin 蛋白"或 "Glyrichin多肽"可互换使用， 都指具有天然抗菌肽 Glyrichin 家族各成员氨基酸序列（序列表 1中序列 1、 序列 3 - 14)的蛋白或多肽。 它 们包括含有或不含起始甲硫氨酸的天然抗菌肽 Glyrichin家族成员， 以及 含有或不含有信号肽的 Glyrichin成员蛋白。
术语 "Glyrich结构域"特指序列表中序列 1〜13中最为保守的 一段多肽， 其氨基酸长度在 59- 68之间， 等电点大于 7， pH7. 0条件 下带正电荷， Glycine含量在 20种氨基酸中含量为最高， 并且含有至 少一个疏水区的一段多肽。 它们的序列及结构特点如图 3， 图 4和表 2所界定。
术语 "Glyrichin家族"是指具有 Glyrich结构域且在该结构域 区段氨基酸同源性相互间在 30%以上的多肽， 包括本发明中已经提到 的 13个多肽和其他尚未报道的多肽。 以进化生物学的观点， 它们来 自于同一个祖先。
本发明还提供了包含 Glyrichin多肽或其片段的融合蛋白。 除了 几乎全长的多肽外， 本发明还包括了 Glyrichin多肽的可溶性片段。
通常，该片段具有 Glyrichin多肽序列中一定数目的连续氨基酸序列。 应理解， 本发明的多肽并不限于上述例举的代表性的多肽。
术语 "修饰" (通常不改变一级结构）包括： 体内或体外的多肽 的化学形式如乙酰化或羧基化。 修饰还包括糖基化， 如那些在多肽的 合成和加工或进一步加工步骤中进行糖基化修饰而产生的多肽。 这种 修饰可以通过将多肽暴露于进行糖基化的酶（如哺乳动物的糖基化酶 或去糖基化酶） 而完成。 修饰形式还包括具有磷酸化氨基酸残基 （如 磯酸丝氨酸、 磷酸苏氨酸） 的序列。 还包括被修饰从而提高了其抗蛋 白水解性能或优化了溶解性能的多肽。
在获得人源 Glyrichin 多核苷酸序列后， 发明人以人源和鼠源 Glyrichin为例， 对该基因家族的功能开展研究， 结果证明人源和鼠 源 Glyrichin是一个在人和小鼠多种组织中广泛表达的具有抗菌活性 的天然免疫分子， 是一种天然存在的抗菌肽， 有望发展成为在医药工 业及所有需要防治细菌感染的不同领域。 这也是 Glyrichin家族各个 成员的基本功能和活性。 下面以人源 Glyrichin (人 Glyrichin, hGlyrichin) 基因的克隆、 表达、 纯化和抗菌活性测试实施例为例， 进一步阐述本发明。 应理解， 这 些实施例仅用于说明本发明而不用于限制本发明的范围。 下列实施例中未 注明具体条件的实验方法， 通常按照常规条件如 Sambrook等人的分子克 隆： 实验室手册（New York : Cold Spring Harbor Laboratory Press, 1989 ) 中所述的条件， 或按照制造厂商所建议的条件。 实施例 1、 人 Glyrichin基因的获得及检测
1、 人 Glyrichin基因完整 0RF序列的获得
根据鼠源 Glyrichin基因设计如下一对简并引物并以人胎肝 RNA为模 板进行 RT- PCR反应：
5, 引物 Pa: 5-CGATGCCGGTGGCCGTGGGTCCCT-3
3， 引物 Pb: 5-TTAGCATCGTATGCCCATTCCA-3
PCR反应体系： PCR反应体系为分子生物学常规体系。 PCR扩增条件 为： 94°C、 4分钟， 1循环； 94°C、 40秒， 60°C、 50秒， 72°C、 1分钟， 30循环； 。C、 7分钟， 1循环。 PCR产物经过 Winzard PCR preps purification kit (购自 Promega公司）纯化， 用 T4连接酶重组到 pGEM-T 载体中得到重组质粒 pGEM-T/hGlyrichin， 然后转化大肠杆菌 JM109, 测 序鉴定。 结果表明人 Glyrichin基因具有序列表中序列 2的核苷酸序列， 编码序列具有表中序列 1的氨基酸残基序列的富含甘氨酸蛋白人
Glyrichin。 .
2、 Northern blot分析
培养人 4种肿瘤细胞株 HepG2、HeLa、J"urket和 HEK293,然后用 Winzard plus RNA purification kit (购自 Promega公司） 提取总 RNA。 各取 20 微克总 RNA， 在 1. 2%甲醛变性琼脂糖凝胶上分离， 并转到 Hybond-N+尼龙 膜。 HGlyrichin完整 0RF为探针， 用 Promega公司的 Prime- a- gene试剂 盒标记。 杂交结果如图 5A所示， 表明 人 Glyrichin基因在所测试的 4种 不同组织来源的肿瘤细胞株中都有表达， 提示它可能是一种广泛表达的天 然抗菌肽。 同时揭示只存在一个转录本， 且大小约为 600bp。
3、 体外转录和翻译试验
以 pGEM- T/hGlyrichin质粒为模板， 利用以下引物： 5, 引物： 5， - CGGGATCCCGATGCCGGTGGCCGTGGGTCCCT- 3， 和 3, 引物：
5' -gctcgagttagcatcggatgcccatcc-3' PCR扩增 得到 hGlyrichin基因全 长的 0RF。 其中 PCR反应体系： 采用常规 (参考精编分子生物学指南) PCR 反应体系。 PCR扩增条件为： 94°C、 4分钟， 1循环； 94°C、 40秒， 60°C、 50秒， 72°C、 1分钟， 30循环； 72°C、 7分钟， 1循环。
然后用 BamHI和 Sacl酶切 PCR扩增产物； 将 pT7载体（购自 Promega 公司）用同样的酶切割，然后用 T4 DNA连接酶连接，构建成 pT7- hGlyrichin 质粒。 构建完成的质粒在如下体系中进行转录和翻译反应：
25 μ 1体系：
兔网织红细胞裂解液： 12. 5 μ 1
反应缓冲液： Ι. Ο μ Ι
0. 5 μ 1
SS3355--MMeett : 1. 0 μ 1 (50 μ li)
Figure imgf000012_0001
RNase 抑制剂： 0. 5 μ 1
T7 DNA聚合酶: 0. 5 μ 1
去离子水： 7. 0 μ 1
总总体体积积：： 25. Ο μ ΐ 30 °C 反应 90分钟。 反应结束后取 5 μ 1样品， 加入 ΙΟ μ Ι Loading Buffer, 进行 SDS-PAGE电泳， 固定液中固定 30分钟， 干燥剂中浸泡 5分 钟， 用干燥架固定凝胶， 干燥凝胶过夜。放射自显影， - 2CTC压片 24小时， 洗片， 结果如图 5B所示， 表明 hGlyrichin基因编码的蛋白体外翻译后的 大小约为 8. 8kDa， 同理论推断的结果一致， 说明该基因可以在体外正常地 转录和翻译。 反应体系中所用材料来自 Promega的 TNT试剂盒， S35-Met 购自 Amersham Bioscience公司。 图中， 1为分子量标准， 2为无关基因 UBF (GenBankUBF-fl AF294842; 中国应用生理学杂志， 20: 66， 2004) 的翻译产物， 3为 hGlyrichin基因翻译产物， 4为试剂盒提供的阳性对照 翻译产物。
实施例 2、 内源性诱导表达的 hGlyrichin (mGlyrichin)对大肠杆菌 BL21的生长抑制试验
1、 pET- 22b- hGlyrichin的构建
以 pGEM- T/hGlyrichin为模板， 以引物 1 : 5， -GGAATTCCATATGCCGGT GGCCGTGGGTC-3 ' 和弓 |物 2： 5， - CCGCTCGAGTTAGCATCGGATGCCCATC- 3， 常规 PCR扩增 hGlyrichin基因。其 PCR反应体系（除引物外）和反应条件同实 施例 1的步骤 3。
将得到的 hGlyrichin基因扩增产物用 Nde I和 Xho I酶切后，用 T4 DNA 连接酶连入经 Nde I和 Xho I酶切的表达载体 pET-22b (+)的 Nde I和 Xho I位点之间， 转化大肠杆菌 BL21， 通过酶切鉴定出阳性克隆 1和 8 (含有 质粒 pET-22b- hGlyrichin) 。
2、 对照质粒 pET- 22b ( + ) - UBF和 pET- 22b ( + ) - PTP的构建 将 UBF基因 （GenBankUBF- fl AF294842; 中国应用生理学杂志， 20: 66， 2004)和 PTP基因（甲状旁腺激素的 34肽基因序列）（GenBank讓 000315 ) 分别克隆入 pET-22b ( + ) 的多克隆位点得到对照质粒 pET- 22b ( + ) -UBF 和 pET- 22b ( + ) -PTPo
3、 生长抑制试验
挑取阳性克隆， 接种到 AMP抗性的液体 LB培养基 （其中氨苄青霉素 为 50ug/ml ) 中， 于 37°C， 250rpm的摇床培养 12h; 以 1 : 100的体积比 接入试管， 继续培养至 0D值为 0. 03时， 向每只试管中加入 IPTG至终浓 度为 0. 5mM， 阴性对照组则加入相应体积的 PBS ; 于 30°C、 250rpm摇菌， 每隔 45分钟取出 1毫升菌液测 OD600值， 连续测 10次以上。 以时间为横 轴， 0D600对数值为纵轴做生长曲线。 结果如图 6A， 图 6B， 图 6C和图 6D 所示， 图 6A表明单转空载体 pET- 22b ( + )的菌株在加入和不加入 IPTG两 种情况下， 细菌的生长曲线基本吻合； 图 6B表明在单转两个已知不具有 抗菌活性基因 UBF和 PTP的情况下， 加入和不加入 IPTG两种情况下， 细 菌的生长同样不受影响； 图 6C和图 6D表明， 在转入 Glyrichin后， 加入 和不加入 IPTG两种情况下细菌的生长曲线明显不同， 在前一种情况下细 菌的生长明显受到抑制， 从图中还可以看出， 随着培养时间延长 IPTG消 耗殆尽， 细菌的生长又得以恢复， 而其他测试基因都没有此现象。 上述结 果充分说明这种抑菌效应是 Glyrichin蛋白本身的作用。
实施例 3、 游离 hGlyrichin表达产物的纯化及杀菌活性检测 ί豳小鼠 Glyrichin的 cDNA i lJi殳计弓 1物 5， ~CGGGATCCCGATGCCGGTGGCC
GTGGGTCCCT- 3，禾口 5， "GG T CI AGCATCGTATGamTCCA-3， ， 以 台肝 进行 RT-PCR扩增 （其 PCR反应体系 （除引物外） 和反应条件同实施例 1的步 骤 1 ) ， 纯化后 PCR产物经测序和限制性内切酶 BamHI和 EcoRI消化后在 T4 DNA连接酶的作用下， 插入到用同样内切酶消化后的 PGEX4- 4Τ2 (构自 Pharmcia) 原核表达载体中， 经转化 JM109 大肠杆菌后， 以 0. 5mM IPTG 进行体外诱导表达 5小时， 收集菌体； 用 PBS重悬， 反复冻融裂解； 于 4 V、 12000rpm离心 20分钟，取上清进行 SDS- PAGE电泳。结果如图 7所示， 表明 GST-Glyrichin融合蛋白在上清中表达， 其分子量为 34KD。图中泳道 1为蛋白质分子量标准，泳道 2为 GST - hGlyrichin融合蛋白诱导表达产物， 泳道 3为未经诱导 GST_hGlyrichin融合蛋白表达产物， 泳道 4为 GST蛋 白诱导表达产物， 泳道 5为未诱导 GST蛋白表达产物。
诱导表达得到的 GST- Glyrichin融合蛋白， 经 Sepharose 4B GST纯 化柱（Pharmcia Inc. )纯化后用肠激酶 （Enterokinase, Roche ) 切割， 得 到游离的 hGlyrichin蛋白， 利用 96孔板法检测 GST- Glyrichin融合蛋白 和游离的 hGlyrichin蛋白的抗菌（大肠杆菌 DH5 α和枯草杆菌 DB430)活 性， 测定最小抑菌浓度 (MIC)。 在抗菌活性测定中， 稀释菌液的浓度为 104-105CFU/ml , 按每孔 80微升菌液接种于 96孔板中， 将多肽以一定比例 稀释， 每孔加入 5微升； 将 96孔板置于 37Ό培养 12h， 紫外分析仪检测 0D600值， 结果显示， GST-Glyrichin融合蛋白没有抑制细菌生长的活性， 而游离的 Glyrichin 具有在较低浓度下抑制细菌生长的活性。 游离的 Glyrichin蛋白具有在较低浓度下抑制革兰氏阳性菌和革兰氏阴性菌的生 长， 具有抗菌活性 （表 3 ) 。 表 3.游离 hGlyrichin蛋白的最小抑菌浓度 (MIC)
最小抑菌
菌株名称
浓度 (ug/ml)
^氏阴性菌 大肠杆菌 DH5 0. 2
^氏阳性菌 枯草杆菌 DB430 3 实施例 4、 hGlyrichin酵母表达及其表达产物杀菌活性的检测 通过 PCR弓 I物设计（全长上游引物： 5 ' -AGGAATTCATGCCGGTGGCCGTGGG
TCCCTAC- 3， ； 5' 缺失体上游引物： 5， -AGGAATTCATGGGCTTCGTGATGGGTTGC - 3， ； 全长下游引物：5， -AAGGAAAAAAGCGGCCGCTTAGCATCGGATGCCCATCCCA ATG- 3' ) ， 以含 hGlyrichin全长的 pET_22b质粒为模板， 在全长上游引 物和全长下游引物、 5' 缺失体上游引物和全长下游引物的引导下分别 PCR扩增全长 hGlyrichin基因和 5 ' 端缺失序列 2的自 5 ' 端第 1到第 60 碱基的 5 ' 缺失体基因。 其 PCR反应体系 （除引物外） 和反应条件同实施 例 1的步骤 3。
将 PCR扩增获得的全长 hGlyrichin基因和 5 ' -缺失体基因分别 插入到 pPIC9K酵母表达质粒 （Invitrogen) 的 EcoRI和 Notl之间， 经转化大肠杆菌 BL-21工程菌和克隆筛选， 获得构建后分别带有全长 和 5' 缺失的 hGlyrichin基因的质粒。 所获得的高纯度质粒经 Sail 内切酶线性化后，采用电转法转化 GS115酵母菌，经含 G418( 50ug/ml ) 的 MD平板筛选获得阳性克隆。 将阳性克隆接种到含 5ml BMGY培养基 中， 30°C摇床培养至 OD600=2. 0- 6. 0 时， 然后用 BMMY (含终浓度为 1%的甲醇）培养基稀释到 0D600=1. 0, 继续培养。 以后每 24小时补加 甲醇到终浓度为 0. 5%，诱导培养后不同时间取 1ml加入到 Eppendorff 离心管中， 离心 16000转， 留上清作活性测试。 活性测试采用琼脂平 板扩散法， 即在含有 1%琼脂的 LB平板表面均匀涂布测试菌 （大肠杆 菌 BL- 21 ) ， 再在每个小方块中央放置直径约 2mm的滤纸片， 将 15ul 不同克隆上清滴加到纸片上， 20分钟后等量加入， 共加 3次。 37°C培 养 4 小时观察抑菌环的大小以获得阳性克隆。 同时以 lOul 浓度为 100mg/ml的氨苄青霉素作为活性对照。经过活性筛选，获得了表达全 长 hGlyrichin的阳性克隆 (L4克隆） 和表达 5， -缺失 hGlyrichin 的阳性克隆 （S2，S12和 S5克隆） 。 L4, S2， S12和 S5克隆的抑菌效 果如图 8所示， 表明转化了无论含全长还是 5 ' -缺失的 hGlyrichin 基因的阳性克隆（L4，S2，S12 和 S5 )上清具有明显抑制琼脂板表面大 肠杆菌 BL- 21生长的效果。 上述实验结果证实， 酵母表达系统可以表 达 hGlyrinchin蛋白， 而且， 表达产物具有抗菌活性； 全长和 5' - 缺失的基因均表达活性蛋白， 为进一步寻找代表该基因活性的确切结 构奠定了牢固的基础。 根据上述系列实验结果， 业内人员极易利用类 似方法进一步筛选获得具有相似活性的最小蛋白质分子。
实施例 5、 hGlyrichin基因的构-效关系研究
1、 不同缺失体质粒转化受体菌对受体菌生长的影响
发明人利用基因重组技术， 设计了系列 PCR引物：
引物 1 ： 5， -GGAATTCCATATGCCGGTGGCCGTGGGTC-3'
引物 2: 5 , -GGAATTCCATATGGGCTTCGTGATGGGTTGC-3 '
引物 3: 5 ' — CCGGCTC GAG TTA GAA TGT GCC AAA GGT-3 ' 引物 4: 5， -CCGCTCGAGTTAGCATCGGATGCCCATC-3'
以 pET-22b-hGlyrichin为模板， 在不同引物组合的引导下分别
PCR扩增全长 hGlyrichin基因 （引物 1和引物 4组合） 、 5 ' 端缺失 序列 2的自 5 ' 端第 1-60位碱基的 5 ' 缺失体基因 （引物 2和引物 4 组合）和 3 ' 端缺失序列 2的自 5 ' 端第 211- 240位碱基的 3 ' 缺失体 基因 （引物 1和引物 3组合） 。 其 PCR反应体系 （除引物外）和反应 条件同实施例 1的步骤 3。将 PCR扩增获得的不同长度 hGlyrichin基 因片段，按照实施例 2中叙述的方法分别进行基因片段与 pET- 22b (+) 质粒连接、 转化大肠杆菌 BL-21菌、 挑克隆并鉴定。 同样， 按照实验 例 2方法进行上述阳性克隆菌的诱导表达以观察全长及不同缺失体对 转化菌生长的影响。 结果如图 9所示， 表明转化了空载体的大肠杆菌 BL-21无论加或不加 IPTG， 其生长不受影响； 而转化了带全长或部分 缺失的目的基因的质粒后，不加 IPTG诱导，细菌生长不受影响， IPTG 诱导后 5小时内， 转化菌的生长明显被抑制。 图中 X轴为 IPTG诱导 后小时数（括号中）， Y轴为 OD600光吸收值。上述结果证实， hGlyrichin 基因的 5' -端和 3' -端部分核苷酸序列对于发挥抗菌作用不是必需 的。 图 9中各个图例中的 （一）表示未加 IPTG, ( + ) 表示加 0. 5mM IPTGo
工业应用
发明人通过实验， 首次发现了 Glyrichin家族， 得到了其保守序 列， 并证明了 hGlyrichin具有抗细菌的作用。 Glyrichin可以应用于 医药领域和所有需要应用抗生素的不同领域， 有广 ii的应用前景。

Claims

权利要求
1、 富含甘氨酸蛋白， 选自下述蛋白质家族中的至少一种：
1 )人 Glyrichin和小鼠 Glyrichin: 均具有序列表中序列 1的氨基酸 残基序列的蛋白质或将序列表中序列 1的氨基酸残基序列经过 1至 20个氨 基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
2 )斑马鱼 anio rerio) Glyrichin: 具有序列表中序列 3的氨基酸 残基序列的蛋白质或将序列表中序列 3的氨基酸残基序列经过 1至 20个氨 基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
3 )按蚊 ί Anopheles gambiae Glyrichin: 具有序列表中序列 4的氨 基酸残基序列的蛋白质或将序列表中序列 4的氨基酸残基序列经过 1至 20 个氨基酸残基的缺失、插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
4) 果蝇 ( Drosophila melanogas) Glyrichin: 具有序歹瞭中序歹 lj 5 的氨基酸残基序列的蛋白质或将序列表中序列 5的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
5 ) 线虫 i aenorhabditis elegans Glyrichin: 具有序列表中序列
6的氨基酸残基序列的蛋白质或将序列表中序列 6的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
6 ) 线虫 謹 orhabditis elegans) Glyrichin: 具有序列表中序列 7的氨基酸残基序列的蛋白质或将序列表中序列 7的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
7 ) 芽殖酵母 ( Schizosaccharomyces pombe Glyrichin : 具有序列 表中序列 8的氨基酸残基序列的蛋白质或将序列表中序列 8的氨基酸残基 序列经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
8 )酉良酒酵母 ( Sacchromyces cerevisiae Glyrichin: 具有序歹 lj表中 序列 9的氨基酸残基序列的蛋白质或将序列表中序列 9的氨基酸残基序列 经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或 氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
9) 拟南芥 iArabidopsis thai i ana) Glyrichin: 具有序列表中序列 10的氨基酸残基序列的蛋白质或将序列表中序列 10的氨基酸残基序列经 过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨 基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
10 ) 疟原虫 ^Plasmodium falciparum 3DT) Glyrichin: 具有序列表中序 列 11的氨基酸残基序列的蛋白质或将序列表中序列 11的氨基酸残基序列 经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或 氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
11 ) 症原虫 ( Plasmodium yoelii yoelii) Glyrichin: 具有序歹 |J表中 序列 12的氨基酸残基序列的蛋白质或将序列表中序列 12的氨基酸残基序 列经过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 / 或氨基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
12 )稻瘟病菌 i Magnaporthe grisea Glyrichin: 具有序列表中序列 13的氨基酸残基序列的蛋白质或将序列表中序列 13的氨基酸残基序列经 过 1至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨 基末端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质；
13 ) 脉孢菌 Neurospora crass a) Glyrichin: 具有序列表中序列 14 的氨基酸残基序列的蛋白质或将序列表中序列 14的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质。
2、 根据权利要求 1 所述的富含甘氨酸蛋白， 其特征在于： 所述富含 甘氨酸蛋白为人 Glyrichin和小鼠 Glyrichin, 它为具有序列表中序列 1 的氨基酸残基序列的蛋白质或将序列表中序列 1的氨基酸残基序列经过 1 至 20个氨基酸残基的缺失、 插入和 /或取代以及在羧基末端和 /或氨基末 端添加 1至 20个氨基酸残基且具有抗细菌作用的蛋白质。
3、 根据权利要求 1或 2所述的富含甘氨酸蛋白， 其特征在于： 所述 缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加的氨基酸残基的 数目为 1至 10个。
4、 根据权利要求 3所述的富含甘氨酸蛋白， 其特征在于： 所述缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加的氨基酸残基的数目 为 1至 5个。
5、 根据权利要求 4所述的富含甘氨酸蛋白， 其特征在于： 所述缺失、 插入和 /或取代以及在羧基末端和 /或氨基末端添加的氨基酸残基的数目 为 1至 3个。
6、 权利要求 1-5任一所述的富含甘氨酸蛋白的编码基因。
7、 根据权利要求 6所述的基因， 其特征在于： 所述富含甘氨酸蛋白 为人 Glyrichin, 其编码基因具有序列表中 SEQ ID Na : 2的 DNA序列或与 序列表中 SEQ ID No : 2限定的 DNA序列具有 90 %以上同源性， 且编码序 列表中序列 1的氨基酸残基序列的 DNA序列或在高严谨条件下可与序列表 中的序列 2限定的 DNA序列杂交的核苷酸序列。
8、 含有权利要求 6或 7所述基因的表达载体。
9、 含有权利要求 6或 7所述基因的细胞系。
10、 含有权利要求 6或 7所述基因的工程菌。
11、 权利要求 1-5任一所述的富含甘氨酸蛋白及其编码基因在抗细菌 中的应用。
12、 根据权利要求 11所述的应用， 其特征在于： 所述富含甘氨酸蛋白 及其编码基因用于制备预防和 /或治疗人或畜细菌性感染疾病的药物。
13、 根据权利要求 11所述的应用， 其特征在于： 所述富含甘氨酸蛋 白及其编码基因用于制备具有预防和 /或治疗潜在细菌感染的不同种类生 物用制品。
14、 根据权利要求 11所述的应用， 其特征在于： 所述富含甘氨酸蛋 白及其编码基因用于生产抗病虫害的转基因生物。
15、 根据权利要求 11所述的应用， 其特征在于： 所述富含甘氨酸蛋 白及其编码基因用于制备针对所述富含甘氨酸蛋白的衍生物或拮抗剂及 其配体、 抗体。
PCT/CN2004/001435 2003-12-11 2004-12-10 Proteines riches en glycine, leurs sequences codantes et leurs applications WO2005056591A1 (fr)

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