TWI583697B - Novel binding of the cell wall of the peptide sequence - Google Patents

Description

Novel peptide cell wall peptide sequence

The present invention relates to a peptide sequence, and more particularly to a peptide sequence suitable for use in novel adherent cell walls.

The development of animal husbandry is very rapid, especially the scale of intensive farming. The main problem that currently hinders the development of the livestock industry is disease, especially infectious diseases. Vaccination is the most basic and effective way to prevent infectious diseases, but for many reasons, many vaccines are currently not immune enough to provide complete protection for animals. Therefore, a lot of research work has been carried out to try to improve the efficacy of vaccines through different ways, including improving vaccine quality (such as increasing antigen content, enhancing immunogenicity, using high-efficiency adjuvants, optimizing seedling production, etc.) and improving immunity. Methods (such as mucosal immunity, embryo immunization, pre-immune, etc.) and enhance the animal's own immune response (such as the use of immune enhancers, reduce the damage of toxic substances to the immune system, control the infection of immunosuppressive pathogenic microorganisms, provide Good feeding and management conditions, etc.). The research and development of new high-efficiency adjuvants is one of the main means to improve the immune effect.

Moreover, transmucosal immunization is a method for vaccination against IgG and secretory IgA which utilize specific pathogens that induce mucosal surfaces. However, since the immunogen expressed by the bacterial vector is presented to the antigen presenting cells of the immune system in the form of particles, it is less likely to cause tolerance than the soluble antigen. In addition, the presence of a common mucosal immune system allows immunization on a specific mucosal surface, thereby inducing secretion of antigen-specific IgA and inducing other specific immune responses at the distal mucosal site. The disadvantage of this method is that the strain itself may cause inflammatory reactions and bacterial infections, which may cause fever and bacteremia in the animal. Another method avoids the use of attenuated strains which themselves become pathogenic bacteria by selecting recombinant commensal bacteria such as Lactobacillus ssp and Lactococcus ssp as vaccine vectors.

However, the disadvantage of using such recombinant organisms is that they can colonize mucosal surfaces, resulting in prolonged exposure to target antigens expressed and released by these recombinant microorganisms. This long-term exposure can cause immune tolerance. In addition, the fact that such organisms are genetically modified and contains recombinant nucleic acids is facing great public opposition as a whole because of the low general acceptance level of products containing recombinant DNA or RNA. There is also opposition to the use of pathogenic strains, even if they are attenuated, or to the use of proteins or proteins from pathogenic strains. Peptidoglycan particles, formerly known as "shells", still contain bacterial components, such as peptidoglycans, with immunomodulatory properties.

Accordingly, it is an object of the present invention to overcome these limitations. To this end, a novel peptide cell wall peptide sequence has been developed which utilizes the functionality of a non-covalent bond to bind to an immunogenic carrier such as a GEM particle. The peptide sequence of the novel binding cell wall allows any target antigen to be immobilized on the surface of the GEM particle without prior modification. The antigen may be a (poly)peptide, a saccharide, a lipid, DNA, RNA or any other bioorganic compound, or even itself may have particulate properties, such as viral particles.

Therefore, it is not expected to develop a novel peptide cell sequence that can solve the traditional technical problems of increasing the effective method and production cost of antibodies, preventing disease inconsistency, etc., and eagerly developing a vaccine stability. A novel binding cell wall peptide sequence with low, anti-ultraviolet force deficiency, and poor persistence of anti-epidemic efficacy.

In view of the above, the present inventors have diligently studied various possible solutions for solving the problems of the conventional technology, and have developed a problem that not only improves the above-mentioned conventional technology, but also has a health hazard that does not harm the environment and livestock, and has It is an epidemic prevention function that meets the needs of breeding environment and long-lasting and sustainable needs, and can effectively reduce epidemic outbreaks or infections, prevent the spread and growth of viruses or germs, and can be made to improve immunity and stability. The novel novel binding cell wall peptide sequence of the loaded antigen with high sexual and long-term anti-epidemic effects has heretofore completed the present invention. Summary of Invention

In particular, according to one aspect of the present invention, a novel peptide cell wall peptide sequence having binding specificity to the cell wall of bacteria can be provided, comprising the peptide sequence of SEQ ID: NO.

Further, in certain embodiments, the peptide sequences of the present invention which are suitable for use in novel adherent cell walls, wherein the peptide sequence is synthetic.

Further, in certain embodiments, the peptide sequence of the present invention which is suitable for use in the novel cell wall, wherein the gene encoding the peptide sequence is SEQ ID: NO.

Wherein, in certain embodiments, in the present invention, the peptide sequence suitable for use in the novel cell wall, wherein the peptide encoding gene sequence has a size of 294 bases.

Another aspect, in certain embodiments, is a peptide sequence suitable for use in novel adhesion cell walls of the invention, wherein the peptide sequence is capable of non-covalent binding to the cell wall of the bacteria and has binding specificity.

A further aspect, in certain embodiments, is a peptide sequence suitable for use in a novel adhesive cell wall, wherein the amino acid sequence of the peptide sequence, and one or more amino acids in the amino acid sequence, can be further Replacement of amino acids.

Wherein, in certain embodiments, a peptide sequence suitable for use in a novel adhesive cell wall of the invention, wherein the peptide sequence is linked to Lactobacillus acidophilus , Lactobacillus casei , and curly milk Lactobacillus crispatus , Lactobacillus bulgaricus , Lactobacillus delbrueckii , Lactobacillus fermentium , Lactobacillus gasseri , Lactobacillus helveticus , yo Lactobacillus johnsonii , Lactobacillus paracasei , Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus rhamnosus , Lactobacillus salivarius And at least one of Lactococcus lactis subspecies lactis .

Further, according to some embodiments, further may be an adjuvant composition of a vaccine comprising a peptide sequence of a Gram-positive or Gram-negative bacteria bound to the novel adhesive cell wall of the present invention.

Further, according to some embodiments, an immunogenic protein vaccine comprising at least one or a plurality of peptide sequences of SEQ ID: NO.

Further, according to some embodiments, a vaccine composition comprising at least one or a plurality of peptide sequences of SEQ ID: NO.

In the following, the embodiments of the present invention will be described in more detail in the detailed description of the embodiments of the present invention so that the spirit and content of the present invention are more complete and easy to understand; however, those of ordinary skill in the art should understand The invention is of course not limited to these examples, and other similar or equivalent functions and order of steps may be utilized to achieve the invention.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

The term "antigen binding" means the ability to bind a target antigen. The ability is conferred by at least one bifunctional polypeptide.

The term "bifunctional" means that the polypeptide has at least two different functionalities: peptidoglycan binding functionality and antigen binding functionality. The functionality may be multivalent, for example a bifunctional polypeptide may comprise multiple antigen binding sites.

The term "animal" as used herein refers to all non-human animals, including mammals.

The term "bird" in this article means a member of chicken, chicken, turkey, duck, hen, hen, pheasant, chicken, cock, pheasant and avian.

Preferably, the method of the invention is applied to a non-human mammal; the best is an animal.

The term "immunogenic carrier" refers to a moiety that has the ability to increase or modify the immunostimulatory properties of an antigen attached thereto when applied to an article, and thus the immunogenic carrier has adjuvant properties.

In a preferred embodiment, the immunogenic carrier complex is an inactive globular peptidoglycan particle (GEM particle, or "shell") made from Gram-positive bacteria.

Processes for the preparation of GEM particles have been previously described, for example, in the patent applications WO 02/101026 and WO 2004/102199. The method retains a majority of the natural globular structure of the bacterium. Briefly, the method comprises treating Gram-positive bacteria with a solution capable of removing cell wall components (such as proteins, lipocalic acids or sugars) from cell wall material.

The term "effective amount" as used herein refers to the amount of antigen that is sufficient to cause an immune response after administration. The immune response involves, but is not limited to, an innately induced, cellular and/or humoral immune response.

In this context, the numerical values and parameters used to define the scope of the invention intrinsically inevitably contain standard deviations due to individual test methods, and are therefore mostly expressed in approximate numerical values, although in specific embodiments Relevant values that are presented as accurately as possible. As used herein, "about" generally refers to the consideration of those of ordinary skill in the art to which the invention pertains, and generally means that the actual value falls within an acceptable standard error of the average value, for example, the actual value is in one Within ±10%, ±5%, ±1%, or ±0.5% of a particular value or range. "Preparation of Novel Peptide Sequences of Novel Adhesive Cell Walls"

First, use an automated solid phase peptide synthesizer (ABI433A peptide synthesizer, Applied Biosystems Inc., Life Technologies Corp., Foster City, CA, USA) and follow the standard procedure provided by the equipment manufacturer (based on Nα-9- armor) The oxycarbonyl group is used to synthesize the peptide sequence of SEQ ID: NO. 1, and is disclosed in SEQ ID: NO.

The peptide sequence of the present invention SEQ ID: NO. 1 is: MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPTLMKETAAAKFERQHMDSPDLSGSGSGSGSAWSHPQFEKGADISGSGSGSGSGELLEVLFQGPRS.

SEQ encoding gene of the present invention wins the peptide sequence ID: NO.2 Department of: ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCCAACCCTTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGAGCGGCTCTGGATCAGGATCTGGCAGCGCTTGGAGCCACCCGCAGTTCGAAAAAGGCGCCGATATCAGCGGCTCAGGATCTGGATCAGGATCTGGCGAATTGCTTGAAGTCCTCTTTCAGGGACCCAGATCT.

Classification and abbreviations of amino acids referred to in the context of the present invention: Aliphatic includes: alanine (A), isoleucine (I), leucine (Leucine, L), 缬(Valine, V), proline (Proline, P). Aromatic amino acids (Aromatic) include: Phenylalanine (F), Tryptophan (W), Tyrosine (Y). Acidic amino acids (Acidic) include: aspartic acid (D), glutamic acid (Glutamic acid, E). Basic amino acids (Basic) include: arginine (R), histidine (Histidine, H), lysine (Lysine, K). Hydroxylic acids include: serine (Srine), and threonine (Treonine, T). Sulfur containing amino acids include: cysteine (Cysteine, C), methionine (Methionine, M). Amidic amino acids (Amidic) include: Asparagine (N), Glutamine (Q). "Example 1" "Preparation of Gram-positive enhancer matrix, GPEM"

Lactococcus lactis (CICC ^® 20209) used to prepare GPEM is usually chemically pretreated with hydrogen chloride (HCl, pH 1.0) as follows: stable phase culture cells are collected by centrifugation and 0.5 volume of phosphate buffered saline is used. (PBS: 58 mM Na ₂ HPO ₄ , 17 mM NaH ₂ PO ₄ , 68 mM NaCl, pH 7.2) was washed once. The cells were resuspended in 1/5 volume of HCl, pH 1.0 solution and boiled for 30 minutes. Then, the GPEM particles formed in this manner were washed three times with PBS, and resuspended in PBS until an average of 2.5 × 10 ¹⁰ GPEM particles/ml was measured with a Burker-Turk hemocytometer, which is shown in Fig. 1. The resulting GPEM particles can be used immediately for binding to the peptide sequence containing the novel adhesive cell wall of the present invention, or stored in a 1.0 ml aliquot at -80 °C until use. "Preparation of Gram-negative enhancer matrix, GNEM"

Escherichia coli BL21 for the preparation of GNEM was usually chemically pretreated with hydrogen chloride (HCl, pH 1.0) according to the following procedure: stable phase culture cells were collected by centrifugation and 0.5 volume of phosphate buffered saline (PBS: 58 mM Na ₂ HPO _{4 ) was used.} Wash once with 17 mM NaH ₂ PO ₄ , 68 mM NaCl, pH 7.2). The cells were resuspended in 1/5 volume of HCl, pH 1.0 solution and boiled for 30 minutes. Then, the GNEM particles formed in this manner were washed three times with PBS, and resuspended in PBS until an average of 2.5 × 10 ¹⁰ GNEM particles/ml was determined by a Burker-Turk hemacytometer. The resulting GNEM particles can be used immediately for binding to the peptide sequence containing the novel adhesive cell wall of the present invention, or stored in a 1.0 ml aliquot at -80 °C until use.

Further, in the adhesion, 2.5×10 ¹⁰ of 1 unit of GPEM particles and GNEM particles obtained by the above were mixed with 2 ml of the medium containing the peptide sequence of the novel adhesive cell wall of the present invention, and then axially rotated. Incubate for 30 minutes at room temperature in an over-end rotator. After binding, GPEM particles and GNEM particles were collected by centrifugation and washed twice with PBS.

The GPEM particles and GNEM particles collected as described above were analyzed by protein expression analysis by high-speed Western blotting (West-blot), and 1 mg was placed in each sample for 30 seconds of exposure monitoring to analyze protein expression. .

Figure 2 is a graph showing the analysis of protein expression using the high-speed western dot blot method after GPEM and GNEM adhesion of the peptide sequence of the novel adhesive cell wall in a specific embodiment of the present invention.

In FIG. 2, S1 represents protein expression of a phosphate buffered saline solution and GNEM for binding reaction; and S2 represents protein expression of a peptide sequence containing a novel adhesion cell wall of the present invention in a phosphate buffered saline solution and GNEM. S3 is a protein expression in which a phosphate buffered saline solution and a GPEM are subjected to a binding reaction; and S4 is a protein expression in which a peptide sequence containing the novel adhesive cell wall of the present invention is subjected to a binding reaction with GPEM in a phosphate buffered saline solution. "Example 2" "Preparation of Gram-positive enhancer matrix, GPEM"

GPEM for preparing five kinds of particles which is Lactococcus lactis ^{(Lactococcus lactis, CICC ® 20209)} , Bacillus subtilis (CICC ^® 10012), Enterococcus faecalis ^{(Enterococcus faecalis, CICC ® 20062)} , Streptomyces A (Streptomyces lipamanii, CICC ^® 10513), Streptomyces rimosus (CICC ^® 11004), which is usually chemically pretreated with hydrogen chloride (HCl, pH 1.0) as follows: Centrifugal culture cells are collected by centrifugation and 0.5 volume of phosphate buffered saline is used. (PBS: 58 mM Na ₂ HPO ₄ , 17 mM NaH ₂ PO ₄ , 68 mM NaCl, pH 7.2) was washed once. The cells were resuspended in 1/5 volume of HCl, pH 1.0 solution and boiled for 30 minutes. Then, the GPEM particles formed in this manner were washed three times with PBS, and resuspended in PBS until an average of 2.5 × 10 ¹⁰ GPEM particles/ml was measured with a Burker-Turk hemacytometer. The resulting GPEM particles can be used immediately for binding to the peptide sequence containing the novel adhesive cell wall of the present invention, or stored in a 1.0 ml aliquot at -80 °C until use. "Preparation of Gram-negative enhancer matrix, GNEM"

Escherichia coli BL21 for the preparation of GNEM was usually chemically pretreated with hydrogen chloride (HCl, pH 1.0) according to the following procedure: stable phase culture cells were collected by centrifugation and 0.5 volume of phosphate buffered saline (PBS: 58 mM Na ₂ HPO _{4 ) was used.} Wash once with 17 mM NaH ₂ PO ₄ , 68 mM NaCl, pH 7.2). The cells were resuspended in 1/5 volume of HCl, pH 1.0 solution and boiled for 30 minutes. Then, the GNEM particles formed in this manner were washed three times with PBS, and resuspended in PBS until an average of 2.5 × 10 ¹⁰ GNEM particles/ml was determined by a Burker-Turk hemacytometer. The resulting GNEM particles can be used immediately for binding to the peptide sequence containing the novel adhesive cell wall of the present invention, or stored in a 1.0 ml aliquot at -80 °C until use.

Further, in the adhesion, 2.5×10 ¹⁰ of 1 unit of GPEM particles and GNEM particles obtained by the above were mixed with 2 ml of the medium containing the peptide sequence of the novel adhesive cell wall of the present invention, and then axially rotated. Incubate for 30 minutes at room temperature in an over-end rotator. After binding, GPEM particles and GNEM particles were collected by centrifugation and washed twice with PBS.

The GPEM particles and GNEM particles collected as described above were analyzed by protein expression analysis by high-speed Western blotting (West-blot), and 1 mg was placed in each sample for 30 seconds of exposure monitoring to analyze protein expression. .

Figure 3 is a graph showing the analysis of protein expression using a high-speed western dot blot method after GPEM and GNEM adhesion of a peptide sequence of a novel adhesive cell wall in a specific embodiment of the present invention.

In the FIG. 3, S5 are diagrams containing the novel peptides of the present invention bonding sequence in the cell wall of phosphate buffered saline and E. coli (Escherichia coli, ATCC ^® 25922) the reaction of the adhesive protein expression For the GNEM; S6 line represents novel comprising the present invention adhesive peptide sequence of cell walls in phosphate buffered saline and Bacillus subtilis (Bacillus subtilis, CICC ^® 10012) protein expression adhesion reaction of as GPEM; S7 are diagrams wins novel containing the present invention adhesive cell walls peptide sequences in phosphate buffered saline and Enterococcus faecalis (Enterococcus faecalis, CICC ^® 20062) is GPEM adhesion protein expression of the reaction; represents S8 based adhesive of the present invention comprising the novel peptides in the sequence of cell walls and phosphate buffered saline Lactococcus lactis (CICC ^® 20209) is a protein expression for GPEM adhesion reaction; S9 is a peptide sequence containing the novel adhesion cell wall of the present invention in phosphate buffered saline solution and Streptomyces lipamanii (CICC ^{® )} 10513) Protein expression for adhesion reaction of GPEM; S10 expression The present invention comprises novel peptide sequence of bonding cell walls in phosphate buffered saline Streptomyces ^{B (Streptomyces rimosus, CICC ® 11004} ) protein expression adhesion reaction is the sum of GPEM.

The related strains of the Gram-positive bacteria and the Gram-negative bacteria used in the present invention all adopt commercially available strains, and the technical scheme of the present invention does not depend on the specific strain of the strain, and has been tested from various times. Biological products companies can achieve the desired results.

Thus, the peptide sequence of the novel binding cell wall of the present invention can be confirmed, because the peptide sequence of the novel binding cell wall has a strong binding effect with Gram-positive bacteria and Gram-negative bacteria, and thus can be effectively applied to develop immunostimulating properties. Adjuvant. The peptide sequence of the novel binding cell wall of the present invention can be clearly adhered to a bacterial cell wall having an immunological reaction, and can be developed as an adjuvant having an immunostimulating agent effect and related applications.

In summary, the content of the present invention has been exemplified by the above embodiments, but the present invention is not limited to the embodiments. A person skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention; for example, combining or modifying the technical contents exemplified in the foregoing embodiments. As a new embodiment, these embodiments are of course considered as belonging to the present invention. Therefore, the scope of protection to be covered in this case also includes the scope of the patent application described below and the scope defined by it.

no.

1 is a photomicrograph showing the GPEM and GNEM used in the present invention; FIG. 2 is a view showing the use of the high-speed western dot ink method after the peptide sequence of the novel adhesive cell wall of the present invention and the GPEM and GNEM adhesion. Fig. 3 is a graph showing the results of analysis of protein expression by the high-speed western dot blot method after the peptide sequence of the novel adhesive cell wall of the present invention and the GPEM and GNEM adhesion.

<110> Guosheng Biochemical Co., Ltd. <120> The peptide sequence of the novel adhesion cell wall <160>2 <210> SEQ ID NO: 1 <211> 98 <212> PRT <213> artificial sequence <223> peptide sequence < 400〉1 Met Arg Gly Ser His His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Thr Leu Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His 35 40 45 Met Asp Ser Pro Asp Leu Ser Gly Ser Gly Ser Gly Ser Gly Ser Ala 50 55 60 Trp Ser His Pro Gln Phe Glu Lys Gly Ala Asp Ile Ser G Ly Ser Gly 65 70 75 80 Ser Gly Ser Gly Ser Gly Glu Leu Leu Glu Val Leu Phe Gln Gly Pro 85 90 95 Arg Ser <210>SEQ ID NO: 2 <211>294 <212>PRT <213>artificial sequence 〈 223> gene encoding the peptide sequence <400> 2 ATGCGGGGTT CTCATCATCA TCATCATCAT GGTATGGCTA GCATGACTGG TGGACAGCAA ATGGGTCGGG ATCTGTACGA CGATGACGAT AAGGATCCAA CCCTTATGAA AGAAACCGCT GCTGCTAAAT TCGAACGCCA GCACATGGAC AGCCCAGATC TGAGCGGCTC TGGATCAGGA TCTGGCAGCG CTTGGAGCCA CCCGCAGTTC GAAAAAGGCG CCGATATCAG CGGCTCAGGA TCTGGATCAG GATCTGGCGA ATTGCTTGAA GTCCTCTTTC AGGGACCCAG ATCT

Claims (10)

A novel peptide cell wall peptide sequence having binding specificity for the cell wall of bacteria comprising the peptide sequence of SEQ ID: NO. The peptide sequence as claimed in claim 1, wherein the peptide sequence is a synthetic 98 peptide sequence. The peptide sequence as claimed in claim 1, wherein the coding gene of the peptide sequence is SEQ ID: NO. The peptide sequence as recited in claim 1, wherein the size of the coding gene of the peptide sequence is 294 bases. The peptide sequence as recited in claim 1, wherein the peptide sequence is capable of non-covalently binding to the cell wall of the bacteria and has binding specificity. The peptide sequence as claimed in claim 1, wherein the amino acid in the peptide sequence comprises a fatty amino acid, an aromatic amino acid, an acidic amino acid, a basic amino acid, a hydroxy amino acid, a sulfur-containing amino acid, and / or guanamine amino acids. The peptide sequence as recited in claim 1, wherein the peptide sequence is conjugated to Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus crispatus , Lactobacillus bulgaricus Lactobacillus delbrueckii , Lactobacillus fermentium , Lactobacillus gasseri , Lactobacillus helveticus , Lactobacillus johnsonii , Lactobacillus paracasei ( Lactobacillus johnsonii ) Lactobacillus paracasei ), Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus rhamnosus , Lactobacillus salivarius , and Lactococcus lactis Subspecies lactis ) at least one of them. The peptide sequence of any one of claims 1 to 7, which is for use in the preparation of a vaccine adjuvant comprising at least one or a plurality of peptide sequences of SEQ ID: NO. A composition derived from Gram-positive bacteria or Gram-negative bacteria. The peptide sequence of any one of claims 1 to 7 for use in the preparation of an immunogenic protein vaccine, wherein the protein vaccine comprises at least one or a plurality of peptide sequences of SEQ ID: NO. The peptide sequence of any one of claims 1 to 7, which is for use in the preparation of a vaccine composition, wherein the vaccine composition comprises at least one or a plurality of peptide sequences of SEQ ID: NO.