CN1670204A - Streptococcus pneumoniae autolysozyme, its encoding nucleic acid and application thereof - Google Patents

Abstract

The invention discloses new genes for encoding gtreptococcuspneumonias autolytic enzyme, polypeptides encoded by the genes, and antibody of the polypeptides. The invention also discloses the depressor of the polypeptides for screening the gtreptococcuspneumonias autolytic enzyme, the use of polynucleotides as primer or as probe, in particular the use of the polypeptide and polynucleotide as diagnosis reagent kit, vaccine and pharmaceutical composition.

Description

Streptococcus pneumoniae autolysozyme, its encoding nucleic acid and application thereof

technical field

The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-streptococcus pneumoniae autolysozyme and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the application of this polynucleotide and polypeptide.

1. Background technology

Streptococcus pneumoniae is one of the main pathogenic bacteria of common bacterial infections, which can cause various diseases such as pneumonia, meningitis, otitis media, sinusitis and bacteremia. According to the statistics of the World Health Organization, more than 1.1 million people worldwide die from Streptococcus pneumoniae infection every year, most of whom are children under 2 years old and elderly people over 65 years old.

Streptococcus pneumoniae is a Gram-positive coccus with a capsule, which can be divided into 84 serotypes according to the different capsule polysaccharide antigens, of which more than 20 serotypes can cause human diseases. The main pathogenic substances of Streptococcus pneumoniae include capsule, hemolysin 0, autolysin, and neuramidase, among which bacterial proteins play a more important role in pathogenicity. In recent years, the resistance of Streptococcus pneumoniae to penicillin has shown a rapid upward trend worldwide. The resistance rate of clinical isolates to penicillin reaches 30%-89%, and the isolation rate of highly resistant strains is close to or even exceeds that of low-grade strains. resistant strains. Not only that, the continuous emergence of multi-drug resistant strains has brought great difficulties to clinical treatment, forcing us to make major adjustments to the treatment plan for Streptococcus pneumoniae infection. Therefore, the research on drug resistance of Streptococcus pneumoniae and the development of new drugs have become a hot spot in the field of anti-infection.

Autolysin (LytA, autolysin): N-acetylmuramic acid-L alanine amidase, located on the surface of the cell wall of S.pn, linked to the choline molecule of teichoic acid (LTA). When the two are connected, LytA is inactive; when S.pn cell wall synthesis stops or nutrients are deficient or treated with antibiotics, the relationship between LTA and LytA is destroyed, and LytA hydrolyzes the glycan chain and the cell wall contains choline The covalent binding between the peptide chains leads to cell autolysis. After S.pn autolysis, cell wall degradation products are produced, an inflammatory reaction occurs, and Pneumolysin is released from the cytoplasm to cause damage to the host. LytA, Pneumolysin, PspA, PsaA, etc. all belong to the S.pn choline-binding protein family, and the latter is considered to be a family that can induce protective immune responses against S.pn infection.

After extensive and in-depth research, the inventor isolated the DNA encoding Streptococcus pneumoniae autolyse from Streptococcus pneumoniae, expressed and purified the Streptococcus pneumoniae autolyse, and further revealed the application of polynucleotides and polypeptides based on this .

Contents of the invention

One object of the present invention is to provide isolated novel polypeptides - Streptococcus pneumoniae autolysozyme and fragments, analogs and derivatives thereof.

Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

Another object of the present invention is to provide an antibody against the polypeptide of the present invention, Streptococcus pneumoniae autolysin.

Another object of the present invention is to provide a polynucleotide that inhibits the expression of the polypeptide of the present invention.

Another object of the present invention is to provide the polypeptide of the present invention for screening inhibitors of Streptococcus pneumoniae autolysozyme; or for identification of peptide fingerprints.

Another object of the present invention is to provide the application of the nucleic acid molecules of the present invention as primers for nucleic acid amplification reactions, or as probes for hybridization reactions, or for making gene chips or microarrays.

Another object of the present invention is to provide a kit for detecting Streptococcus pneumoniae, which contains primers, antibodies or polypeptides for specifically detecting the autolysozyme of Streptococcus pneumoniae.

Another object of the present invention is to provide a vaccine for preventing Streptococcus pneumoniae infection, which contains the eukaryotic expression vector of Streptococcus pneumoniae autolyse or polynucleotide encoding the enzyme.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of Streptococcus pneumoniae pneumonia, which contains the inhibitor of Streptococcus pneumoniae autolyse enzyme screened by the polypeptide of the present invention.

In the first aspect of the present invention, the present invention relates to an isolated polypeptide, which is derived from Streptococcus pneumoniae, comprising: a polypeptide having an amino acid sequence of SEQ ID No. 2, or a conservative variant thereof, or a biologically active fragment thereof or derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2. More preferably, the polypeptide does not contain a signal peptide sequence.

In a second aspect of the present invention, the present invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No.2;

(b) a polynucleotide complementary to polynucleotide (a);

More preferably, the sequence of the polynucleotide is one selected from the following group: (a) having the sequence of positions 1-957 in SEQ ID NO:1.

In the third aspect of the present invention, the present invention also relates to an antibody capable of specifically binding to the polypeptide of the present invention.

In the fourth aspect of the present invention, there is provided the use of the polypeptide of the present invention itself as a therapeutic drug.

In the fifth aspect of the present invention, the use of the polypeptide and coding sequence of the present invention in diagnosis is provided. Utilize the polypeptide of the present invention as an indicator of the degree of infection, use its specific antibody to detect its level; use the coding sequence or its fragments as primers for nucleic acid amplification reactions, or as probes for hybridization reactions, or for making genes Chip or microarray applications.

In the sixth aspect of the present invention, a kit for detecting Streptococcus pneumoniae is provided, which contains primers, antibodies or polypeptides for specifically detecting the autolyse of Streptococcus pneumoniae.

In the seventh aspect of the present invention, a vaccine for preventing Streptococcus pneumoniae is provided, which contains Streptococcus pneumoniae autolyse or a eukaryotic expression vector of a polynucleotide encoding the enzyme.

In the eighth aspect of the present invention, there is provided a pharmaceutical composition for treating Streptococcus pneumoniae pneumonia, which contains the inhibitor of Streptococcus pneumoniae autolysin screened by the polypeptide of the present invention.

Other aspects of the present invention will be apparent to those skilled in the art due to the disclosure of the technology herein. For example, the present invention may relate to a vector containing a polynucleotide of the present invention, particularly an expression vector; A vector genetically engineered host cell, including a transformed, transduced or transfected host cell; a method for producing a polypeptide of the invention comprising culturing said host cell and recovering an expression product.

Unless otherwise specified, the following terms used in this specification and claims have the following meanings:

"Nucleic acid sequence" means an oligonucleotide, nucleotide or polynucleotide, fragments or portions thereof, and may also refer to genomic or synthetic DNA or RNA, which may be single- or double-stranded, representing the sense strand or antisense strand. Similarly, the term "amino acid sequence" refers to oligopeptide, peptide, polypeptide or protein sequences and fragments or portions thereof. When "amino acid sequence" in the present invention refers to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with said protein molecule .

A protein or polynucleotide "variant" refers to an amino acid sequence or a polynucleotide sequence encoding it having one or more amino acid or nucleotide changes. The alterations may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. A variant may have "conservative" changes in which a substituted amino acid has similar structural or chemical properties to the original amino acid, eg, a leucine is replaced by an isoleucine. Variants may also have non-conservative changes, such as replacing glycine with tryptophan.

"Biologically active" refers to a protein that possesses the structural, regulatory, or biochemical functions of a native molecule. Similarly, the term "immunological activity" refers to the ability of a native, recombinant or synthetic protein and fragments thereof to induce a specific immune response and bind to specific antibodies in a suitable animal or cell.

"Inhibitor" refers to a molecule that blocks or modulates the biological activity of S. pneumoniae autolyse when bound to S. pneumoniae autolyse. Inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that can bind S. pneumoniae autolysozyme.

"Modulation" refers to changes in the function of S. pneumoniae autolysin, including increase or decrease in protein activity, changes in binding properties, and changes in any other biological, functional or immunological properties of S. pneumoniae autolysin.

"Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify S. pneumoniae autolysozyme using standard protein purification techniques. Substantially pure S. pneumoniae autolysozyme yields a single major band on non-reducing polyacrylamide gels. The purity of Streptococcus pneumoniae autolysin polypeptide can be analyzed by amino acid sequence.

"Complementary" or "complementary" refers to the natural association of polynucleotides through base pairing under permissive conditions of salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules can be partial or total. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

"Homology" refers to the degree of complementarity, which may be partial or complete. "Partially homologous"refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization under conditions of reduced stringency (Southern blot or Northern blot, etc.). Substantially homologous sequences or hybridization probes can compete and inhibit binding of a fully homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions of reduced stringency permit non-specific binding, since conditions of reduced stringency require the binding of two sequences to each other for a specific or selective interaction.

"Antisense" refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. "Antisense strand" refers to the nucleic acid strand that is complementary to the "sense strand."

"Derivative" refers to a chemical modification of HFP or the nucleic acid encoding it. Such chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain key biological properties of the native molecule.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fab, F(ab') ₂ and Fv, which can specifically bind to the epitope of the autolysozyme of Streptococcus pneumoniae.

The term "isolated" refers to the removal of a substance from its original environment (eg, its natural environment if naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances with which it coexists in a natural system is isolated. Such polynucleotides may be part of a vector, or such polynucleotides or polypeptides may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, "isolated S. pneumoniae autolysozyme" means that S. pneumoniae autolysozyme is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify S. pneumoniae autolysozyme using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of Streptococcus pneumoniae autolysozyme polypeptide can be analyzed by amino acid sequence.

In the first aspect of the present invention, the present invention provides a new polypeptide - Streptococcus pneumoniae autolysozyme, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of S. pneumoniae autolysozyme. As used in the present invention, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially maintains the same biological function or activity of the Streptococcus pneumoniae autolysozyme of the present invention. Fragments, derivatives or analogs of polypeptides of the present invention may be: (I) one wherein one or more amino acid residues are substituted by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted which may or may not be encoded by the genetic code; or (II) one in which a certain group on one or more amino acid residues is replaced by another group comprising substituents; or (III) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused to the mature polypeptide ( Such fragments, derivatives and analogs are considered to be within the purview of those skilled in the art from the description herein such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence.

In a second aspect of the present invention, the present invention provides an isolated polynucleotide consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO:2. The polynucleotide sequence of the present invention comprises the nucleotide sequence of SEQ ID NO:1. The polynucleotides of the present invention were discovered from a full-length cDNA library of Streptococcus pneumoniae. The full length of the polynucleotide sequence it contains is 957 bases, and its open reading frame encodes 318 amino acids (such as SEQ ID NO: 2). According to the amino acid sequence homologous comparison, it is found that the polypeptide has 99% homology with the autolysozyme of the international standard strain of Streptococcus pneumoniae, and it can be deduced that the protein is the autolysozyme of Streptococcus pneumoniae.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used in the present invention, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein or polypeptide having SEQ ID NO: 2, but differs from the sequence of the coding region shown in SEQ ID NO: 1.

A polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: the coding sequence for the mature polypeptide only; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences. coding sequence.

The term "polynucleotide encoding a polypeptide" is intended to include polynucleotides encoding the polypeptide and polynucleotides including additional coding and/or non-coding sequences.

The present invention also relates to variants of the polynucleotides described above, which encode polypeptides having the same amino acid sequence as those of the present invention or fragments, analogs and derivatives of polypeptides. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

The present invention also relates to polynucleotides that hybridize to the sequences described above. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with Use a denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 95%, more Preferably hybridization occurs above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, the length of "nucleic acid fragment" contains at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides glycosides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding S. pneumoniae autolysozyme.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

The specific polynucleotide sequence encoding Streptococcus pneumoniae autolysozyme of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, 2) antibody screening of expression libraries to detect cloned polynucleotides with common structural features Fragment, 3) polynucleotide fragment isolated by expressed sequence tag (EST) technology.

The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolation of double-stranded DNA sequence from genomic DNA; 2) chemical synthesis of DNA sequence to obtain double-stranded DNA of the polypeptide.

Of the methods mentioned above, isolating genomic DNA is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The method of choice more often is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to generate a plasmid or phage full-length cDNA library. There are many mature technologies for the method of extracting mRNA, and the kit is also available from commercial sources (Qiagene). And constructing a cDNA library is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). When combined with polymerase reaction technology, even minimal expression products can be cloned.

These cDNA libraries can be screened for the gene of the present invention by a conventional method. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) appearance or loss of marker gene function; (3) determination of the transcript level of Streptococcus pneumoniae autolysozyme; (4) ) Detection of protein products expressed by genes by immunological techniques or assays of biological activity. The above methods can be used alone or in combination with multiple methods.

In the (1) method, the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probes used here are usually DNA sequences chemically synthesized based on the gene sequence information of the present invention. The genes themselves or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, luciferin or enzymes (such as alkaline phosphatase) and the like.

In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the autolysozyme gene of Streptococcus pneumoniae.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain the full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein. selected and synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The polynucleotide sequences of the genes of the present invention obtained as described above, or various DNA fragments, etc., can be determined by conventional methods such as the dideoxy chain termination method. Such polynucleotide sequence determination can also use commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones before splicing into a full-length cDNA sequence.

The present invention also relates to a vector comprising the polynucleotide of the present invention, as well as a host cell produced by genetic engineering using the vector of the present invention or directly using the coding sequence of Streptococcus pneumoniae autolysin, and a recombinant technology to produce the polypeptide of the present invention method.

In the present invention, the polynucleotide sequence encoding Streptococcus pneumoniae autolyse can be inserted into the vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Vectors applicable in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria; pMSXND expression vectors expressed in mammalian cells and baculovirus-derived vectors expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational regulatory elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding S. pneumoniae autolysozyme and appropriate transcription/translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Inserting an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.

Those of ordinary skill in the art will know how to select appropriate vectors/transcriptional regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

In the present invention, the polynucleotide encoding Streptococcus pneumoniae autolysozyme or the recombinant vector containing the polynucleotide can be transformed or transfected into host cells to form genetically engineered host cells containing the polynucleotide or recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma cells, etc.

Transformation of host cells with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. An alternative is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant Streptococcus pneumoniae autolyse. Generally speaking, there are the following steps:

(1). Use the polynucleotide (or variant) encoding Streptococcus pneumoniae autolysin of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Cultivate host cells in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In step (2), the medium used in the culture can be selected from various conventional mediums according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

In step (3), the recombinant polypeptide can be encapsulated in the cell, expressed on the cell membrane, or secreted out of the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. These methods include but are not limited to: conventional refolding treatment, protein precipitant treatment (salting out method), centrifugation, osmotic destruction, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cut physically, chemically or enzymatically, and then subjected to one-dimensional, two-dimensional or three-dimensional gel electrophoresis analysis, and better mass spectrometry analysis.

In the third aspect of the present invention, the present invention provides methods for producing antibodies using polypeptides, fragments and derivatives thereof as antigens. These antibodies can be polyclonal or monoclonal. The invention also provides the antibody against the antigenic determinant of Streptococcus pneumoniae autolysozyme. These antibodies include, but are not limited to: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by a Fab expression library.

The production of polyclonal antibodies can be obtained by direct injection of Streptococcus pneumoniae autolyse to immunize animals (such as rabbits, mice, rats, etc.), and various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant wait. The techniques for preparing monoclonal antibodies against Streptococcus pneumoniae autolysozyme include, but are not limited to, hybridoma technology, trioma technology, human B-cell hybridoma technology, EBV-hybridoma technology, and the like. Chimeric antibodies combining human constant regions and nonhuman variable regions can be produced using existing techniques. However, the existing technology for producing single-chain antibodies can also be used to produce single-chain antibodies against Streptococcus pneumoniae autolysozyme.

Antibodies against S. pneumoniae autolysin can be used in immunohistochemical techniques to detect the tissue distribution of S. pneumoniae autolysin secretion in biopsy specimens.

Monoclonal antibodies that bind to the autolysozyme of Streptococcus pneumoniae can also be labeled with radioactive isotopes and injected into the body to track their location and distribution. The antibody in the invention can be used to prevent or block the pathological changes caused by the autolysozyme of Streptococcus pneumoniae.

In the fourth aspect of the present invention, the polypeptide of the present invention can be directly used for the treatment of Streptococcus pneumoniae pneumonia.

The autolysozyme of Streptococcus pneumoniae belongs to the choline-binding protein family, which has a highly conserved choline-binding region. Its non-covalent combination with teichoic acid or lipoteichoic acid on the cell surface is the basis for bacteria to play a pathogenic role. No corresponding coding gene has been found in humans. Therefore, the autolysozyme of Streptococcus pneumoniae may be an ideal drug target. Drugs that can destroy its structure or prevent the choline-binding protein from binding to the bacterial cell wall can be developed based on the characteristics of the choline-binding region of the autolysozyme molecule. In addition, because there are active and inactive forms of autolysin molecules, and under certain conditions, the inactive form can be converted into an active form. The activated autolytic enzyme can make bacteria autolyze and die, and has a bactericidal effect, so it can also be considered to directly use itself as an antibacterial drug.

In the fifth aspect of the present invention, the polypeptide, the gene encoding the polypeptide and the polypeptide-specific antibody of the present invention can be directly applied to the diagnosis of pneumonia caused by Streptococcus pneumoniae. In unfavorable growth environment of Streptococcus pneumoniae, the autolytic enzyme is activated to autolyze the bacteria, releasing autolytic enzyme and other antigenic components or bacterial toxins into the infected tissue of the host, causing pathological damage. Therefore, the level of autolysozyme in affected tissues reflects the extent of S. pneumoniae infection.

The invention also relates to a diagnostic test method for quantitatively and locatingly detecting the autolysozyme level of Streptococcus pneumoniae. These assays are well known in the art and include assays of specific enzyme activity and immunological assays. The level of autolysin of Streptococcus pneumoniae detected in the test can be used as an indicator of the infection degree of Streptococcus pneumoniae.

The polynucleotide encoding Streptococcus pneumoniae autolyse can be used for the diagnosis of Streptococcus pneumoniae. The polynucleotide encoding the S. pneumoniae autolyse can be used to detect the presence of S. pneumoniae and the expression level of the autolyse. For example, design primers based on the polynucleotide sequence, use polymerase chain reaction (PCR) to extract nucleic acid templates from the eggs of fecal samples for gene amplification, and determine pneumonia according to whether the size of the amplified polynucleotide fragment meets the predetermined value. Streptococcus infection. The technology is highly sensitive and specific, and can overcome the missed and false detections that are prone to occur in conventional morphological detection methods. The method of genetic testing can not only detect the presence of Streptococcus pneumoniae infection, but also type Streptococcus pneumoniae and determine its subspecies or strains. The DNA sequence encoding the autolyse of Streptococcus pneumoniae can also be used to hybridize the biopsy specimen to determine the expression status of the autolyse of Streptococcus pneumoniae. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all open and mature technologies, and relevant kits are available from commercial sources. Part or all of the polynucleotides of the present invention can be used as probes to be immobilized on microarrays (Microarray) or DNA chips (also known as "gene chips") for analysis of differential expression of genes in tissues and gene diagnosis. The transcripts of Streptococcus pneumoniae autolysin can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification with primers specific for S. pneumoniae autolysin.

In the sixth aspect of the present invention, an immunological diagnostic kit for Streptococcus pneumoniae is provided. There are two main types: enzyme-linked immunosorbent assay kit (ELISA) and rapid immune colloidal gold kit (ICT). The diagnosis of pneumococcal disease can be done by detecting circulating antigens in serum or urine to determine current infection, and can also be quickly screened by detecting specific antibodies in serum or saliva. At present, antibody detection kits are mainly used clinically for auxiliary diagnosis. There are two methods for detecting antibodies. One is to use labeled secondary antibody as a detection reagent to combine with the antibody captured by the antigen coated on the reaction plate or reaction membrane, and the other is to use the labeled antigen as a detection reagent. As a detection reagent, the secondary antibody can generally detect only one antibody, and has relatively high non-specific binding; as a detection reagent, the labeled antigen can detect multiple antibodies with higher specificity.

In the present invention, the purified Streptococcus pneumoniae autolyse can be used to prepare an ELISA detection kit, and the purified Streptococcus pneumoniae autolyse can also be used to prepare a colloidal gold immunochromatography detection kit.

In the seventh aspect of the present invention, the Streptococcus pneumoniae autolysozyme or its gene of the present invention is used to develop a vaccine. The currently used Streptococcus pneumoniae vaccines are all polysaccharide vaccines for each serotype, and autolysin, as an antigenic protein, is highly conserved among various serotypes, and its amino acid sequence has a consistency of more than 98%. Vaccines protect against all serotypes. This vaccine contains a eukaryotic expression vector of the S. pneumoniae autolysozyme or a polynucleotide encoding the enzyme. For example, to make a protein vaccine, clone the cDNA of the LYTA gene into the prokaryotic expression plasmid pET30a(+), induce expression in the prokaryotic, and then purify it with a Ni-TED affinity chromatography column to obtain a purified (His)6 fusion protein, which can be used as a protein vaccine. Balb/c mice were immunized with intranasal drops: (LytA 50ug+CPG10ug) (10u1)/time, once every 2 weeks×3 times; challenged by intraperitoneal injection of Streptococcus pneumoniae. Alternatively, to make a DNA vaccine, clone the cDNA of the LYTA gene into the eukaryotic expression plasmid pEGFP-N1; extract a large amount of the plasmid according to the alkaline lysis method described in the "Molecular Cloning Experiment Guide"; after immunizing the mice with the eukaryotic recombinant plasmid DNA, Take a peritoneal cell smear and observe the fluorescence under a fluorescence microscope to detect the expression of the recombinant antigen. If fluorescence is detected, the recombinant plasmid can express the target protein in vivo and can be used for DNA vaccines. After three consecutive immunizations with CpG adjuvant suitable for the corresponding animal, the infection was challenged by intraperitoneal injection of Streptococcus pneumoniae, and the protective effect was evaluated. Observe the survival rate of the mice, measure the changes of IgG, secretory IgA and cytokines, and evaluate the immune protection.

In the eighth aspect of the present invention, the present invention also provides a pharmaceutical composition for the treatment of Streptococcus pneumoniae pneumonia, which contains the inhibitor of Streptococcus pneumoniae autolysin screened by the polypeptide of the present invention, and the screened inhibitor is The safe and effective dose and pharmaceutically acceptable excipients form a pharmaceutical composition for treating pneumonia caused by Streptococcus pneumoniae. These carriers can be water, dextrose, ethanol, salts, buffers, glycerol and combinations thereof.

The inhibitor pharmaceutical composition may be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes of administration. The amount and dosage range administered to a patient will depend on many factors, such as the mode of administration, the health condition of the subject to be treated, and the judgment of the diagnosing physician.

Description of drawings

Fig. 1 is the polyacrylamide gel electrophoresis pattern (SDS-PAGE) of the isolated Streptococcus pneumoniae autolysozyme, 36.5kDa is the molecular weight of the protein, and the arrow points to the isolated protein band.

Figure 2 is a schematic diagram of colloidal gold immunochromatography

label

1 Test line 2 Quality control line 3 Glass fiber sheet

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Embodiment 1: Cloning of Streptococcus pneumoniae autolysozyme

Through bioinformatics technology, the author found the complete coding reading frame sequence of the registered nucleic acid sequence encoding the autolysozyme of Streptococcus pneumoniae (NCBI accession number M13812.1) and designed 2 primers:

P1: 5'ATGGAAATTAATGTGAGTAAATTAAG 3' (SEQ ID NO: 3)

P2: 5'TTATTTACTGTAATCAAGCCATCTG 3' (SEQ ID NO: 4)

Use a small amount of bacterial genomic DNA extraction kit to extract the genomic DNA of Streptococcus pneumoniae 19F isolate, the 50μl reaction system contains 2-3μg of Streptococcus pneumoniae genomic DNA extracted by the above method, 25mmol/L MgCl23μl, 10mmol/L mixed 2.4 μl of dNTP, 1 μl of each 20 pmol/μl primer, 0.25 μl of 5 U/μl ExTaq enzyme. Pre-denaturation at 950C for 3 minutes, followed by 34 cycles of amplification: denaturation at 940C for 1 minute, annealing at 560C for 2 minutes, extension at 720C for 2 minutes, and extension at 720C for 10 minutes after the final cycle. PCR products were identified by 1% agarose electrophoresis and sequenced. Sequencing results show that the length of the amplified product is 957 (SEQ ID NO: 1), which is a complete open reading frame, and the homology with the international standard strain reaches 99%, which encodes 318 amino acids (such as SEQ ID NO: 2 ).

Example 2: Homology retrieval of cDNA clones

The sequence of the Streptococcus pneumoniae autolysozyme of the present invention and the protein sequence encoded thereof, with the Blast program (BasiclocalAlignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], in Genbank, Swissport and other databases were used for homologous search. The gene with the highest homology to the polypeptide of the present invention is a known autolysozyme of Streptococcus pneumoniae, and its accession number in Genbank is M13812.1. Blastx results showed that the identity of the two was 99%.

Example 3: Northern blot analysis of the expression of Streptococcus pneumoniae autolysozyme gene:

Total RNA was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159]. The method includes acid guanidine thiocyanate phenol-chloroform extraction. That is, use 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) to homogenate the tissue, add 1 times the volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49:1 ), mixed and centrifuged. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. 20 µg of RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transfer to nitrocellulose membrane. ³² P-labeled DNA probes were prepared by the random primer method using α- ³² P dATP. The DNA probe used is the PCR-amplified sequence of the autolysozyme coding region (1 bp to 954 bp) of Streptococcus pneumoniae shown in FIG. 1 . The 32P-labeled probe (approximately 2×10 ⁶ cpm/ml) was hybridized to the RNA-transferred nitrocellulose membrane at 42° C. overnight in a solution containing 50% formamide-25 mM KH ₂ PO ₄ ( pH7.4)-5*SSC-5*Denhardt's solution and 200 μg/ml salmon sperm DNA. After hybridization, the filters were washed in 1×SSC-0.1% SDS at 55° C. for 30 min. Then, analysis and quantification were performed with Phosphor Imager.

Example 4: In vitro expression, isolation and purification of recombinant Streptococcus pneumoniae autolysozyme

According to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:

Primer3: 5'-GGG GTCGAC ATGGAAATTAATGTGAGTAAATTAAG-3' (Seq ID No: 5)

Primer4: 5'-CAC CTCGAG TTATTTTACTGTAATCAAGCCATCTG-3' (Seq ID No: 6)

The 5' ends of these two primers contain Sal I and Xho I restriction sites respectively, followed by the coding sequences of the 5' and 3' ends of the target gene respectively, and the NdeI and BamHI restriction sites correspond to the expression vector plasmid pET- Selective endonuclease site on 30a(+) (product of Novagen, Cat. No. 69865.3). Using the extracted DNA as a template, carry out PCR reaction. The PCR reaction conditions were as follows: 20 pg of library DNA contained in a total volume of 50 μl, 10 pmol of primers Primer-5 and Primer-6 respectively, and 1 μl of Advantagepolymerase Mix (product of Clontech Company). Cycle parameters: 94°C for 20s, 54°C for 30s, 72°C for 2min, a total of 25 cycles. The amplified product and plasmid pET-30a(+) were digested with Sal I and Xho I respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into Escherichia coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method, and after culturing overnight on an LB plate containing kanamycin (final concentration 50 μg/ml), positive clones were screened by colony PCR method, and sequenced. Select positive clones with the correct sequence, and culture the host strain BL21 (pET-LYTA) at 37°C in LB liquid medium containing kanamycin (final concentration 30 μg/ml) to the logarithmic growth phase, and add IPTG to the final concentration 0.5mmol/L, continue culturing at 28°C for 12 hours. Bacteria were collected by centrifugation, broken by ultrasonic waves, supernatant was collected by centrifugation, and chromatographically performed with an affinity chromatography column His.Bind Quick Cartridge (product of Novagen Company) capable of binding to 6 histidines (6His-Tag), to obtain The purified target protein Streptococcus pneumoniae autolysozyme. After SDS-PAGE electrophoresis, a single band was obtained at 36.5kDa (Figure 1). Transfer the band to PVDF membrane and use Edams hydrolysis method to analyze the N-terminal amino acid sequence. As a result, the N-terminal 15 amino acids are completely identical to the N-terminal 15 amino acid residues shown in SEQ ID NO: 2.

The physicochemical properties of embodiment 5 Streptococcus pneumoniae autolysozyme

The Streptococcus pneumoniae autolysozyme purified by affinity chromatography was excised with Throbinenterokinase enzyme after the carrier sequence, and then subjected to SDS-PAGE electrophoresis and standard molecular weight curve, the molecular weight was measured to be about 36.5kDa; the protein contains acidic amino acid residues 46, 30 basic amino acid residues, using Amerham isoelectric focusing electrophoresis apparatus, through pH3-10 and pH4-7 gradient fixed gel isoelectric focusing electrophoresis, the measured isoelectric point is 5.07. The protein molecule is spherical in aqueous solution environment and is easily soluble in water. Its half-life is 30 hours in mammalian mitochondria, more than 20 hours in yeast cells, and more than 10 hours in E. coli. The structure of the protein is stable.

The generation of embodiment 6 anti-streptococcus pneumoniae autolysozyme antibody

Rabbits were immunized with 4 mg of Streptococcus pneumoniae autolysin purified by affinity chromatography plus complete Freund's adjuvant, and then boosted with the protein plus incomplete Freund's adjuvant for a booster immunization once 15 days later. The titer of antibody in rabbit serum was determined by ELISA using a titer plate coated with 15 μg/ml Streptococcus pneumoniae autolysozyme. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a Sepharose4B column activated by cyanogen bromide, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation proved that the purified antibody could specifically bind to the autolysozyme of Streptococcus pneumoniae. The antibody is a polyclonal antibody, and the titer with the recombinant antigen is above 1:32 in the agar immunological double expansion test, and the titer with the crude antigen of the parasite is above 1:16. The antibody was able to detect LytA circulating antigen in severely infected S. pneumoniae patients.

Embodiment 7: the application of polynucleotide fragment of the present invention as hybridization probe

Selecting suitable oligonucleotide fragments from the polynucleotides of the present invention has many uses as hybridization probes, such as using the probes to hybridize with bacterial culture genomes in respiratory mucosal secretions to identify whether Containing the polynucleotide sequence of the present invention and the detected homologous polynucleotide sequence, so as to identify whether there is Streptococcus pneumoniae infection. From the polynucleotide SEQ ID NO:1 of the present invention, select oligonucleotide fragments as hybridization probes, the following principles and several aspects to be considered should be followed: the preferred range of probe size is 18-50 nucleotides ; GC content is 30%-70%, if it exceeds, non-specific hybridization will increase; there should be no complementary region inside the probe; those that meet the above conditions can be used as primary probes, and then further computer sequence analysis, including the primary probes Carry out homology comparison with its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complementary regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases If the bases are exactly the same, the primary probe should not be used generally; in addition, the replacement mutant sequence based on the designed probe fragment or its complementary fragment is used as the second probe.

Probe 1 (probe1), belonging to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (40Nt):

5'-GACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAG-3' (Seq ID No: 7)

Probe 2 (probe2), belonging to the second type of probe, is equivalent to the substitution mutation sequence (40Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

5'-GACTACCGCCTTTATATCGGACTCTTACGCAATCTAGCAG-3' (Seq ID No: 8)

Nucleic acid probes usually use filter hybridization methods, including dot blotting, Southern blotting, Northern blotting, and copying methods. They all use basically the same steps to hybridize after immobilizing the polynucleotide sample to be tested on the filter membrane. .

For other unlisted common reagents and their preparation methods related to the following specific experimental steps, please refer to the literature: DNAPROBES G.H.Keller; M.M.Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experimental manuals such as "Molecular Cloning Experiments" Guide" (Second Edition in 1998) [US] Sam Brook et al., Science Press.

Steps: 1) Prepare DNA with a small amount of bacterial genome DNA extraction kit.

2) Preparation of sample membranes: Take 4×2 nitrocellulose membranes (NC membranes) of appropriate size, and lightly mark the spotting position and sample number on them with a pencil. Each probe needs two NC membranes. In order to wash the membrane with high-intensity conditions and intensity conditions in subsequent experimental steps. Take 15 microliters each of the control and the control, spot on the sample membrane, and dry at room temperature. Place on filter paper soaked with 0.1mol/L NaOH, 1.5mol/LNaCl for 5 minutes (twice), dry and place on filter paper soaked with 0.5mol/L Tris-HCl (pH7.0), 3mol/LNaCl for 5 minutes (twice) and let dry. Put it in clean filter paper, wrap it with aluminum foil, and dry it under vacuum at 60-80°C for 2 hours.

3) ^The probe was labeled with ³² P by phosphokinase and passed through a Sephadex G-50 column, and the amount of isotope was monitored by a liquid scintillation instrument. γ- ^32P -dATP).

4) Pre-hybridization: put the sample membrane in a plastic bag, add 3-10mg of pre-hybridization solution (10×Denhardt’s; 6×SSC, 0.1mg/ml CT DNA (calf thymus DNA).), seal the bag , shake in a water bath at 68°C for 2 hours.

5) Hybridization: Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and shake in a water bath at 42°C overnight.

6) Membrane washing:

High strength wash film:

Take out the hybridized sample membrane; wash in 2×SSC, 0.1% SDS, 40°C for 15 minutes (twice); 0.1×SSC, in 0.1% SDS, wash at 40°C for 15 minutes (twice); 0.1×SSC, In 0.1% SDS, wash at 55°C for 30 minutes (twice), and dry at room temperature.

Low intensity wash:

Take out the hybridized sample membrane; wash in 2×SSC, 0.1% SDS, 37°C for 15 minutes (twice); 0.1×SSC, in 0.1% SDS, wash at 37°C for 15 minutes (twice); 0.1×SSC, Wash in 0.1% SDS at 40°C for 15 minutes (twice), and dry at room temperature.

7) X-ray autoradiography:

-70°C, X-ray autoradiography (the pressing time depends on the radioactivity of the hybridization spots).

Experimental results:

In the hybridization experiment carried out by using low-intensity washing conditions, the radioactive intensity of the hybridization spots of the above four probes was not significantly different; while in the hybridization experiment carried out by using low-intensity washing conditions, the radioactive intensity of the hybridization spots of probe 1 was significantly stronger than that of The radioactive intensity of the other three probe hybridization spots. Probe 1 can thus be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.

Embodiment 8: Preparation of diagnostic kit

In addition to routine pathogenic biological examinations, the main way to diagnose pneumococcal disease is to use immunological diagnostic kits. There are two main types: enzyme-linked immunosorbent assay kit (ELISA) and rapid immune colloidal gold kit (ICT). The diagnosis of pneumococcal disease can be done by detecting circulating antigens in serum to determine current infection, or by detecting specific antibodies in serum or saliva for rapid screening. In general, the diagnosis of S. pneumoniae infection is based on detection of circulating antigens in the patient.

Preparation of ELISA detection kit: Prepare polyclonal antibody and monoclonal antibody from purified Streptococcus pneumoniae, and use carbonate coating buffer (pH9.6) to prepare 0.1mg/ml autolytic enzyme monoclonal antibody, 96-well enzyme standard Add 50ul to each well, overnight at 4°C. After pouring off the coating solution, use 100ul of PBS-Tween20 solution containing 10% skimmed milk powder to block the excess protein binding sites on the well wall, and pour off the blocking solution after blocking overnight at 4°C. Add 50 ul of the serum to be tested into the reaction well of the microplate plate, shake gently for 5 minutes, let it stand in a wet box at 37°C for 30-60 minutes, and wash with PBS-Tween20 washing solution repeatedly for 3 times, each time for 3 minutes; Horseradish peroxidase or alkaline phosphatase-labeled polyclonal antibody (1:1000 dilution 50ul), repeat the above binding and washing process, then add substrate and chromogenic reagent for color development, when the color development is obvious, add 2% sulfuric acid solution to stop the reaction. Visually observe or measure the reaction results with a microplate reader.

Preparation of colloidal gold immunochromatography detection kit: Common immunochromatography systems include dipstick, card and cassette. The cassette immunochromatographic system has the same basic principle and structure as shown in Figure 2: it consists of a chromatographic system (the chromatographic membrane is connected to one or two absorbing layers) a solid-phase immune reaction system (the antigen or antibody is linearly coated At a specific position on the chromatographic membrane as a test line or a quality control line), the sample and colloidal gold labeling reagent are added from one end, and under the capillary action of the membrane, they move along the membrane surface to the other end and are absorbed by the absorbing layer. The components to be tested in the sample and the colloidal gold detection reagent undergo an immunological binding reaction with the capture reagent bound to the membrane during the chromatography process, and stay on the coated line (including test line 1 and quality control line 2), showing A red reaction line. When the sample does not contain the component to be tested, there will be no color at the test line 1, while the quality control line 2 as a positive control will be red. In the dipping strip detection kit, add the lower end of the test strip to the sample solution to be tested and the labeled colloidal gold solution; in the card type and box type detection kits, the labeled colloidal gold is stored in a dry solid form on a glass fiber sheet at one end 3. For the card type, you only need to drop the solution to be tested and the buffer solution directly onto the glass fiber sheet, close the card, and the result can be judged by the observation window on the cover in a few minutes; for the box type, put the test strip into a small plastic box , the end of the colloidal gold corresponds to the circular sample hole, the sample and buffer solution are added from the sample hole, and the result is judged from the rectangular observation window after a few minutes.

In this detection kit, the chromatographic membrane is a mixed cellulose membrane with a pore size of 8 μm, the purified Streptococcus pneumoniae autolysozyme monoclonal antibody is coated on the far end of the absorption layer as the detection line, and the recombinant Streptococcus pneumoniae LytA is coated on the absorption layer. The proximal end of the layer was used as the quality control line, and the colloidal gold solution of the polyclonal antibody against Streptococcus pneumoniae was added dropwise to the glass fiber sheet, dried, and placed on the other end of the chromatographic membrane.

Preparation and labeling of colloidal gold: make chloroaurate salt into a concentration of 0.01% with deionized ultrapure water, after boiling, quickly add 1% trisodium citrate solution (add 2ml per 100ml), keep it on fire quickly Shake and mix well, cook until the color of the solution turns wine red, remove from the fire, and make up the original volume with ultrapure water. The average particle size of the prepared colloidal gold is about 20nm. Adjust the prepared colloidal gold solution to pH 8.0 with 0.25 mol/L potassium carbonate solution, add purified autolysin polyclonal antibody in an amount of 20 μg/mL under stirring, react for 10 minutes, and centrifuge at 15000g for 30 minutes , the pellet was resuspended with 1/10 of the original volume of PBS (pH 6.0).

Embodiment 9: Preparation of vaccine

1. DNA vaccine

The cDNA of LYTA gene was cloned into the eukaryotic expression plasmid pEGFP-N1; a large number of plasmids were extracted according to the alkaline lysis method described in the "Molecular Cloning Experiment Guide"; after the eukaryotic recombinant plasmid DNA was immunized in mice, peritoneal cell smears were taken, Observe the fluorescence under a fluorescence microscope to detect the expression of the recombinant antigen. If the fluorescence is detected, the recombinant plasmid can express the target protein in vivo and can be used for DNA vaccines. After three consecutive immunizations with CpG adjuvant suitable for the corresponding animal, the infection was challenged by intraperitoneal injection of Streptococcus pneumoniae, and the protective effect was evaluated.

2. Protein vaccines

The cDNA of the LYTA gene was cloned into the prokaryotic expression plasmid pET30a(+), and after prokaryotic induction and expression, it was purified by Ni-TED affinity chromatography to obtain the purified (His) ₆ fusion protein, which can be used as a protein vaccine. Balb/c mice were immunized with nasal drops: (LytA 50ug+CPG10ug) (10ul)/time, once every 2 weeks×3 times; after intraperitoneal injection of Streptococcus pneumoniae for challenge, the survival rate of the mice was observed, Changes of IgG, secretory IgA and cytokines were measured to evaluate their immune protection.

Embodiment 10: preparation medicine

After the prokaryotic expression product of Streptococcus pneumoniae autolyse is purified by affinity chromatography, it is inhaled into the patient's respiratory tract in the form of aerosol, and the recombinant autolyse exerts its function of lysing bacteria, replacing antibiotics to dissolve Streptococcus pneumoniae or other targets. Lambert-positive bacteria, which is very important for the treatment of multi-drug resistant strains.

Sequence Listing

<110> Sun Yat-Sen University

<120> Streptococcus pneumoniae autolysozyme, its encoding nucleic acid and application thereof

<130> LytA

<160>8

<170>PatentIn version 3.1

<210>1

<211>957

<212>DNA

<213>Streptococcus pneumoniae

<400>1

atggaaatta atgtgagtaa attaagaaca gatttgcctc aagtcggcgt gcaaccatat 60

aggcaagtac acgcacactc aactgggaat ccgcattcaa ccgtacagaa tgaagcggat 120

tatcactggc ggaaagaccc agaattaggt tttttctcgc aattgttgg gaacggttgc 180

atcatgcagg taggacctgt tgataatggt gcctgggacg ttgggggcgg ttggaatgct 240

gagacctatg cagcggttga actgattgaa agccattcaa ctaaagaaga gttcatgacg 300

gactaccgcc tttatatcga actcttacgc aatctagcag atgaagcagg tttgccgaaa 360

acgcttgata cagggagttt agctggaatt aaaacgcacg agtattgcac gaataaccaa 420

ccaaacaacc actcagacca tgtggatcca tacccttact tggcaaaatg gggcattagc 480

cgtgagcagt ttaagtatga tattgagaac ggcttgacga ttgaaacagg ctggcagaag 540

aatgacactg gctactggta cgtacattca gacggctctt atccaaaaga caagtttgag 600

aaaatcaatg gcacttggta ctactttgac agttcaggct atatgcttgc agaccgctgg 660

aggaagcaca cagacggcaa ctggtactgg ttcgacaact caggcgaaat ggctacaggc 720

tggaagaaaa tcgctgataa gtggtactat ttcaacgaag aaggtgccat gaagacaggc 780

tgggtcaagt acaaggacac ttggtactac ttagacgcta aagaaggcgc catggtatca 840

aatgccttta tccagtcagc ggacggaaca ggctggtact acctcaaacc agacggaaca 900

ctggcagaca agccagaatt cacagtagag ccagatggct tgattacagt aaaataa 957

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<212>PRT

<213>Streptococcus pneumoniae

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Met Glu Ile Asn Val Ser Lys Leu Arg Thr Asp Leu Pro Gln Val Gly

1 5 10 15

Val Gln Pro Tyr Arg Gln Val His Ala His Ser Thr Gly Asn Pro His

20 25 30

Ser Thr Val Gln Asn Glu Ala Asp Tyr His Trp Arg Lys Asp Pro Glu

35 40 45

Leu Gly Phe Phe Ser His Ile Val Gly Asn Gly Cys Ile Met Gln Val

50 55 60

Gly Pro Val Asp Asn Gly Ala Trp Asp Val Gly Gly Gly Trp Asn Ala

65 70 75 80

Glu Thr Tyr Ala Ala Val Glu Leu Ile Glu Ser His Ser Thr Lys Glu

85 90 95

Glu Phe Met Thr Asp Tyr Arg Leu Tyr Ile Glu Leu Leu Arg Asn Leu

100 105 110

Ala Asp Glu Ala Gly Leu Pro Lys Thr Leu Asp Thr Gly Ser Leu Ala

115 120 125

Gly Ile Lys Thr His Glu Tyr Cys Thr Asn Asn Gln Pro Asn Asn His

130 135 140

Ser Asp His Val Asp Pro Tyr Pro Tyr Leu Ala Lys Trp Gly Ile Ser

145 150 155 160

Arg Glu Gln Phe Lys Tyr Asp Ile Glu Asn Gly Leu Thr Ile Glu Thr

165 170 175

Gly Trp Gln Lys Asn Asp Thr Gly Tyr Trp Tyr Val His Ser Asp Gly

180 185 190

Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr

195 200 205

Phe Asp Ser Ser Gly Tyr Met Leu Ala Asp Arg Trp Arg Lys His Thr

210 215 220

Asp Gly Asn Trp Tyr Trp Phe Asp Asn Ser Gly Glu Met Ala Thr Gly

225 230 235 240

Trp Lys Lys Ile Ala Asp Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Ala

245 250 255

Met Lys Thr Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp

260 265 270

Ala Lys Glu Gly Ala Met Val Ser Asn Ala Phe Ile Gln Ser Ala Asp

275 280 285

Gly Thr Gly Trp Tyr Tyr Leu Lys Pro Asp Gly Thr Leu Ala Asp Lys

290 295 300

Pro Glu Phe Thr Val Glu Pro Asp Gly Leu Ile Thr Val Lys

305 310 315

<210>3

<211>26

<212>DNA

<213>Streptococcus pneumoniae

<400>3

atggaaatta atgtgagtaa attaag 26

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<213>Streptococcus pneumoniae

<400>4

ttatttact gtaatcaagc catctg 26

<210>5

<211>35

<212>DNA

<213>Streptococcus pneumoniae

<400>5

gggtcgaca tggaaattaa tgtgagtaaa ttaag 35

<210>6

<211>35

<212>DNA

<213>Streptococcus pneumoniae

<400>6

cacctcgagt tattttactg taatcaagcc atctg 35

<210>7

<211>40

<212>DNA

<213>Streptococcus pneumoniae

<400>7

gactaccgcc tttatatcga actcttacgc aatctagcag 40

<210>8

<211>40

<212>DNA

<213>Streptococcus pneumoniae

<400>8

gactaccgcc tttatatcgg actcttacgc aatctagcag 40

Claims (10)

1. An isolated polypeptide - Streptococcus pneumoniae autolysozyme, characterized in that it comprises: a polypeptide of the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog or derivative thereof. 2. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof; (b) A polynucleotide complementary to polynucleotide (a). 3. The polynucleotide according to claim 2, characterized in that the sequence of said polynucleotide comprises the sequence of positions 1-957 in SEQ ID NO:1. 4. An antibody capable of binding to a polypeptide, characterized in that said antibody is capable of specifically binding to said Streptococcus pneumoniae autolyse. 5. A class of polynucleotides that regulate polypeptide expression, characterized in that they are polynucleotides that inhibit the expression of the Streptococcus pneumoniae autolysozyme, and have a polynucleotide sequence or a fragment thereof shown in SEQ ID NO: 1 Antisense sequence or oligomeric single-stranded RNA identical to its sequence. 6. The application of the polypeptide according to claim 1, characterized in that it is used for screening inhibitors of Streptococcus pneumoniae autolysin; or for identification of peptide fingerprints. 7. The application of the polynucleotide according to any one of claims 2-3, characterized in that it is used as a primer for nucleic acid amplification reactions, or as a probe for hybridization reactions, or for making genes chip or microarray. 8. A kit for detecting Streptococcus pneumoniae, characterized in that it contains primers for specifically detecting the autolysin of Streptococcus pneumoniae, the antibody of claim 4, any one of the polypeptides of claim 1 or a combination thereof. 9. A vaccine for preventing Streptococcus pneumoniae, characterized in that it contains the polypeptide according to claim 1 or the eukaryotic expression vector containing the polynucleotide according to claim 2 or 3. 10. A pharmaceutical composition for treating Streptococcus pneumoniae pneumonia, which is characterized by containing the inhibitor of Streptococcus pneumoniae autolyase selected by the polypeptide of claim 1.