WO2013040840A1 - 预防、诊断、治疗、预后全身性细菌感染疾病的方法和试剂 - Google Patents

预防、诊断、治疗、预后全身性细菌感染疾病的方法和试剂 Download PDF

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WO2013040840A1
WO2013040840A1 PCT/CN2011/084641 CN2011084641W WO2013040840A1 WO 2013040840 A1 WO2013040840 A1 WO 2013040840A1 CN 2011084641 W CN2011084641 W CN 2011084641W WO 2013040840 A1 WO2013040840 A1 WO 2013040840A1
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crispld2
lccl
raaih
domain
protein
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PCT/CN2011/084641
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English (en)
French (fr)
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王志勤
江宏铨
张新
霍夫曼•M.•K.
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上海人类基因组研究中心
上海南方基因科技有限公司
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Publication of WO2013040840A1 publication Critical patent/WO2013040840A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the present invention belongs to the fields of biotechnology and medicine, and in particular, the present invention provides a systemic bacterial infection and a protein associated with sepsis and septic shock-CRISPLD2 protein, and its use in sepsis and infectivity
  • the sepsis caused by bacterial infection is still an acute syndrome that endangers human health. Sepsis is currently the 10th leading cause of death in the United States. The incidence and mortality of sepsis in China is not optimistic. . There are at least 18 million sepsis cases per year worldwide, accounting for 0.3% of the world's total population, and our country estimates an estimated 3 million cases per year. Conservative predictions of severe sepsis and septic shock in our country were 1.74 million patients.
  • the main cause of sepsis caused by Gram-negative and positive bacteria is the infiltration of bacteria or bacterial toxins into the bloodstream. After the invasion of pathogens, healthy people usually only show transient bacteremia. The bacteria can be quickly eliminated by the body's immune defense system, and it does not cause obvious symptoms.
  • a single microbial infection means that an isolated bacterial organism can be detected in the culture of microorganisms; a plurality of bacterial infections means that more than one bacterial body, that is, a plurality of bacterial bodies, can be detected in the culture of microorganisms.
  • sepsis caused by multiple bacterial infections is associated with a higher risk of complications, a longer course of disease, and a higher mortality rate.
  • LPS lipopolysaccharide
  • DNA bacterial nucleic acid
  • PPN glycopeptide
  • LTA phospholipidic acid
  • CRISPLD2 cysteine-enriched secreted protein 2
  • R3H LCCL-RAAIH
  • the medicament is for:
  • LPS lipopolysaccharide
  • LPS lipopolysaccharide
  • the medicament is for controlling a series of pathological reactions induced by lipopolysaccharide (LPS) from a source, and modifying the immunoregulatory function of single-stranded DNA.
  • LPS lipopolysaccharide
  • cysteine-enriched secreted protein 2 comprising the LCCL-RAAIH (R3H) domain is prevented, mediated by binding to bacterial single-stranded DNA (ssDNA) and/or lipopolysaccharide (LPS). Or treatment of sepsis, severe sepsis or septic shock.
  • ssDNA single-stranded DNA
  • LPS lipopolysaccharide
  • cysteine-enriched secreted protein 2 comprising the LCCL-RAAIH (R3H) domain or a gene encoding the same for the diagnosis or prognosis of sepsis, severe sepsis A reagent or kit for blood or septic shock.
  • the sepsis, severe sepsis, and septic shock are sepsis-negative bacteria and sepsis caused by various microbial infections, severe sepsis, and septic shock.
  • the bacterial infection is a Gram-negative bacterium and a plurality of microbial infections, and may comprise a Gram-positive bacterium.
  • the bacterial infection is a mixed bacterial infection.
  • the mixed bacterial infection and the plurality of microbial infections include endogenous Gram-negative and positive bacterial mixed infections or exogenous Gram-negative infections.
  • the bacterium is selected from, but not limited to, Escherichia coli, Staphylococcus, Salmonella, Klebsiella, Acinetobacter baumannii.
  • a reagent for specifically recognizing a cysteine-enriched secreted protein 2 or a gene encoding the same comprising a LCCL-RAAIH (R3H) domain is provided for the preparation of a diagnostic or prognostic bacterial infection pus Kit for toxemia, severe sepsis, and septic shock.
  • said specifically recognizing cysteine enrichment comprising a LCCL-RAAIH (R3H) domain
  • the agent for secreting protein 2 or its encoding gene is selected from (but not limited to)
  • An antibody or ligand that specifically binds to a cysteine-enriched secreted protein 2 comprising the LCCL-RAAIH (R3H) domain comprising the LCCL-RAAIH (R3H) domain.
  • the reagent for specifically recognizing a cysteine-rich secreted protein 2 comprising the LCCL-RAAIH (R3H) domain is a cysteine resistant to the LCCL-RAAIH (R3H) domain.
  • kits for the diagnosis or prognosis of sepsis, severe sepsis, and septic shock which comprises:
  • a container for specifically recognizing a cysteine-rich secreted protein 2 or a gene encoding the same, comprising a LCCL-RAAIH (R3H) domain; preferably, a reagent for detecting a serum concentration of CRISPLD2 , such as polyclonal antibodies.
  • a reagent for detecting a serum concentration of CRISPLD2 such as polyclonal antibodies.
  • a method for preventing or treating a systemic bacterial infection or a bacterial infection-related disease comprising the steps of: increasing cysteine enrichment of a LCCL domain in a serum of a mammalian subject in need thereof The concentration of secreted protein 2.
  • a method for preventing, ameliorating or treating a bacterial infection or a bacterial infection-related disease comprising: administering a cysteine enrichment comprising a LCCL-RAAIH (R3H) domain to a bacterial infected person Secreted protein 2.
  • Figure 1 shows the standard curve for ELISA detection.
  • FIG. 2 shows the results of the identification of recombinant CRISPLD2. among them,
  • the lanes are as follows: 1, 4 and 6 are CRISPLD2 in the medium; 2 is the molecular weight standard; 3 and 5 are the control medium.
  • E. col i LPS E. coli-LPS
  • CRISPLD2 CRISPLD2
  • C E-coli glycolipid (PGN) and CRISPLD2 combined dissociation sensorgram.
  • PPN E-coli glycolipid
  • CRISPLD2 combined dissociation sensorgram.
  • D Synthetic bacterial single-stranded GPC-DNA CODN2006) Sensing map of dissociation with CRISPLD2.
  • FIG. 4 Time kinetics of serum CRISPLD2 concentration after cecal ligation and perforation-induced mixed bacterial infection with sepsis and low-dose LPS intraperitoneal injection.
  • A is the change of serum CRISPLD2 concentration in rats after cecal ligation and perforation induced low and moderate mixed bacterial infection.
  • B is the change of serum CRISPLD2 concentration in mice after intraperitoneal injection of low dose LPS.
  • Recombinant CRISPLD2 blocks the binding of LPS to target cell receptors and inhibits the release of inflammatory factors from target cells.
  • A-B recombinant CERISPLD2 protein blocks E. coli LPS and Salmonella (S. minnesota; S.m) LPS binding to target cell receptors.
  • MFI is the median of fluorescence intensity.
  • C-D Recombinant CERISPLD2 protein reduces E. coli LPS-induced inflammatory factor release.
  • A recombinant human CRISPLD2 protects mice from endotoxin shock.
  • CRISPLD2 serum concentration was positively correlated with LPS-induced endotoxin shock survival (mouse peritoneal (i.p.) injection of sublethal dose of E. coli LPS 0.2mg).
  • CRISPLD2 The normal distribution curve of CRISPLD2 in mouse serum is shown, which indicates the relationship between the susceptibility change of endotoxin shock and the level of CRISPLD2.
  • the threshold value of CRISPLD2 associated with endotoxin shock susceptibility is determined as the mean value of normal serum CRISPLD2 concentration.
  • the ordinate is the serum CRISPLD2 level; the horizontal and vertical coordinates are the survival rate or the mouse count.
  • Figure 10 Intravenous or endogenous activation of recombinant CRISPLD2 protein up-regulates serum CRISPLD2 levels to protect mice infected with concentrated bacteria from toxic shock.
  • Figure 1 According to the CRISPLD2 level threshold and the PCT level threshold (2 ⁇ g/ml), sepsis patients can be divided into three subtypes (A, B, C), of which CRISPLD2-low/PCT-high mortality Correlation (A); while the other two subtypes were associated with low mortality (B, C) 0 normal population serum CRISPLD2 levels and normal distribution of PCT levels (D). Correlation between subtypes and patient mortality in serum of patients with sepsis in different severity (E).
  • CRISPLD2-High/PCT-High In consecutive patient sera, CRISPLD2 levels remain above their threshold when PCT levels are above the PCT threshold.
  • a natural immunoregulatory protein _ _ cysteine-rich secretory prote in LCCL containing the LCCL-RAAIH (R3H) domain cyste ine-rich secretory prote in LCCL) Domain containing 2, CRISPLD2
  • R3H LCCL-RAAIH
  • CRISPLD2 cyste ine-rich secretory prote in LCCL
  • CRISPLD2 protein as a target molecule, serum CRISPLD2 concentration can be detected in individuals, and the sensitivity of individuals to bacterial infections (such as toxic shock) can be predicted, and severe sepsis caused by bacterial infection of serum level of CRISPLD2 can be traced and concentrated. Toxic shock.
  • CRISPLD2 protein by increasing the concentration of CRISPLD2 protein in serum, it is possible to prevent and treat bacterial infections causing severe sepsis and toxic shock. The present invention has been completed on this basis.
  • a cDNA sequence for CRISPLD2 can be found in accession number NM_031476 (length 4607 bp) or SEQ ID NO: 1;
  • the genomic sequence can be found in accession number NC_000016.
  • the amino acid sequence can be found in the sequence number P_1 13664 or SEQ ID NO: 2.
  • CRISPLD2 protein CRISPLD2 polypeptide
  • CRISPLD2 polypeptide Cysteine-enriched secreted protein 2 containing LCCL-RAAIH (R3H) domain
  • human severe sepsis and toxic shock The related protein CRISPLD2” is used interchangeably and refers to a protein or polypeptide having a human sepsis and a nucleic acid sequence of the toxic shock-associated protein CRISPLD2. They include the CRISPLD2 protein with or without the starting methionine.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state of living cells are not isolated and pure.
  • the same polynucleotide or polypeptide, if separated from other substances present in the natural state, is isolated and purified.
  • isolated CRISPLD2 protein or polypeptide means that the CRISPLD2 polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art will be able to purify the CRISPLD2 protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, either a chemically synthesized product, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • the invention also encompasses fragments, derivatives and analogs of the CRISPLD2 protein.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the native CRISPLD2 protein of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (eg, a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or A fusion protein for the formation of an antigenic IgG fragment).
  • conservative amino acid residues preferably conservative amino acid residues
  • CRISPLD2 protein refers to a polypeptide having the sequence of SEQ ID NO: 2 of CRISPLD2 protein activity.
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 2 which have the same function as the CRISPLD2 protein. These variants include (but are not limited to;): a number of amino acid deletions (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10), Insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • a number of amino acid deletions usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10
  • Insertion and/or substitution and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the CRISPLD2 protein.
  • these variants include the LCCL-RAAIH (R3H) domain of the CRISPLD2 protein.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to CRISPLD2 DNA under high or low stringency conditions, And a polypeptide or protein obtained using an antiserum against the CRISPLD2 polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising a CRISPLD2 polypeptide or a fragment thereof.
  • the present invention also encompasses soluble fragments of the CRISPLD2 polypeptide.
  • these variants include the LCCL-RAAIH of the CRISPLD2 protein. (R3H) domain.
  • the invention also provides analogs of the CRISPLD2 protein or polypeptide.
  • the difference between these analogs and the native CRISPLD2 polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known in molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, guanidino-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above. Typically, these analogs include the CRISPLD2 protein.
  • Modifications include: chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to enhance their anti-proteolytic properties or to optimize solubility properties.
  • chemically derived forms of the polypeptide such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptid
  • CRISPLD2 protein conservative variant polypeptide means up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of SEQ ID NO: 2. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the gene (polynucleotide) encoding the CRISPLD2 protein may be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either a coding strand or a non-coding strand.
  • polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the full-length nucleotide sequence of CRISPLD2 of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method of amplifying DNA/RNA using a PCR technique is preferably used to obtain the gene of the present invention.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art.
  • Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method; centrifugation, osmotic bacteriolysis, super treatment, ultracentrifugation, molecular sieve chromatography (condensation) Glue filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • a protein precipitant salting method; centrifugation, osmotic bacteriolysis, super treatment, ultracentrifugation, molecular sieve chromatography (condensation) Glue filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, which are specific for a polypeptide encoded by CRISPLD2 DNA or a fragment thereof.
  • the invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; Or a chimeric antibody, such as an antibody that has murine antibody binding specificity but still retains an antibody portion from a human.
  • immunologically active antibody fragments such as Fab' or (Fab) 2 fragments
  • antibody heavy chains such as antibody light chains; genetically engineered single chain Fv molecules;
  • a chimeric antibody such as an antibody that has murine antibody binding specificity but still retains an antibody portion from a human.
  • Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, a purified CRISPLD2 gene product or a fragment thereof having antigenicity can be administered to an animal to induce production of a polyclonal antibody. Similarly, cells expressing the CRISPLD2 protein or its antigenic fragment can be used to immunize an animal to produce antibodies.
  • the antibody of the invention may also be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology.
  • Antibodies against the CRISPLD2 protein can be used in immunohistochemistry to detect CRISPLD2 protein in biopsy specimens (especially in serum samples).
  • monoclonal antibodies that bind to the CRISPLD2 protein can also be labeled with a radioisotope that can be injected into the body to track its location and distribution.
  • polyclonal antibodies can be used to immunize animals such as rabbits, mice, rats, etc. with CRISPLD2 protein or polypeptide.
  • CRISPLD2 protein or polypeptide A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • substances which interact with the CRISPLD2 protein such as receptors, inhibitors, agonists or antagonists, can be screened by various conventional screening methods.
  • the agonist of CRISPLD2 includes a stabilizer, an accelerator, an upregulator, and the like.
  • Any substance which can increase the activity of CRISPLD2 protein, maintain the stability of CRISPLD2 protein, promote the expression of CRISPLD2 protein, prolong the effective action time of AC CRISPLD2hE protein, promote transcription and translation of CRISPLD2 gene, and the like, can be used in the present invention as a prophylaxis, relief Or a substance that treats a bacterial infection or a disease associated with a bacterial infection.
  • the CRISPLD2 protein and its agonist (also called agonist) and the like can prevent or treat sepsis and septic shock when administered (administered) therapeutically.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • the polypeptide of the present invention can be directly used for the treatment and prevention of bacterial infection or bacterial infection-related diseases, especially for the prevention and treatment of severe sepsis and toxic shock.
  • CRISPLD2 protein of the present invention is used, other therapeutic agents for the same condition can be used at the same time.
  • the present invention also provides a composition (including a pharmaceutical composition) which contains a safe and effective amount (eg, 0.001-99. 9 wt% ⁇
  • a safe and effective amount eg, 0.001-99. 9 wt% ⁇
  • the CRISPLD2 polypeptide of the invention or an agonist thereof and a (pharmaceutically acceptable carrier or excipient).
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • the invention also relates to a diagnostic test method for the quantitative detection of CRISPLD2 protein levels.
  • assays are well known in the art and include ELISA, FISH assays, and radioimmunoassays. Therefore, after understanding the relationship between the protein level of CRISPLD2 and disease, it is easy for people in the field to diagnose or prognose a disease associated with bacterial infection or bacterial infection.
  • the level of CRISPLD2 protein detected in the assay can be used to explain the importance of the CRISPLD2 protein in regulating bacterial antigen responses and the susceptibility to disease diagnosis.
  • One method for detecting the presence of CRISPLD2 protein in a sample is to recognize the CRISPLD2 protein using a specific antibody.
  • the main applications of the invention include:
  • CRISPLD2 protein as a target molecule, detecting the serum CRISPLD2 concentration in an individual, predicting the sensitivity of the individual to sepsis and toxic shock, providing a basis for clinicians, and may cause sudden sepsis and shock.
  • the patient takes the necessary preventative treatment.
  • a threshold can be determined based on clinical experience to make a warning of the disease when the CRISPLD2 protein is below this threshold.
  • the recombinant CRISPLD2 protein was used for intervention in patients with severe sepsis.
  • the invention also provides a kit for monitoring serum concentration of CRISPLD2, comprising a container, and a cysteine-rich secreted protein 2 (CRISPLD2) in the container for specifically recognizing a LCCL-RAAIH (R3H) domain or
  • the reagent encoding the gene preferably, an agent for detecting the serum concentration of CRISPLD2, such as a polyclonal antibody.
  • the reagents in the kit can be used to track the concentration of the innate immune regulatory molecule CRISPLD2 in serum samples to assess the patient's natural immune regulation homeostasis. This imbalance in homeostasis directly reflects disease severity and possible shock mortality.
  • RAAIH R3H sequences, which bind ssDNA ( SEQ ID NO: 2 sequence, SEQ ID NO: NP_113664, number of amino acid residues 497 aa):
  • Primer 1 (amplification of cDNA corresponding to l-497aa of CRISPLD2 protein):
  • F1 5'- GCTGTCGCCGCTGCTACCGC (SEQ ID NO: 3);
  • Rl 5'-GACGCCCCTTCTCCCCTGGT (SEQ ID NO: 4).
  • the PCR product was inserted into the plasmid pGEM-T (Promega) as a template for expression vector construction.
  • the cDNA encoding full-length CRISPLD2 was inserted into the multiple cloning site of pcDNA3.1A (InviyroGen) plasmid, and then transferred into CHO cells (ATCC) to express recombinant protein with c-myc tag and 6 His recombinant CRISPLD2 protein.
  • Primer 2 (amplification of cDNA corresponding to CRISPLD2 protein 257-497aa):
  • F2 5'-TGGAATTCCGAGAAGAAACCTACACTC (SEQ ID NO: 5)
  • R2 5'- TGCTCGAGATTCACTGCCTGACAGCA (SEQ ID NO: 6)
  • the PCR product was inserted into the plasmid pGEM-T CPromega) as a template for expression vector construction.
  • the cDNA encoding the CRISPLD2 protein fragment (SEQ ID NO: 2, 257-497aa) was inserted into the multiple cloning site of plasmid pGEX-4T-1 (GE Healthcare) and transferred into CHO cells (ATCC) to obtain recombinantly expressed GST- CRISPLD2 fusion protein. This fusion protein is used to immunize rabbits and obtain antisera.
  • Anti-CRISPLD2 polyclonal antibody (anti-CRISPLD2 IgG) was purified from antisera using Protein G Agarose.
  • the plasmid was transfected into CHO cells using Lipofectamine (Invitrogen;), and then transfected positive cells were selected using antibiotic G418.
  • CHO cells stably expressing human CRISPLD2 were cultured while maintaining G418, and further segregation was used to selectively isolate and select highly expressed CHO cell clones.
  • CHO cells expressing recombinant CRISPLD2 protein were cultured in serum-free medium. The supernatant was collected to contain a high concentration of recombinant CRISPLD2 protein. The recombinant protein in the culture solution is purified by affinity chromatography.
  • Partial phosphate buffer dialysis (containing recombinant protein), SDS-PAGE electrophoresis, Coomassie blue staining SDS-PAGE gel, purity, and BCA assay kit protein quantification
  • the culture medium or serum was appropriately diluted with sodium carbonate buffer (pH 9.5), and coated in a 96-well plate (Nunc MaxiSorp, Denmark) at 4 ° C overnight.
  • the 96-well plate was washed twice with phosphate buffer, incubated with phosphate buffer containing 0.4% casein for 1 hour, and then 8% (v/v) fetal bovine serum and anti-CRISPLD2 polyclonal antibody (10 ug / ml). Incubate in phosphate buffer for 2 hours, wash 4 times with phosphate buffer, add horseradish peroxidase-conjugated secondary antibody, and wash 4 times with phosphate buffer.
  • Protein samples were diluted in loading buffer, separated by 10% SDS-PAGE gel, and protein purity was determined by Coomassie blue staining.
  • Western blotting SDS-PAGE The protein on the electrophoresis gel was transferred to a nitrocellulose membrane (GE Healthcare). The nitrocellulose membrane was incubated with the buffer containing the primary antibody for 2 hours at 37 ° C, washed, and the fluorescein-labeled secondary antibody. (ROCKLAND) Incubate for 1 hour at 37 °C. ODYSSEY infrared image system
  • Example 3 bacterial binding ability of CRISLD2 Binding of bacterial antigen to CRISPLD2 was quantified by Surface plasmon resonance (SPR) using a BIAcore TlOO biosensing detector.
  • SPR Surface plasmon resonance
  • the bacterial antigens are as follows:
  • E. coli LPS purchased from Sigma;
  • Staphylococcus aureus LTA purchased from InvivoGen;
  • E-coli glycolipid purchased from InvivoGen;
  • Synthetic single-stranded GPC-DNA (ODN2006): purchased from InvivoGen, the sequence is: 5 '- tcg tcg ttt tgt cgt ttt gtc gtt -3 ' (24 mer);
  • Synthetic single-stranded CPG-DNA (ODN2006(Ctr)) : purchased from InvivoGen, or its sequence is: 5 '- tgc tgc ttt tgt get ttt gtg ctt -3 ' (24 mer);
  • Synthetic double-stranded RNA (PolyI: C): purchased from InvivoGen.
  • Recombinant CRISPLD2 protein and IgG (0.18 mg/ml dissolved in acetic acid buffer, pH 4.5) were coupled to two channels of a sensor chip (GE Healthcare) using reagents supplied by the instrument company. Different concentrations (concentrations: 0, 0.3, 1.0, 3.0, 10 and 30 ⁇ ) Bacterial antigens were dissolved in phosphate buffer and measured for injection while flowing through two channels of the sensor chip (20 ⁇ /min) .
  • the binding reaction unit (RU) of the bacterial antigen obtained from the recombinant CRISPLD2 protein channel was subtracted from the IgG channel signal (RU); the unbound bacterial antigen was washed away in phosphate buffered saline (PBS); then the bound bacterial antigen was washed away with 50 mM NaOH. , Regenerate the chip.
  • PBS phosphate buffered saline
  • K D can be calculated by k. Ff /k. n The ratio of the two constants is obtained.
  • RU maximum binding reaction unit
  • CRISLD2 binds to ssDNA and LPS, but does not bind to S. aureus LTA, synthetic E. coli PGN or synthetic double-stranded RNA (CdsRNA).
  • Example 4 Threshold levels of CRISPLD2 in blood samples and individual susceptibility to endotoxin shock. Serum and blood plasma CRISPLD2 concentrations (100 normal adults, 5 neonates) were determined as follows: For 100 normal newborns with serum and blood plasma, 5 newborns were cord blood, and their CRISPLD2 concentrations were analyzed by immunoblotting.
  • the concentration of serum and plasma CRISPLD2 was calculated and quantified by the ODYSSEY infrared image scanning analysis system.
  • Semi-quantitative immunoblotting was compared with competitive-ELISA (Table 2) and the results were consistent.
  • Table 2 Competitive-ELISA detection of 100 normal healthy human serum and 5 newborn cord blood.
  • CLP cecal ligation and perforation
  • balb/c mice Three groups of balb/c mice were intraperitoneally injected with 400 ⁇ g of E. coli-LPS (with PBS as solvent). At the same time, the normal control group was intraperitoneally injected with PBS (21 mice), and the experimental group was intraperitoneally injected with 1 mg of recombinant CRISPLD2 (17 Mouse), the experimental control group was intraperitoneally injected with 1 mg of recombinant CRISP3 (7 mice) (the protein belongs to the CRISP family but lacks LPS) Combined area). Survival rates are counted and plotted.
  • recombinant human CRISPLD2 protected mice from endotoxin shock.
  • mice (20 mice) were intraperitoneally injected with a non-toxic dose of E. coli-LPS (30 ⁇ g/mouse). On the tenth day, blood was drawn from the tail vein with 4 normal mice. CRISPLD2 was tested by ELISA, and 6 hours later. All mice were randomized intraperitoneally with different lethal doses of E. coli-LPS. On the thirty-second day, the surviving mice were again bleeding from the tail vein, serum 1 :
  • CRISPLD2 protein is a target molecule that predicts Gram-negative bacterial sepsis and individual sensitivity to endotoxin.
  • Example 8 long-term treatment with broad-spectrum antibiotics caused a decrease in serum CRISPLD2 concentration in mice
  • Vancomycin 1 mg / ml + neomycin (Neo) 0.5 mg / ml dissolved in drinking water to feed mice
  • mice killed the symbiotic bacteria in the body. At different time points, the mice were bled in the tail vein, serum 1:1000 dilution, and CRISPLD2 was quantitatively detected by ELISA. As a result, as shown in Fig. 7A, long-term use of vancomycin plus neomycin resulted in a decrease in serum CRISPLD2 concentration in mice.
  • mice The normal control group of normal drinking water, the 4-day group and the 15-day group containing broad-spectrum antibiotics in drinking water, the three groups of mice were intraperitoneally injected with 200 ⁇ g E.coli-LPS. Normal mice will have a 70-80% mortality rate by intraperitoneal injection of 400 micrograms of E. coli-LPS. Inhalation of 200 micrograms of E. coli-LPS in mice given broad-spectrum antibiotics for 15 consecutive days compared with normal controls. The rate was significantly increased, whereas mice taking broad-spectrum antibiotics for 4 consecutive days did not change significantly compared to normal controls. Statistical methods and statistics have been plotted on the map. The results are shown in Figure 7B, indicating that long-term use of vancomycin plus neomycin increased the sensitivity of mice to endotoxin shock (32 mice).
  • Example 9 Serum concentration of CRISPLD2 for predicting susceptibility to toxic shock
  • Figure 8A shows three experimental results similar to Figure 7, combined data of 42 mice, and analyzed the correlation between serum CRISPLD2 levels and endotoxin shock survival to determine the serum CRISPLD2 concentration threshold.
  • the survival rate of E. coli-LPS sublethal dose was significantly decreased in mice with serum CRISPLD2 concentrations below the mean; in contrast, mice with serum CRISPLD2 concentrations above the mean were tolerant to E. coli. - LPS sublethal dose (200 ⁇ g/mouse), a significant increase in survival.
  • the mean serum CRISPLD2 concentration can be used as a threshold for susceptibility to toxic shock.
  • FIG. 8C is based on the results of the above experimental analysis, the clinical relevance of sepsis will apply this threshold: normal serum
  • the mean concentration of CRISPLD2 is used as a threshold for individual susceptibility to endotoxic shock.
  • Example 10 intravenous injection of recombinant CRISPLD2, or pre-injection of low-dose LPS, to improve serum CRISPLD2 levels to protect moderate cecal ligation and perforation (CLP-Moderate) mice with mixed bacterial infection
  • Figure 9 shows three different severity CLP-induced mixed bacterial infections in a sepsis model applied to the study (mild, moderate, severe sepsis rats, respectively, Figures, B, and C;).
  • Serum Crispld2 levels were inversely correlated with PCT levels.
  • Crispld2 is up-regulated with low PCT concentrations, with a relative mortality rate of 20%; in moderate sepsis, Crispld is up-regulated with an increase in PCT, with a 50% mortality rate; in severe sepsis, low Crispld2 with high PCT , the relevant mortality rate is 100%.
  • FIGS 9A and 9B show that serum Crispld2 levels are significantly up-regulated in mild to moderate sepsis and not up-regulated in severe sepsis (C;).
  • Recombinant CRISPLD2 was injected intravenously to increase serum CRISPLD2 levels by 25%, and moderate cecal ligation and perforation (CLP-moderate) mice with mixed bacterial infection (survival rate increased from 40% to 73%), as shown in Figures 10A and B; Low-dose LPSC50 ⁇ g/mouse) After 8 days of pre-injection, serum CRISPLD2 levels were increased by 67%, and moderate cecal ligation and puncture (CLP-moderate) mice with mixed bacterial infection were well protected, as shown in Figure 10C-D. . This protective capacity shown in Figure 10 correlates with serum CRISPLD2 levels.
  • the murine CLP sepsis model simulates acute mixed bacterial infections, with pathological principles and clinical symptoms approaching human acute bacterial infections with sepsis.
  • the ligation of different parts of the animal cecum, the size of the cecal penetrating needle and the number of cecal perforations determine the severity of sepsis in the mixed bacterial infection of the animal.
  • the CRISPLD2 concentration was found to be able to prognose severe sepsis and septic shock caused by systemic bacterial infection by tracking CRISPLD2 serum levels. It has also been shown that increasing the concentration of CRISPLD2 protein in serum can prevent and treat severe sepsis and toxic shock caused by systemic bacterial infection. Of the 78 patients, 30 patients with local bacterial infections and non-infectious diseases were also investigated. CRISPLD2 water in the serum of patients with local bacterial infections. Flat detection can be used to assess the ability of CRISPLD2 to regulate and control immune responses. The changes of serum CRISPLD2 levels in patients with toxic shock were completely different from those in patients with hemorrhagic shock.
  • CRISPLD2 concentration of CRISPLD2 in the patient's body fluid (serum;) was analyzed.
  • PCT body fluid
  • serum samples with low PCT/CRISPLD2 levels appear in consecutive serum samples to reflect the severity and mortality of patients with sepsis.
  • Serum CRISPLD2 levels are indicated for the diagnosis and prognosis of patients with severe sepsis and septic shock, as well as for the assessment of CRISPLD2 in immune response in patients with mild bacterial infections.
  • PATIENTS 48 patients with severe acute trauma and other causes of gram-negative and positive mixed bacterial infections, and critically ill patients entered the ICU ward. The diagnosis of sepsis is based on the international ACCP/SOOM standard.
  • Serum CRISPLD2 and sepsis marker molecular detection The average test was performed once/four days, and the serum CRISPLD2 and serum procalcitonin (PCT, Procalcitonie) concentrations were observed continuously for 28 days.
  • Threshold definition The mean value of serum CRISPLD2 level is 220 ⁇ g/ml, which is considered to be lower than CRISPLD2, and is considered to be high in CRISPLD2; procalcitonin (PCT) sepsis serum PCT threshold is 2.0 Ng/ml, normal value is 0.01-0.03 ng/ml, up to hundreds ng/ml. In other infectious diseases, serum PCT is generally below the 2.0 ng/ml level.
  • PCT procalcitonin
  • CRISPLD2 levels and PCT levels can be defined as the following three categories:
  • CRISPLD2-High, Low/PCT-Low In consecutive patient sera, CRISPLD2 levels fluctuate across its threshold, while PCT levels are consistently below the PCT threshold.
  • CRISPLD2-High/PCT-High In consecutive patient sera, CRISPLD2 levels remain above their threshold when PCT levels are above the PCT threshold.
  • CRISPLD2-low/PCT-high PCT high and continuous PCT serum, when PCT levels are above the PCT threshold, PCT is high
  • CRISPLD2 is low (less than 222 ⁇ g / ml) at the same time one or several times.
  • Patient clinical severity index Clinical composite index APACHEII; Acute Physiology and Chronic Health Evaluation II Acute physiology and chronic health status score.
  • FIG 11 shows that sepsis patients can be divided into three subtypes (A, B, C), of which more than 50% of patients are
  • FIG. 11E and 11F show: severity of the APACHE II index 13-22 In septic patients, 35% of patients had CRISPLD-high/PCT-high characteristics, and patients with this characteristic had low mortality. This CRISPLD-high/PCT-high appearance may only reflect CRISPLD2 regulatory host in patients. The immune response reduces the damaging effects of excessive inflammatory reactions.
  • Figure 10 shows the results of experiments in animals that up-regulated CRISPLD2 serum levels to protect systemic bacterial infections.
  • Figures 11B and 11C show that low mortality (3-7%) occurs in patients with partial sepsis with CRISPLD2-high, low/PCT-low characteristics, and CRISPLD-high/PCT-high serum characteristics, for reasons not yet clear.
  • This part of the patient may cover a part of Gram-positive bacteria infected with sepsis because of patients with gram-positive bacterial sepsis PCT serum levels (range 0.1-8.9 ng/ml) span the 2 ng/ml PCT limit in serum, while molecules of the gram-positive bacteria such as ssDNA, deRNA, PGN can upregulate CRISPLD2 production in immune cells. And secretion.
  • Figures 11E and 11F also show that in patients with low severity (APACHE II index less than 10), the presence of CRISPLD2-low/PCT-high serum samples was not associated with mortality (A). Serum in patients with mild bacterial infections
  • CRISPLD2 and PCT levels can be used to assess the extent to which CRISPLD2 regulates immune responses and host immune responses, and is useful for diagnosis and prevention of disease progression.
  • patients with mild systemic bacterial infections may have a sharp drop in serum CRISPLD2 levels. According to the patient's condition, it is possible to judge the possible changes that may occur, prompting the doctor to take necessary measures to prevent the disease from worsening.
  • patients with non-sepsis include: urinary tract infections, bronchitis, acute cholangitis, traumatic pancreatitis, and gastrointestinal bleeding and hemorrhagic shock.
  • serum CRISPLD2 levels were upregulated/PCT levels below 3 ng/ml.
  • Hemorrhagic shock was completely different from systemic bacterial septic shock.
  • serum CRISPLD2 was up-regulated rather than decreased, and PCT levels were lower than 2 ng/ml (mortality 20%).
  • the secreted protein CRISPLD2 is a target molecule for predicting severe sepsis and septic shock sensitivity.
  • the present invention shows that the highly conserved cysteine-rich secretory protein CRISPLD2 (Cysteine - rich secretory protein LCCL domain containing 2) Is a high serum concentration of ssDNA and P LPS binding protein, which possesses a RAAIH (R3H) (RAAIH) domain that binds to ssDNA; two LCCL domains that bind to LPS and have high affinity with ssDNA and LPS combination.
  • RAAIH RAAIH
  • CRISPLD2 Unlike the known emergency protein, rat intestinal perforation mixed bacterial infection and the second day (acute phase) of intraperitoneal injection of non-toxic dose of LPS in mice, serum CRISPLD2 concentration slowly increased, peaking on the fifth day. In contrast, preliminary experimental data showed that CRISPLD2 concentration in the exudate of the infected site of the intestinal perforated mixed bacterial infection increased rapidly within a few hours after infection. Statistical analysis found that the serum concentration of this protein was positively correlated with the lethal dose of endotoxin. CRISPLD2 protects mixed-bacterial-infected animals from their serum concentrations, suggesting that CRISPLD2 is a prognostic septic shock, a target for the treatment of severe sepsis and septic shock.
  • CRISPLD2 protein can protect mice and increase the survival rate of septic shock, which can prevent the high expression of septic shock in human, mouse and rat heart, lung, small intestine, placenta and granulocytes and monocytes.
  • CRISPLD2 In in vitro experiments, the inventors also found that most white blood cells (including granulocytes, monocytes) naturally release the ssDNA and LPS binding proteins.
  • the protein can block the binding of LPS to the receptor in the normal physiological concentration range, and inhibit the release of LPS-induced inflammatory factors, including tumor necrosis factor TNF- a release; the recombinant protein can protect the mouse by intraperitoneal injection, and greatly reduce endotoxin shock.
  • Mortality, intravenous injection of the recombinant protein reduces the mortality of mixed bacterial infections, exogenous or endogenously up-regulating the serum level of the protein 24-76% resulting in a significant reduction in mixed bacterial infection mortality.
  • CRISPLD2 Exogenous and endogenous up-regulation of serum CRISPLD2 levels, protection of mixed bacterial infection in CLP mice suggests: 1. In mixed bacterial infection, endotoxin (LPS) is the main cause of several known bacterial antigens causing septic shock One of the antigens; 2. CRISPLD2 is a key serum regulatory protein in the pathological response of LPS. 3. Reactions caused by bacterial antigens, which may include abnormalities in the coagulation system, excessive immune response, tissue and organ failure, and other pathological reactions of blood stasis; the regulatory mechanism of CRISPLD2 protein may occur before the pathogen molecule binds to its cellular receptor.
  • sepsis patients can be divided into three subtypes, of which 50-60% of patients are CRISPLD2-low/PCT-high, associated with high mortality; and CRISPLD2-high/PCT - High subtype and CRISPLD2-high-low/PCT-low associated with low mortality.
  • CRISPLD2-low/PCT-high associated with high mortality
  • CRISPLD2-high/PCT - High subtype and CRISPLD2-high-low/PCT-low associated with low mortality.
  • a serum sample with a low concentration of CRISPLD2/high PCT concentration may indicate the severity and prognosis of the patient.
  • Serum CRISPLD2 levels are indicated for the diagnosis and prognosis of patients with severe sepsis and septic shock.
  • CRISPLD2 regulates the body's immune response and the extent of host inflammatory response.
  • the presence of CRISPLD2-low/PCT-high in serum can prompt the physician to take action and predict future changes in the patient.
  • Recombinant CRISPLD2 can be used to treat and protect patients with severe sepsis from reducing mortality.

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Abstract

本发明公开了预防、诊断、治疗和预后细菌感染相关疾病的方法和试剂。具体而言，本发明公开了含有LCCL-RAAIH（R3H）结构域的半胱氨酸富集的分泌蛋白2或其编码的基因在制备预防、治疗全身性细菌感染相关疾病的药物中的用途，本发明还公开了特异性识别所述蛋白或基因的试剂在制备诊断或预后相关疾病的试剂盒中的用途。

Description

预防、 诊断、 治疗、 预后全身性细菌感染疾病的方法和试剂 技术领域
本发明属于生物技术和医学领域， 具体地说， 本发明提供了一种全身性细菌感染以 及其引起的脓毒症和感染性休克相关的蛋白 -CRISPLD2蛋白，及其在脓毒症和感染性休 克诊断分型、 预后和治疗中的用途， 通过检测血清 CRISPLD2蛋白浓度对严重脓毒症进 行分型和预后， 通过提高血清 CRISPLD2蛋白浓度， 可以预防、 治疗严重脓毒症和感染 性休克。 背景技术
细菌感染引起的脓毒血症至今仍然是危害人类健康的急性综合症， 脓毒血症目前是 美国排位第 10的主要死亡原因， 在中国脓毒血症的发病率和死亡率也不容乐观。 全球 每年至少有 1 800万例脓毒症发生， 占世界总人口的 0. 3%， 我国估计为 300万例 /年计 算。 保守预测我国严重脓毒症和脓毒性休克病人为 174万病人。 革兰氏阴性以及阳性细 菌引起的脓毒血症主要原因是由于细菌或细菌毒素侵入血流引起。 健康者在病原菌入侵 后， 一般仅表现为短暂的菌血症， 细菌可被人体的免疫防御系统迅速消灭， 并不引起明 显症状； 但各种免疫防御功能缺陷者 (包括局部和全身屏障功能的丧失)， 都易诱发脓毒 血症。 放射治疗、 广谱抗菌素、 细胞毒类药物的应用， 以及各种大手术致严重的开放 性创伤等都是脓毒血症的重要诱因。
单一微细菌感染是指微生物培养中可以检出一个孤立的细菌体； 多种细菌感染是指 微生物培养中可以检出超过一个的细菌体， 即多个细菌体。 与单一细菌感染相比较， 多 种细菌感染引起的脓毒血症伴有较高并发症风险， 较长的病程以及较高死亡率。
全身性细菌感染引起的脓毒血症以及浓毒性休克的诊断、 预防治疗工作仍然面临许 多挑战， 监测血流中的微量细菌和细菌毒素都是可行的诊断方法但存在严重缺点， 而且 缺乏统一的标准。
近 20多年， 发现一些可以应用于诊断的分子靶点的人血清分子可以应用于细菌感染 的诊断， 例如 B型尿肽 (B-type natriuretic peptide；)、 降血钙素原 (Procalcitonin；)、 细菌聚脂 多糖结合蛋白 (LBP；)、 细菌渗透性增强蛋白 (BPi;>、 可溶性 CD14 (sCD14;>、 Endocan等。 其中降血钙素原 (Procalcitonin)已被广泛应用于脓毒血症、 严重脓毒血症、 浓毒性休克的 分类以及诊断， 但它们都不能准确、 有效预测脓毒性休克的发生， 以及应用于严重脓毒 症及脓毒性休克预后有待改善。
目前感染免疫学研究认为，引起脓毒血症以及浓毒性休克的病源分子主要是革兰氏 阴性菌的脂多糖 (LPS)、 细菌核酸 (DNA)、 糖肽 (PGN)以及磷脂壁酸 (LTA); 又发现细菌 的单链 DNA(ssDNA)可以保护个体， 降低混合细菌感染的死亡率。 革兰氏阴性菌感染往 往伴随其它细菌的感染。多种抗原的病理协同作用还有待进一步研究，其原理有待揭示。 因此， 本领域迫切需要发现新的病理机制、 开发的新的、 有效地对严重脓毒血症以及浓 毒性休克进行预后、 预防和治疗的方法。 发明内容
本发明的目的在于提供预防、 诊断、 治疗、 预后全身性细菌感染以及多种微生物感染 相关疾病的方法和试剂。
在本发明的第一方面，提供 含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋 白 2 (CRISPLD2)及其编码基因的用途， 用于制备预防、缓解或治疗细菌感染或细菌感染 相关疾病的药物。
在一个优选例中， 所述的药物用于：
阻断脂多糖 (LPS)与靶细胞受体结合； 或
降低脂多糖 (LPS)诱导的炎症因子 （如肿瘤坏死因子） 释放。
在另一优选例中， 所述的药物用于从源头上控制脂多糖 (LPS)诱导的一系列病理反 应， 以及修饰单链 DNA的免疫调控功能。
在另一优选例中， 所述的含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2通过结合细菌单链 DNA(ssDNA)和 /或脂多糖 (LPS) 预防、 缓解或治疗脓毒血症、 严重 脓毒血症或感染性休克。
在本发明的另一方面， 提供含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋 白 2或其编码基因的用途， 用于制备诊断或预后脓毒血症、 严重脓毒血症或感染性休克 的试剂或试剂盒。
在另一优选例中， 所述的脓毒血症、 严重脓毒症以及感染性休克是格兰氏阴性细菌 及多种微生物感染引起的脓毒血症、 严重脓毒症以及感染性休克。
在另一优选例中， 所述的细菌感染是格兰氏阴性细菌及多种微生物感染， 可以包含 格兰氏阳性细菌。
在另一优选例中， 所述的细菌感染是混合细菌感染。
在另一优选例中， 所述的混合细菌感染以及多种微生物感染包括内源性格兰氏阴性 和阳性细菌混合感染或外源性格兰氏阴性感染。
在另一优选例中， 所述的细菌选自 (但不限于)： 大肠杆菌， 葡萄球菌， 沙门氏菌， 克 雷伯氏菌， 鲍曼不动杆菌。
在本发明的另一方面， 提供特异性识别含 LCCL-RAAIH (R3H)结构域的半胱氨酸富 集的分泌蛋白 2或其编码基因的试剂的用途， 用于制备诊断或预后细菌感染脓毒血症、 严重脓毒血症以及感染性休克的试剂盒。
在另一优选例中， 所述的特异性识别含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集 的分泌蛋白 2或其编码基因的试剂选自 (但不限于
特异性扩增含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2基因的引物； 特异性识别含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2基因的探针； 或
特异性结合含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2的抗体或配 体。
在另一优选例中， 所述的特异性识别含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集 的分泌蛋白 2的试剂是抗含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2的 抗体， 如多克隆抗体。
在本发明的另一方面， 提供一种用于诊断或预后脓毒血症、 严重脓毒血症以及感染 性休克的试剂盒， 它包括：
容器， 以及位于容器中的特异性识别含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集 的分泌蛋白 2或其编码基因的试剂； 较佳地， 为用于检测 CRISPLD2血清浓度的试剂， 如多克隆抗体。
在本发明的另一方面， 提供一种预防或治疗全身性细菌感染或细菌感染相关疾病的 方法， 包括步骤： 提高需要的哺乳动物对象的血清中含 LCCL结构域的半胱氨酸富集的 分泌蛋白 2的浓度。
在本发明的另一方面， 提供一种预防、 缓解或治疗细菌感染或细菌感染相关疾病的 方法， 该方法包括： 给予细菌感染者含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分 泌蛋白 2。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1、 显示了 ELISA检测的标准曲线。
图 2、 显示了重组 CRISPLD2的鉴定结果。 其中，
A、 10% SDS-PAGE 胶分离纯化重组 CRISPLD2分子， 考马斯蓝鉴定；
B、 抗 CRISPLD2抗体免疫印迹；
C、 抗 c-myc-标记抗体免疫印迹。 各泳道如下： 1， 4和 6 为培养基中的 CRISPLD2; 2为分子量标准； 3和 5为对照培养基。
图 3、应用 BIAcore 技术测定的分子相互作用； CRISPLD2 与 LPS和单链 DNA(OND) 结合。 其中，
A、 E. col i LPS(E.coli-LPS)与 CRISPLD2结合解离的传感图。
B、 金黄色葡萄球菌 (S. aureus) LTA(Aureus-LTA)与 CRISPLD2 结合解离的传感图。
C、 E-coli糖脂 (PGN)与 CRISPLD2 结合解离的传感图。 D、 人工合成细菌单链 GPC-DNA CODN2006) 与 CRISPLD2 结合解离的传感图。
E、 人工合成细菌单链 CPG-DNA (ODN2006(Ctr )与 CRISPLD2 结合解离的传感图。
F、 人工合成双链 RNA(PolyI:C;)与 CRISPLD2 结合解离的传感图。
图 4、 盲肠结扎手术和穿剌诱导混合细菌感染脓毒血症以及低剂量 LPS腹腔注射后 血清 CRISPLD2浓度的时间动力学曲线。 A为大鼠盲肠结扎手术和穿剌诱导低度及中度 混合细菌感染后血清 CRISPLD2浓度变化。 B为低剂量 LPS腹腔注射后小鼠血清 CRISPLD2浓度变化。
图 5、 重组 CRISPLD2阻断 LPS与靶细胞受体结合， 抑制靶细胞炎症因子释放。 其 中， A-B :重组 CERISPLD2蛋白阻断大肠杆菌 LPS及沙门氏菌 (S. minnesota; S.m) LPS 与靶细胞受体结合。 其中， MFI是荧光强度的中值。 C-D: 重组 CERISPLD2蛋白降低 大肠杆菌 LPS诱导的炎症因子释放。
图 6、 重组人 CRISPLD2 保护小鼠免于内毒素休克致死， 血清 CRISPLD2 浓度与 E.coli-LPS致死剂量相关性分析。
A、 重组人类 CRISPLD2 保护小鼠免于内毒素休克致死。
B、 小鼠血清 CRISPLD2浓度与 E.coli-LPS致死剂量正相关。
图 7、 抗菌素长时间治疗引起小鼠血清 CRISPLD2浓度下降， 小鼠对内毒素休克的 敏感性增加。 其中，
A、 长时间服用万古霉素加新霉素导致小鼠血清 CRISPLD2 浓度下降。
B、 长时间服用万古霉素加新霉素增加了小鼠对内毒素休克的敏感性。
图 8、 CRISPLD2血清浓度与 LPS诱导的内毒素休克的易感性呈正相关， 以及
CRISPLD2血清浓度相关内毒素休克易感性的阈值确定。
合并三例类似图 7抗菌素长时间治疗的实验数据绘图并统计分析：
A、CRISPLD2血清浓度关与 LPS诱导的内毒素休克的存活率呈正相关 (小鼠腹腔 (i.p.) 注射亚致死剂量的大肠杆菌 LPS 0.2mg)。
B、显示小鼠血清 CRISPLD2水平正态分布曲线， 提示了相应的内毒素休克的易感性 变化与 CRISPLD2水平的关系，初步确定内毒素休克易感性相关的 CRISPLD2水平阈值 为正常血清 CRISPLD2浓度的均值。 纵坐标是血清 CRISPLD2水平； 横纵坐标是存活率 或小鼠计数。
C、 显示人类血清 CRISPLD2水平正态分布曲线以及人类血清 CRISPLD2浓度平均 值。
图 9、 在轻度、 中度、 重度盲肠结扎穿孔脓毒症模型中， 血清 Crispld2和 PCT水平 与死亡率相互关系。
图 10、 重组 CRISPLD2蛋白静脉注射或内源性激活上调血清 CRISPLD2水平保护 混合细菌感染小鼠免于浓毒性休克。 图 1 1、 根据 CRISPLD2水平阈值和 PCT水平阈值 （2微克 /毫升）， 脓毒症病人可以 分为三个亚型 （A、 B、 C) , 其中 CRISPLD2-低 /PCT-高于高死亡率相关 (A) ; 而其他两个 亚型与低死亡率相关 (B、 C) 0正常人群血清 CRISPLD2水平和 PCT水平的正态分布 (D)。 在不同严重程度脓毒症病人血清中， 各亚型与病人死亡率的相关性 (E)。在不同严重程度 脓毒症病人中， 各亚型病人百分比的变化规律 (F)。 15例严重脓毒症及脓毒性休克病人 血清中， 各亚型与病人存活率时间曲线的相关性 (G)。 APACHE II综合指数 4-14为轻度 病人; 15-23为重度病人； 23-39为严重病人。 病例 48， 标本数 246。 15例严重脓毒症 及脓毒性休克病人包括在 48例脓毒症病人之内。
CRISPLD2-高,低 /PCT-低 (A): 连续的病人血清中， CRISPLD2水平波动跨越其阈值， 而 PCT水平始终低于 PCT阈值。
CRISPLD2-高 /PCT-高 (B) : 连续的病人血清中， 当 PCT水平高于 PCT阈值时， CRISPLD2水平保持高于其阈值。
CRISPLD2-低 /PCT-高 (C) : 连续的病人血清中， PCT水平高于 PCT阈值时， PCT 高与 CRISPLD2低 (低于 222微克 /毫升）同时出现一次或数次。 具体实施方式
本发明人经过深入而广泛的研究， 首次证明了一种天然免疫调节蛋白 _ _含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2(cyste ine-rich secretory prote in LCCL domain containing 2， CRISPLD2)可结合细菌的单链 DNA和 LPS , 其血清浓度与 细菌感染或细菌感染相关疾病 （如严重脓毒血症以及浓毒性休克） 的发生密切相关。 一 方面， 以 CRISPLD2蛋白作为靶分子， 可检测个体血清 CRISPLD2浓度， 预测个体对细 菌感染 （如浓毒性休克） 的敏感性， 并通过跟踪血清水平 CRISPLD2预后细菌感染引起 的严重脓毒血症以及浓毒性休克。 另一方面， 通过提高血清中 CRISPLD2蛋白的浓度， 可以预防和治疗细菌感染引起严重脓毒血症以及浓毒性休克。 在此基础上完成了本发 明。
一种 CRISPLD2的 cDNA序列可参见登录号 NM_031476 (长度 4607 bp)或 SEQ ID
NO: 1， 其基因组序列可参见登录号 NC_000016. 9， 其氨基酸序列可参见序列号 P_1 13664或 SEQ ID NO: 2。
在本发明中，术语" CRISPLD2蛋白" 、" CRISPLD2多肽" 、 "含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2 " 或 "人严重脓毒血症以及浓毒性休克相关蛋白 CRISPLD2 "可互换使用，都指具有人严重脓毒血症以及浓毒性休克相关蛋白 CRISPLD2 氨基酸序列的蛋白或多肽。 它们包括含有或不含起始甲硫氨酸的 CRISPLD2蛋白。
如本文所用， "分离的" 是指物质从其原始环境中分离出来 (如果是天然的 物质， 原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯 化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其它物质中分开， 则为分离 纯化的。
如本文所用， "分离的 CRISPLD2蛋白或多肽" 是指 CRISPLD2多肽基本上不含天 然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质 纯化技术纯化 CRISPLD2蛋白。基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一 的主带。
本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选重组多肽。 本 发明的多 肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从 原核或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞） 中产生。 根据重组生产方案所用 的宿主， 本发明的多肽可以是糖基化的， 或可以是非糖基化的。
本发明还包括 CRISPLD2蛋白的片段、衍生物和类似物。如本文所用，术语"片段"、 "衍生物" 和"类似物" 是指基本上保持本发明的天然 CRISPLD2蛋白相同的生物学功 能或活性的多肽。 本发明的多肽片段、 衍生物或类似物可以是 (i) 有一个或多个保守或 非保守性氨基酸残基 (优选保守性氨基酸残基)被取代的多肽， 而这样的取代的氨基酸残 基可以是也可以不是由遗传密码编码的， 或 (i i)在一个或多个氨基酸残基中具有取代基 团的多肽， 或 (i i i)成熟多肽与另一个化合物 (比如延长多肽半衰期的化合物， 例如聚乙 二醇)融合所形成的多肽， 或 (iv)附 加的氨基酸序列融合到此多肽序列而形成的多肽 (如 前导序列或分泌序列或用来 纯化此多肽的序列或蛋白原序列， 或与抗原 IgG片段的形 成的融合蛋白）。 根据本文的教导， 这些片段、 衍生物和类似物属于本领域熟练技术人 员公知的范围。
在本发明中， 术语 " CRISPLD2蛋白" 指具有 CRISPLD2蛋白活性的 SEQ ID NO: 2序列的多肽。 该术语还包括具有与 CRISPLD2蛋白相同功能的、 SEQ ID NO: 2序列 的变异形式。 这些变异形式包括 (但并不限于；)： 若干个 (通常为 1-50 个， 较佳 地 1-30 个， 更佳地 1-20个， 最佳地 1-10个)氨基酸的缺失、 插入和 /或取代， 以及在 C末端 和 /或 N末端添加或缺失一个或数个 (通常为 20个以内， 较佳地为 10个以内， 更佳 地 为 5个以内)氨基酸。 例如， 在本领域中， 用性能相近或相似的氨基酸进行取代时， 通 常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加或缺失一个或数个氨基 酸通常也不会改变蛋白质的功能。该术语还包括 CRISPLD2蛋白的活性片段和活性衍生 物。 通常， 这些变异形式包括 CRISPLD2蛋白的 LCCL-RAAIH (R3H)结构域。
该多肽的变异形式包括： 同源序列、 保守性变异体、 等位变异体、 天然突变体、 诱 导突变体、 在高或低的严紧度条件下能与 CRISPLD2 DNA杂交的 DNA所编码的蛋白、 以及利用抗 CRISPLD2多肽的抗血清获得的多肽或蛋白。本发明还提供了其它多肽， 如 包含 CRISPLD2多肽或其片段的融合蛋白。 除了几乎全长的多肽外， 本发明还包括了 CRISPLD2多肽的可溶性片段。通常，这些变异形式包括 CRISPLD2蛋白的 LCCL-RAAIH (R3H)结构域。
发明还提供 CRISPLD2蛋白或多肽的类似物。 这些类似物与天然 CRISPLD2多肽的 差别可以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者兼而 有之。 这些多肽包括天然或诱导的遗传变异体。 诱导变异体可以通过各种技术得到， 如 通过辐射或暴露于诱变剂而产生随机诱变， 还可通过定点诱变法或其它已知分子生物学 的技术。 类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨基酸)的类似物， 以及 具有非天然存在的或合成的氨基酸 (如 β、 Υ -氨 基酸)的类似物。 应理解， 本发明的多肽 并不限于上述例举的代表性的多肽。 通常， 这些类似物包括 CRISPLD2蛋白的
LCCL-RAAIH (R3H)结构域。
修饰 (通常不改变一级结构；)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或 羧基化。 修饰还包括糖基化， 如那些在多肽的合成和加工中或进一步加工步骤中进行糖 基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶 (如哺乳动物的 糖基化酶或去糖基化酶)而完成。 修饰形式还包括具有磷 酸化氨基酸残基 (如磷酸酪氨 酸， 磷酸丝氨酸， 磷酸苏氨酸)的序列。 还包括被修饰从而提高了其抗蛋白水解性能或 优化了溶解性能的多肽。
在本发明中， " CRISPLD2蛋白保守性变异多肽 "指与 SEQ ID NO: 2的氨基酸序列 相比， 有至多 10个， 较佳地至多 8个， 更佳地至多 5个， 最佳地至多 3个氨基酸被性 质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表 1进行氨基 酸替换而产生。
表 1
Figure imgf000008_0001
|Val (V) |lle; Leu; Met; Phe; Ala Leu
编码 CRISPLD2蛋白的基因 （多核苷酸） 可以是 DNA形式或 RNA形式。 DNA形 式包括 cDNA、基因组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA 可以是编码链或非编码链。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。
本发明的 CRISPLD2核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或 人工合成的方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序列， 尤其 是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员已知的常规方 法所制备的 cDNA库作为模板， 扩增而得有关序列。 当序列较长时， 常常需要进行两次 或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离 得到有关序 列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通常， 通 过先合成多个小片段， 然后再进行连接可获得序列很长的片段。
目前， 已经可以完全通过化学合成来得到编码本发明蛋白 (或其片段， 或其 衍生物） 的 DNA序列。 然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子 （或 如载体)和细胞中。 此外， 还可通过化学合成将突变引入本发明蛋白序列中。
应用 PCR技术扩增 DNA/RNA的方法被优选用于获得本发明的基因。 用于 PCR的 引物可根据本文所公开的本发明的序列信息适当地选择， 并可用常规方法合成。 可用常 规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原 核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法 处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可 用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉 淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。
获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据所用的 宿主细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下 进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法 （如温度转换或化学诱 导)诱导选择的启动子， 将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如 果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复性处 理、用蛋白沉淀剂处理 （盐析方法；)、离心、渗透破菌、超处理、超离心、分子筛层析 （凝 胶过滤)、 吸附层析、 离子交换层析、 高效液相层析 （HPLC)和其它各种液相层析技术及 这些方法的结合。
另一方面， 本发明还包括对 CRISPLD2 DNA或是其片段编码的多肽具有特异性的多 克隆抗体和单克隆抗体， 尤其是单克隆抗体。
本发明不仅包括完整的单克隆或多克隆抗体， 而且还包括具有免疫活性的抗体片段， 如 Fab'或 (Fab) 2片段； 抗体重链； 抗体轻链； 遗传工程改造的单链 Fv分子； 或嵌合抗 体， 如具有鼠抗体结合特异性但仍保留来自人的抗体部分的抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。 例如， 纯化的 CRISPLD2基因产物或者其具有抗原性的片段， 可被施用于动物以诱导多克隆抗体的产 生。与之相似的， 表达 CRISPLD2蛋白或其具有抗原性的片段的细胞可用来免疫动物来 生产抗体。 本发明的抗体也可以是单克隆抗体。 此类单克隆抗体可以利用杂交瘤技术 来制备。
抗 CRISPLD2蛋白的抗体可用于免疫组织化学技术中， 检测活检标本 (尤其是 血清 样本)中的 CRISPLD2蛋白。 此外， 与 CRISPLD2蛋白结合的单克隆抗体也可用放射性 同位素标记， 注入体内可跟踪其位置和分布。
多克隆抗体的生产可用 CRISPLD2蛋白或多肽免疫动物， 如家兔， 小鼠， 大鼠等。 多种佐剂可用于增强免疫反应， 包括但不限于弗氏佐剂等。
利用本发明蛋白， 通过各种常规筛选方法， 可筛选出与 CRISPLD2蛋白发生相互作 用的物质， 如受体、 抑制剂、 激动剂或拮抗剂等。
如本文所用， 所述的 CRISPLD2的激动剂包括了稳定剂、 促进剂、 上调剂等。 任何 可提高 CRISPLD2蛋白的活性、维持 CRISPLD2蛋白的稳定性、促进 CRISPLD2蛋白的 表达、 延长 AC CRISPLD2hE蛋白有效作用时间、 促进 CRISPLD2的基因转录和翻译以 及的物质均可用于本发明， 作为对于预防、 缓解或治疗细菌感染或细菌感染相关疾病的 物质。
CRISPLD2蛋白及其激动剂 （也称为促效剂） 等， 当在治疗上进行施用 (给药)时， 可 预防或治疗败血症以及败血症休克。 通常， 可将这些物质配制于无毒的、 惰性的和药 学上可接受的水性载体介质中， 其中 pH通常约为 5-8， 较佳地 pH约为 6-8， 尽管 pH 值可随被配制物质的性质以及待治疗的病症而有所变化。 配制好的药物组合物可以通 过常规途径进行给药， 其中包括 (但并不限于)： 口服、 肌内、 腹膜内、 静脉内、 皮下、 皮内、 或局部给药。
本发明的多肽可直接用于细菌感染或细菌感染相关疾病的治疗和预防， 尤其是防治 严重脓毒血症以及浓毒性休克。在使用本发明 CRISPLD2蛋白时， 还可同时使用其它用 于同一病症的治疗剂。
本发明还提供了一种组合物 (包括药物组合物)， 它含有安全有效量 (如 0. 001-99. 9wt%^ 本发明 CRISPLD2多肽或其激动剂以及 (药学上)可接受的载体或赋形剂。 这类 载体包括 (但并不限于)： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。 药物制 剂应与给药方式相匹配。 本发明的药物组合物可以被制成针剂形式， 例如用生理盐水或 含有葡萄糖和其它辅剂的水溶液通过常规方法进行制备。 诸如片剂和胶囊之类的药物组 合物， 可通过常规方法进行制备。 药物组合物如针剂、 溶液、 片剂和胶囊宜在无菌条件 下制造。 活性成分的给药量是治疗有效量， 例如每天约 1微克 /千克体重-约 5毫克 /千 克体重。 此外， 本发明的多肽还可与其它治疗剂一起使用。
本发明还涉及定量检测 CRISPLD2蛋白水平的诊断试验方法。 这些试验是本领域所 熟知的， 包括 ELISA、 FISH测定和放射免疫测定。 因此， 在了解了 CRISPLD2蛋白水 平与疾病的关系后， 本领域人员易于进行细菌感染或细菌感染相关疾病的诊断或预后。 试验中所检测的 CRISPLD2 蛋白水平， 可以用作解释 CRISPLD2蛋白在调控细菌抗原 反应中的重要性和用于诊断疾病的易感性。
一种检测样品中是否存在 CRISPLD2蛋白的方法是利用特异性抗体识别 CRISPLD2 蛋白。 本发明的主要应用包括：
1. 将 CRISPLD2蛋白作为靶分子， 检测个体血清 CRISPLD2浓度， 预测诊断个体 内对脓毒血症以及浓毒性休克的敏感性， 为临床医生提供依据， 对可能突发脓毒血症并 引起休克的病人采取必要的预防治疗措。可根据临床的经验确定一个阈值，当 CRISPLD2 蛋白低于该阈值时， 作出疾病的警示。
2. 将重组 CRISPLD2蛋白用于病人严重脓毒血症时干预治疗。
3. 连续跟踪血清样品中先天免疫调节分子 CRISPLD2浓度， 以评估患者的天然免疫 调节和病原体反应之间的动态平衡。 作为预防和治疗感染性休克的措施， 医源性调节血 清 CRISPLD2水平， 修正天然免疫调控和病原体反应之间的不平衡。
本发明还提供了血清 CRISPLD2浓度监测的试剂盒， 其中包含容器， 以及位于容器 中的特异性识别含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2 (CRISPLD2) 或其编码基因的试剂 （较佳地， 为用于检测 CRISPLD2血清浓度的试剂， 如多克隆抗 体)。 试剂盒中的试剂可用于跟踪血清样品中先天免疫调节分子 CRISPLD2浓度， 以评 估患者的天然免疫调控动态平衡状况， 这一动态平衡的失衡直接反映疾病严重程度和可 能的休克死亡率。
临床上， 严重脓毒血症患者血清 CRISPLD2浓度连续监测中， 血清样本中出现 CRISPLD2浓度低于正常平均值， 而常规临床血清感染指标高于严重脓毒血症警戒线的 血清标本直接指示浓毒性休克的可能。 该指标与疾病严重性、 死亡率相关性： 严重脓毒 血症患者血清样本中， CRISPLD2低于正常人平均值的样本出现频率直接反映病人的死 亡几率。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 科学出版社， 2002中所述的条件， 或 按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按重量计算。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。
实施例 1、 质粒构建、 细胞转染和蛋白表达纯化
1. 质粒构建
CRISPLD2的氨基酸序列 （SEQ ID NO: 2) 下， 其中结合 LPS的二个 LCCL结构域 位于第 286-370位和第 387-460位；第 333-337位为 RAAIH (R3H) 序列，结合 ssDNA (SEQ ID NO: 2序列， 序列号 NP_113664， 氨基酸残基数 497 aa):
1 MSCVLGGVIP LGLLFLVCGS QGYLLPNVTL LEELLSKYQH NESHSRVRRA IPREDKEEIL 61 MLHNKLRGQV QPQASNMEYM TWDDELEKSA AAWASQCIWE HGPTSLLVSI GQNLGAHWGR 121 YRSPGFHVQS WYDEVKDYTY PYPSECNPWC PERCSGPMCT HYTQIVWATT NKIGCAVNTC 181 RKMTVWGEVW ENAVYFVCNY SPKGNWIGEA PYKNGRPCSE CPPSYGGSCR NNLCYREETY 241 TPKPETDEMN EVETAPIPEE NHVWLQPRVM RPTKPKKTSA VNYMTQVVRC DTKMKDRCKG
301 STCNRYQCPA GCLNHKAKIF GTLFYESSSS IC|RAA啤 GI LDDKGGLVDI TRNGKVPFFV
361 KSERHGVQSL SKYKPSSSFM VSKVKVQDLD CYTTVAQLCP FEKPATHCPR IHCPAHCKDE
421 PSYWAPVFGT NIYADTSSIC KTAVHAGVIS NESGGDVDVM PVDKKKTYVG SLRNGVQSES
481 LGTPRDGKAF RIFAVRQ 应用 PCR从人类混合组织 mRNA中获得部分或全长 CRISPLD2阅读框架 (ORF) cDNA。
引物 1(扩增 CRISPLD2蛋白第 l-497aa对应的 cDNA):
F1: 5'- GCTGTCGCCGCTGCTACCGC (SEQ ID NO: 3)；
Rl: 5'-GACGCCCCTTCTCCCCTGGT (SEQ ID NO: 4)。
PCR产物插入质粒 pGEM-T (Promega) 作为表达载体构建的模板。
将编码全长 CRISPLD2的 cDNA插入 pcDNA3.1A (InviyroGen) 质粒的多克隆位点， 然后， 转入 CHO细胞 (ATCC), 重组表达产生蛋白带有 c-myc标记和 6个 His的重组 CRISPLD2蛋白。
引物 2(扩增 CRISPLD2蛋白第 257-497aa对应的 cDNA):
F2: 5'-TGGAATTCCGAGAAGAAACCTACACTC (SEQ ID NO: 5)
R2: 5'- TGCTCGAGATTCACTGCCTGACAGCA (SEQ ID NO: 6) PCR产物插入质粒 pGEM-T CPromega) 作为表达载体构建的模板。
将编码 CRISPLD2蛋白片段 （SEQ ID NO: 2中第 257-497aa) 的 cDNA插入质粒 pGEX-4T-l (GE Healthcare) 的多克隆位点， 转入 CHO细胞 (ATCC), 获得重组表达的 GST-CRISPLD2融合蛋白。 这一融合蛋白用来免疫兔子， 获得抗血清。 应用 Protein G Agarose 从抗血清中纯化获得抗 CRISPLD2多克隆抗体 （抗 CRISPLD2 IgG)。
2. 细胞转染和蛋白表达纯化
应用 Lipofectamine (Invitrogen；)， 将质粒转染 CHO细胞， 然后应用抗菌素 G418选 择转染阳性细胞。 稳定表达人类 CRISPLD2的 CHO细胞培养时保持 G418 , 应用有限 稀释法进一步分离、 选择高表达的 CHO细胞克隆。
无血清培养液培养表达重组 CRISPLD2蛋白的 CHO细胞。收集上清液内含高浓度重 组 CRISPLD2蛋白。 应用亲和层析， 纯化培养液中的重组蛋白。
收集部分磷酸缓冲液透析 (含有重组蛋白）， 用 SDS-PAGE电泳分离， 考马斯蓝 coomass ie blue)染色 SDS-PAGE胶， 鉴定纯度， 并用 BCA 测定试剂盒蛋白定量
(Pierce)。
3. 免疫酶联反应法 (ELISA)测定 CRISPLD2浓度
用碳酸钠缓冲液 （PH 9.5) 适当稀释培养液或血清， 包被 96孔板 (Nunc MaxiSorp, Denmark), 4°C过夜。磷酸缓冲液洗 96孔板两遍，用含 0.4%酪蛋白的磷酸缓冲液孵育 1 小时， 然后用含 8%(v/v)胎牛血清和抗 CRISPLD2多克隆抗体 (10 u g /ml)的磷酸缓冲液 孵育 2小时， 磷酸缓冲液洗 4遍， 加入辣根过氧化酶偶联的第二抗体， 再用磷酸缓冲 液洗 4遍， 商业 TMB检测反应试剂 96孔板显色反应， 检测波长为 450nm-570nm。 以 重组 CRISPLD2 (0.03- 4.00微克 /ml)为标准， 可以定量样本中 CRISPLD2的含量。 获得 的 ELISA标准曲线如图 1所示。 实施例 2、 蛋白纯度的鉴定
蛋白标本稀释在上样缓冲液中， 由 10% SDS-PAGE 电泳胶分离， 直接考马斯蓝染 色鉴定蛋白纯度。 免疫印迹法： SDS-PAGE 电泳胶上的蛋白质转移到硝酸纤维 膜 (GE Healthcare), 硝酸纤维膜与含有第一抗体的缓冲液一起培养 2 小时 37°C， 清洗， 荧光 素标记的第二抗体 (ROCKLAND) 培养 1小时 37°C。 ODYSSEY红外线图像系统
(Infrared Imaging System) 扫描硝酸纤维膜获得图像。结果如图 2所示， 可见获得了纯化 的蛋白。 实施例 3、 CRISLD2的细菌结合能力 应用 BIAcoreTlOO生物传感检测仪， 通过 Surface plasmon resonance (SPR) 方法定 量测定细菌抗原与 CRISPLD2的结合。
细菌抗原分别如下：
E. coli LPS : 购自 Sigma;
金黄色葡萄球菌 LTA ： 购自 InvivoGen;
E-coli糖脂 (PGN) : 购自 InvivoGen;
人工合成单链 GPC-DNA(ODN2006) :购自 InvivoGen,其序列为： 5 '- tcg tcg ttt tgt cgt ttt gtc gtt -3 ' (24 mer);
人工合成单链 CPG-DNA (ODN2006(Ctr)):购自 InvivoGen,或其序列为： 5 '- tgc tgc ttt tgt get ttt gtg ctt -3 ' (24 mer)；
人工合成双链 RNA(PolyI:C): 购自 InvivoGen。
用仪器公司提供的试剂分别将重组 CRISPLD2蛋白和 IgG (0.18 mg/ml溶解于醋酸缓 冲液， pH 4.5) 偶联到传感芯片 （GE Healthcare) 的两个通道。 不同浓度 （浓度分别为： 0， 0.3， 1.0， 3.0， 10禾 Ρ 30 μΜ) 细菌抗原溶于磷酸缓冲液， 并衡量注入， 同时流过传感 芯片 (20 μ ΐ/min)的两个通道。 从重组 CRISPLD2蛋白通道获得的细菌抗原的结合反应单 位 (RU)减去 IgG通道的信号 (RU) ； 磷酸缓冲液 (PBS)洗去未结合的细菌抗原； 然后 50 mM NaOH洗去结合的细菌抗原， 对芯片进行再生。
KD可以通过计算 k。ff /k。n两个常数的比率得到。 解离比率常数 (dissociation rate constant, k。ff)可以用公式： Rt = RtQ e tQ)。 结合比率常数 （association rate constant, 。„)) 1^,从1^值推导， 公式为： Rt = Rmax [l-e - (k°n e + k°ff) (t - tQ)] 这里 C为细菌抗原浓度， 是 细菌抗原与 CRISPLD2结合最大结合反应单位 (RU)， Rt 是时间 t.时 细菌抗原与 CTRISPLD2结合的量。
结果如图 3所示。 因此， CRISLD2能与 ssDNA和 LPS结合， 但不结合金黄色葡萄 球菌 LTA、 人工合成大肠杆菌 PGN或人工合成双链 RNA CdsRNA)。 实施例 4、血液样品中 CRISPLD2的浓度以及个体对内毒素休克易感性的阈值水平。 血清、 血桨 CRISPLD2的浓度 (100例正常成人， 5例新生儿)测定如下： 对于 100例 正常成人的血清和血桨， 5例新生儿的脐带血， 用免疫印迹法分析其 CRISPLD2浓度。 参照重组 CRISPLD2标准浓度， 血清、血浆 CRISPLD2浓度由 ODYSSEY红外线图像扫 描分析系统计算定量。 成人血清与新生儿脐带血 CRISPLD2 比较， 并用 T-test进行显 著性分析。 P = 0.025。 将半定量免疫印迹法与竞争性 -ELISA (表 2) 进行比较， 结果相符 表 2、 竞争性 -ELISA检测 100例健康正常人血清以及 5例新生儿的脐带血
分子 标本 例数 (n) 平均浓度 ( g/ml) 范围（ g/ml) CRISPLD2 正常成人血清 100 221 55-437
CRISPLD2 脐带血桨 5 55 21-103 实施例 5、 盲肠结扎穿孔混合细菌感染模型以及不同剂量 LPS腹腔注射后血清
CRISPLD2浓度
低度、 中度盲肠结扎穿孔 (CLP)混合细菌感染脓毒血症模型的制备： 动物盲肠不同部 位的结扎， 盲肠穿剌针头的大小和盲肠穿孔次数的多少决定了动物混合细菌感染脓毒血 症的严重程度。 盲肠结扎穿孔手术前 12小时禁食。 腹腔戊巴比妥 4毫克 /公斤麻醉， 下 腹部去毛消毒。 下腹部中线手术切开 1厘米切口， 从肠系膜分离盲肠， 进行盲肠结扎及 穿孔； 低度脓毒血症结扎 10%盲肠； 中度脓毒血症结扎 50%盲肠； 穿孔使用 21号针头。 假手术组开下腹以后无盲肠结扎和穿孔。 关闭创口， 补充体液 (50毫升 /公斤生理盐水)。 动物可以进食和饮水。
低度、 中度盲肠结扎穿孔 (CLP)混合细菌感染大鼠模型， 以假手术为阴性对照； 小鼠 腹腔注射不同非毒性剂量 E.coli-LPS， 细菌脂蛋白和磷脂壁酸 LTA， 不同时间点小鼠尾 静脉抽血， 血清 CRISPLD2由 ELISA检测。 盲肠结扎手术和穿剌诱导混合细菌感染脓 毒血症以及低剂量 LPS腹腔注射后血清 CRISPLD2浓度的时间动力学曲线如图 4 A为盲 肠结扎手术和穿剌诱导低度、 中度混合细菌感染后血清 CRISPLD2浓度变化。 图 4B为 低剂量 LPS腹腔注射后血清 CRISPLD2浓度变化。 实施例 6、重组 CRISPLD2阻断 LPS与靶细胞受体结合，抑制靶细胞炎症因子释放
5 X 106个 /毫升人类外周单个核细胞悬浮在 RPMI 1640培养液中（0.1%FCS)， 加入重 组 CERISPLD2蛋白和 Cy2标记的 LPS , 4°C， 20分钟。 流式细胞仪分析， 单核细胞与 淋巴细胞由点阵图分离 （Dot-plot, SSC versus FSC), 分别分析 FITC-LPS在这两群细胞 上的荧光强度。 重组 CRISP-3蛋白 (购自 R&DM乍为对照。 结果如图 5A和 5B所示， 重 组 CERISPLD2蛋白可阻断大肠杆菌 LPS(Ec LPS)及沙门氏菌 LPS与靶细胞受体结合。
5 X 106个 /毫升人类外周单个核细胞悬浮在 RPMI 1640培养液中（0.1%FCS)， 加入重 组 CERISPLD2蛋白和 LPS, 37°C、 18小时。 培养液中 TNF-a、 IL-6由 ELISA测定。 结 果如图 5C和 5D所示，重组 CERISPLD2蛋白可降低大肠杆菌 LPS诱导的炎症因子释放。 实施例 7、 重组人 CRISPLD2保护小鼠免于内毒素休克致死， 血清 CRISPLD2浓度 与内毒素休克存活率相关
三组 balb/c小鼠都腹腔注射 400微克 E.coli-LPS (以 PBS为溶剂)， 同时， 正常对照 组腹腔注射 PBS (21个小鼠)， 实验组腹腔注射 1毫克重组 CRISPLD2 (17个小鼠）， 实 验对照组腹腔注射 1毫克重组 CRISP3 (7个小鼠) (该蛋白同属 CRISP家族，但缺失 LPS 结合区域)。 存活率计数并制图。
结果如图 6A所示， 重组人 CRISPLD2保护小鼠免于内毒素休克致死。
小鼠 (20个小鼠)腹腔注射非毒性剂量 E.coli-LPS (30微克 /小鼠)，第十天时与 4只正 常小鼠一起尾静脉抽血， CRISPLD2进行 ELISA检测， 6小时后对全部小鼠进行不同致 死剂量 E.coli-LPS随机腹腔注射。第三十二天时，存活的小鼠再次尾静脉抽血，血清 1 ：
1000稀释， CRISPLD2进行 ELISA检 测， 6小时后再次随机腹腔注射不同致死剂量
E.coli-LPS。每一小鼠的致死剂量 E.coli-LPS与当时的血清 CRISPLD2浓度数据相对应， 组成数据矩阵， 并应用 Graphpad Prism 5软件进行统计学分析， P值 =0.0009。 Excel的 logarithmic trendline是数据矩阵的最佳匹配。
结果如图 6B所示， 小鼠血清 CRISPLD2浓度与 E.coli-LPS致死剂量正相关。
上述结果提示， CRISPLD2蛋白是一个预测革兰氏阴性细菌败血症以及个体对内毒 素敏感性的靶分子。 实施例 8、 广谱抗菌素长时间治疗引起小鼠血清 CRISPLD2浓度下降
万古霉素 （Van) 1毫克 /毫升 +新霉素 （Neo) 0.5毫克 /毫升溶解于饮用水中喂食小鼠
(27个小鼠)杀灭体内共生菌，不同时间点小鼠尾静脉抽血，血清 1： 1000稀释， CRISPLD2 进行 ELISA定量检测。结果如图 7A所示， 长时间服用万古霉素加新霉素导致小鼠血清 CRISPLD2浓度下降。
正常饮水的正常对照组， 饮水中含广谱抗菌素的 4天组以及 15天组， 三组小鼠都腹 腔注射 200微克 E.coli-LPS。 正常小鼠腹腔注射 400微克 E.coli-LPS才会有 70-80%死 亡率，连续 15天服用广谱抗菌素的小鼠与正常对照相比，腹腔注射 200微克 E.coli-LPS 导致的死亡率显著增加，而连续 4天服用广谱抗菌素的小鼠与正常对照相比无显著变化。 统计方法和统计数据已标在图上。 结果如图 7B所示， 表明长时间服用万古霉素加新霉 素增加了小鼠对内毒素休克的敏感性 (32个小鼠)。 实施例 9、 预测浓毒性休克易感性的血清 CRISPLD2浓度阈值
图 8A所示合并三个类似图 7的实验结果， 42个小鼠的组合数据，分析血清 CRISPLD2 水平与内毒素休克存活率相关性， 以确定具有应用价值的血清 CRISPLD2浓度阈值。
图 8B所示合并三个类似图 7的实验结果， 血清 CRISPLD2浓度成正态分布。
血清 CRISPLD2浓度在平均值以下的小鼠由 E.coli-LPS亚致死剂量 (200微克 /小鼠） 导致的存活率显著下降；反之，血清 CRISPLD2浓度在平均值以上的小鼠耐受 E.coli-LPS 亚致死剂量 (200微克 /小鼠）， 存活率显著增加。 血清 CRISPLD2浓度平均值可以作为浓 毒性休克易感性的阈值。
图 8C根据以上实验分析结果， 脓毒症临床相关研究将应用这一阈值： 即正常血清 CRISPLD2浓度的均值作为个体对内毒素休克易感性的阈值。 实施例 10、重组 CRISPLD2静脉注射，或小剂量 LPS预注射，提高血清 CRISPLD2 水平保护混合细菌感染的中度盲肠结扎穿孔 (CLP-Moderate)小鼠
图 9显示三种不同严重程度 CLP诱导的混合细菌感染脓毒症模型应用于研究 (轻度、 中度、 严重脓毒血症大鼠， 分别为图 、 B和 C；)。 血清 Crispld2水平与 PCT水平呈负 相关。 在轻度脓毒症， Crispld2上调伴随 PCT浓度低下， 相关死亡率 20%; 在中度脓 毒症， Crispld上调伴随 PCT增加， 相关死亡率 50%; 在重度脓毒症， 低 Crispld2伴随 高 PCT， 相关死亡率 100%。
图 9 三个点阵图显示： Crispld2水平低 /PCT水平高的血清样本出现频率和死亡率相关 (右下象限）。
图 9A和 9B显示： 在轻度、 中度脓毒症， 血清 Crispld2水平显著上调， 而在重度脓 毒症中未见上调 （C；)。
重组 CRISPLD2静脉注射， 提高血清 CRISPLD2水平 25%， 保护混合细菌感染的中 度盲肠结扎穿孔 (CLP-中度)小鼠 (存活率从 40%上升至 73%)， 如图 10A和 B所示； 小剂 量 LPSC50微克 /小鼠)预注射 8天以后， 提高血清 CRISPLD2水平 67%， 很好地保护混合 细菌感染的中度盲肠结扎穿孔 （CLP-中度） 小鼠， 如图 10C-D所示。 图 10所示这一保 护能力与血清 CRISPLD2水平相关。
动物模型模说明： 鼠类 CLP脓毒血症模型模拟急性混合细菌感染， 病理学原理和临 床症状接近人类急性细菌感染脓毒血症。 1. 轻度脓毒血症模型， 死亡率 10-20%; 2. 中 度严重脓毒血症模型， 死亡率 40-60%; 3. 重度严重脓毒血症模型， 死亡率 90-100%。 动物盲肠不同部位的结扎， 盲肠穿剌针头的大小和盲肠穿孔次数的多少决定了动物混合 细菌感染脓毒血症的严重程度。 盲肠结扎穿孔手术前 12小时禁食。 腹腔戊巴比妥 4毫 克 /公斤麻醉， 下腹部去毛消毒。 下腹部中线手术切开 1厘米切口， 从肠系膜分离盲肠， 进行盲肠结扎及穿孔； 低度脓毒血症结扎 10%盲肠； 中度脓毒血症结扎 50%盲肠； 高度 脓毒血症结扎 75%盲肠； 穿孔使用 21号针头。 假手术组开下腹以后无盲肠结扎和穿孔。 关闭创口， 补充体液 (50毫升 /公斤生理盐水；)。 动物可以进食和饮水。 实施例 11、 脓毒血症与血清 CRISPLD2水平临床相关性
调查了 78例病人， 其中 48例脓毒症人， 以 CRISPLD2蛋白为靶分子检测个体血清
CRISPLD2浓度， 发现通过跟踪 CRISPLD2血清水平可以预后全身性细菌感染引起的严 重脓毒血症以及脓毒性休克。 并且证明提高血清中 CRISPLD2蛋白的浓度， 可预防和治 疗全身性细菌感染引起的严重脓毒血症以及浓毒性休克。 78例病人中的 30例局部细菌 感染病人和非感染性疾病的病人也进行了调查， 局部细菌感染病人血清中 CRISPLD2水 平检测可以用来评估 CRISPLD2调节控制免疫反应的能力。 浓毒性休克病人血清中 CRISPLD2水平变化规律与出血性休克病人血清中其变化规律完全不同。
在临床上， 分析病人体液中 (血清;) CRISPLD2浓度情况。 严重脓毒血症病人 PCT高， 但 PCT高 /CRISPLD2浓度低下的血清样本在连续血清样本中出现反映脓毒血症病人的 严重程度及死亡率。血清 CRISPLD2水平适用于严重脓毒症及脓毒性休克病人诊断和预， 也适用于轻度细菌感染相关病人中评估 CRISPLD2调控免疫反应。
病人 : 48例 急症严重创伤及其他原因引起的格兰氏阴性和阳性混合细菌感染病人， 重症病人进入 ICU病房。 脓毒症诊断根据国际 ACCP/SOOM 标准。
血清 CRISPLD2以及脓毒血症标志分子检测： 平均检测 1次 /4天， 28天连续观察血 清 CRISPLD2以及血清降钙素原 (PCT， Procalcitonie)的浓度。
阈值界定： 血清 CRISPLD2水平的均值 220微克 /毫升为阈值， 低于这一水平被认定 为是 CRISPLD2低，反之被认定为 CRISPLD2高； 降钙素原 (PCT) 脓毒血症血清 PCT 阈 值为 2.0纳克 /毫升， 正常值为 0.01-0.03纳克 /毫升， 最高可达数百纳克 /毫升。 在其他感 染性疾病， 血清 PCT—般低于 2.0纳克 /毫升水平。
在脓毒症病人中， 我们发现 CRISPLD2水平与 PCT水平的关系可定义为以下三类：
CRISPLD2-高,低 /PCT-低： 连续的病人血清中， CRISPLD2水平波动跨越其阈值， 而 PCT水平始终低于 PCT阈值。
CRISPLD2-高 /PCT-高：连续的病人血清中，当 PCT水平高于 PCT阈值时， CRISPLD2 水平保持高于其阈值。
CRISPLD2-低 /PCT-高： 连续的病人血清中， PCT水平高于 PCT阈值时， PCT高与
CRISPLD2低 (低于 222微克 /毫升）同时出现一次或数次。
病人临床严重程度指标： 临床综合指数 APACHEII; Acute Physiology and Chronic Health Evaluation II急性生理学及慢性健康状况评分。 APACHE II指数 4-12为轻度病人； 13-22为较重病人； 23-39为严重病人。
图 11显示脓毒症病人可以分为三个亚型 （A、 B、 C), 其中 50% 以上的病人为
CRISPLD2-低 /PCT-高， 与高死亡率相关 (A); 而其他两个亚型与低死亡率相关 (B、 C)0 图 11E和 11F显示： 在 APACHE II 指数 13-22严重程度的脓毒症病人中， 35%病 人血清具有 CRISPLD-高 /PCT-高特征， 并且具有这一特征的病人死亡率低， 这种 CRISPLD-高 /PCT-高表象可能仅反映了患者体内 CRISPLD2调控宿主免疫反应， 降低了 过度炎症反应造成的损害性效应。 图 10显示上调 CRISPLD2血清水平， 保护全身性细 菌感染的动物实验结果佐证了这一推测。
图 11B和 11C显示： 低死亡率 (；3-7%)发生在具有 CRISPLD2-高,低 /PCT-低特征以及 CRISPLD-高 /PCT-高血清特征的部分脓毒血症病人中， 原因尚未明确。 这部分病人可能 覆盖了一部分格兰氏阳性细菌感染脓毒症病人， 因为在格兰氏阳性细菌脓毒血症病人 PCT血清水平 (范围 0.1-8.9纳克 /毫升)， 横跨了血清中 2纳克 /毫升 PCT这一界限， 而兰 氏阳性细菌的分子例如 ssDNA、 deRNA、 PGN都可以上调免疫细胞的 CRISPLD2产生 和分泌。
图 11E和 11F还显示：在严重程度低 (APACHE II指数小于 10)的病人中， CRISPLD2- 低 /PCT-高血清样本的出现与死亡率无相关性 (A)。 轻度细菌感染相关病人中血清
CRISPLD2和 PCT水平的检测可以用来评估 CRISPLD2调控免疫反应以及宿主免疫反应 的程度， 对病情诊断和预防病情恶化有一定帮助， 例如轻度全身性细菌感染相关病人血 清中 CRISPLD2水平急剧下降可以提示医生， 根据病人病情判断接踵而来可能发生的变 化， 提示医生采取必要的措施防止病情恶化。
另外， 非脓毒症病人 30例 包括： 尿路感染、 支气管炎、 急性胆管炎、 外伤性胰 腺炎、 还有消化道出血和失血性休克。
其中细菌感染相关的 18例尿路感染和支气管炎病人出现血清 CRISPLD2水平上调 /PCT水平低于 3ng/ml 。
其他 7例急性胆管炎及外伤性胰腺炎， 血清 CRISPLD2水平略有上调 /PCT水平低于 2ng/mL
失血性休克与全身性细菌感染性休克情况完全不同， 5例消化道出血和失血性休克病 人中血清 CRISPLD2上调而不是下降， PCT水平低于 2ng/ml (死亡率 20%)。
总之， 非脓毒症病人血清 CRISPLD2水平略有上升， 而血清 CRISPLD2与 PCT无负 相关性。 讨论
1. 分泌蛋白 CRISPLD2是一个预测严重脓毒血症及脓毒性休克敏感性的靶分子 本发明显示， 非常保守的半胱氨酸富集的分泌蛋白 CRISPLD2 (Cysteine - rich secretory protein LCCL domain containing 2) 是一个血清浓度较高的 ssDNA禾 P LPS结合 蛋白， 该蛋白拥有一个 RAAIH (R3H) (RAAIH)结构域与 ssDNA结合； 两个与 LPS结合 的 LCCL结构域， 并具有较高的亲和力与 ssDNA和 LPS结合。 不同于已知应急蛋白， 大鼠肠穿孔混合细菌感染以及小鼠腹腔注射非毒性剂量 LPS的第二天 (急性期)， 血清 CRISPLD2浓度缓慢上升， 第五天峰值。 相反， 初步实验数据显示肠穿孔混合细菌感染 大鼠的感染部位组织渗出液中 CRISPLD2浓度感染后数小时内快速上升。统计学分析发 现该蛋白血清浓度与内毒素致死剂量正相关。 CRISPLD2保护混合细菌感染动物与其血 清浓度相关， 这提示 CRISPLD2蛋白是一个预后脓毒性休克敏感性、 治疗严重脓毒血症 及脓毒性休克的靶分子。
2. CRISPLD2蛋白可以保护小鼠， 增加脓毒性休克的存活率， 可以预防脓毒性休克 人类、 小鼠和大鼠的心脏、 肺脏、 小肠、 胎盘以及粒细胞、 单核细胞都高表达 CRISPLD2。 体外实验中本发明人还发现大部分白细胞 (包括粒细胞、 单核细胞） 自然释 放该 ssDNA和 LPS结合蛋白。该蛋白在正常生理浓度范围内可以阻断 LPS与受体结合， 并抑制 LPS诱导的炎症因子释放， 包括肿瘤坏死因子 TNF- a释放； 该重组蛋白腹腔注 射可以保护小鼠， 大幅降低内毒素休克的死亡率， 静脉注射该重组蛋白降低混合细菌感 染死亡率， 外源性或内源性上调该蛋白血清水平 24-76%导致大幅降低混合细菌感染死 亡率。
外源性和内源性上调血清 CRISPLD2水平，保护混合细菌感染 CLP小鼠的结果提示： 1. 混合细菌感染中， 内毒素 (LPS)是引起脓毒性休克的几种已知细菌抗原中的主要抗原 之一； 2. CRISPLD2是 LPS病理学反应的关键血清调控蛋白。 3. 由细菌抗原引起的反 应， 可能包括凝血系统异常， 过度的免疫反应， 组织器官衰竭以及其它血崩式的病理学 反应； CRISPLD2蛋白的调控机制可以发生在病原分子与其细胞受体结合之前。
3. 临床应用
根据以上理论基础， 临床研究发现： 脓毒症病人可以分为三个亚型， 其中 50-60% 以 上的病人为 CRISPLD2-低/ PCT-高， 与高死亡率相关； 而 CRISPLD2-高 /PCT-高亚型和 CRISPLD2-高-低 /PCT-低与低死亡率相关。 严重脓毒血症病人 CRISPLD2浓度低下 /PCT 浓度高的血清样本出现， 可以提示该病人的严重程度和预后。 血清 CRISPLD2水平检测 适用于严重脓毒症及脓毒性休克病人诊断和预后。
脓毒血症病人中血清出现 CRISPLD2-高,低 /PCT-低或者 CRISPLD2-高 /PCT-高的特 征， 提示医生注意该病人临床检验细菌培养的结果， 排除格兰氏阳性细菌感染脓毒血症 可能性。
而其他细菌感染相关病人血清中 CRISPLD2与 PCT水平的检测可以用来评估
CRISPLD2调控身免疫反应以及宿主炎症反应的程度， 血清中 CRISPLD2-低 /PCT-高的 一旦出现可以提示医生采取措施， 并对病人将来可能的变化做出预测。
在出血性休克病人中 CRISPLD2水平与 PCT水平变化规律与细菌感染性休克情况完 全不同。
重组 CRISPLD2可以用于治疗， 保护严重脓毒症病人降低死亡率。
本发明人还根据实验结果和严重脓毒血症病人临床研究， 提出了应用临床预后的血 清 CRISPLD2浓度阈值标准， 以及预防、 干预治疗的方法。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人 员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所 限定的范围。

Claims

权 利 要 求
1. 含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2或其编码基因的用 途， 用于制备预防、 缓解或治疗全身性细菌感染以及相关疾病的药物。
2. 如权利要求 1所述的用途， 其特征在于， 所述的药物用于：
阻断脂多糖与靶细胞受体结合； 或
抑制脂多糖诱导的炎症因子释放。
3. 如权利要求 1所述的用途， 其特征在于， 所述的含 LCCL-RAAIH (R3H) 结构域 的半胱氨酸富集的分泌蛋白 2通过结合细菌单链 DNA和 /或脂多糖预防、 缓解或治疗脓 毒血症、 严重脓毒血症或感染性休克。
4. 含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2或其编码基因的用 途， 用于制备诊断或预后脓毒血症、 严重脓毒血症或感染性休克的试剂或试剂盒。
5. 特异性识别含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2或其编 码基因的试剂的用途， 用于制备诊断或预后细菌感染脓毒血症、 严重脓毒血症或感染性 休克的试剂盒。
6. 如权利要求 5所述的用途，其特征在于，所述的特异性识别含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2或其编码基因的试剂选自：
特异性扩增含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2基因的引 物；
特异性识别含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2基因的探 针； 或
特异性结合含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2的抗体或 配体。
7. 如权利要求 5或 6所述的用途，其特征在于，所述的特异性识别含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2的试剂是抗含 LCCL-RAAIH (R3H) 结构域 的半胱氨酸富集的分泌蛋白 2的抗体。
8. 一种用于诊断或预后脓毒血症、 严重脓毒血症或感染性休克的试剂盒， 其特征在 于， 它包括：
容器， 以及位于容器中的特异性识别含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富 集的分泌蛋白 2或其编码基因的试剂。
9. 如权利要求 8所述的试剂盒， 其特征在于， 所述的特异性识别含 LCCL-RAAIH (R3H) 结构域的半胱氨酸富集的分泌蛋白 2的试剂是抗含 LCCL-RAAIH (R3H)结构域的 半胱氨酸富集的分泌蛋白 2的抗体。
10. 一种预防、 缓解或治疗细菌感染或细菌感染相关疾病的方法， 其特征在于， 该 方法包括：给予细菌感染者含 LCCL-RAAIH (R3H)结构域的半胱氨酸富集的分泌蛋白 2。
PCT/CN2011/084641 2011-09-19 2011-12-26 预防、诊断、治疗、预后全身性细菌感染疾病的方法和试剂 WO2013040840A1 (zh)

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CN101766806A (zh) * 2008-12-29 2010-07-07 上海人类基因组研究中心 Crispld2蛋白在炎症方面的应用
CN102028935A (zh) * 2009-09-29 2011-04-27 上海南方基因科技有限公司 败血症以及败血症休克的预测、预防和治疗方法及试剂盒

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