CN102741272B - Peptides inhibiting adhesion of aspergillus fumigatus to cornea - Google Patents
Peptides inhibiting adhesion of aspergillus fumigatus to cornea Download PDFInfo
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Abstract
Provided are peptides inhibiting the adhesion of Aspergillus fumigatus to cornea, comprising: (1) peptide with the sequence of SEQ ID NOs: 1-10 respectively; (2) peptide having 70% or above homology to the peptide of (1) respectively, whose function is the same or similar with that of the peptide of (1). Also provided are polynucleotides. The polynucleotides are 1) a nucleotide of any peptide with the sequence of SEQ ID NOs: 1-10; 2) nucleotides of the peptides having 70 % or more homology with the peptide described in (1), and the encoded peptides have the same or the similar functions with the peptide described in (1). 10 peptides selected by the invention have competitive inhibitory effect on the interaction of the Aspergillus fumigatus and epithelial cells of the cornea in different degrees, can specifically block the adherence between ligands on the surface of the Aspergillus fumigatus and the epithelial cells of the cornea, and become new target spots for designing treatment drugs of fungal keretitis, thereby providing new ideas for clinical treatment thereof.
Description
Technical field
The invention belongs to the biological prevention field of infectious eye disease, be specifically related to suppress the polypeptide that aspergillus fumigatus adheres to cornea.
Background technology
Keratitis is the modal frequently-occurring disease of eye, causes that the pathogenic micro-organism of keratitis comprises fungi, bacterium, virus and Acanthamoeba etc.In recent years, along with the abuse of Broad spectrum antibiotics and immunosuppressor, and generally the wearing of contact lens, the incidence probability of keratitis increases greatly.Fungal keratitis is more common in and is take agricultural population as main developing country, in China, there is the trend increasing year by year, relevant statistics shows that fungal keratitis proportion in the infectious keratopathy of China, up to 34.8%～61.9%, has become the first cause of corneal blindness in some areas.Fungal keratitis is often not clear because infecting Pseudomonas or bacterial strain, and drug susceptibility is differed, and treats comparatively thorny.Once patient's diagnosis and treatment danger that just has blinding not prompt enough.So it is vital that corneal inflammation is carried out effectively treatment at the initial stage of a disease.For this reason, carry out the repercussion study of fungi and corneal epithelial cell, can be more early to finding, block sooner, more by force the action target spot of fungi infestation process, there is important clinical meaning.
The pathogenic course of pathogenic bacteria is the interactional result of itself and host.Pathogenic agent, the interactional research of host cell are often conceived to single molecule or a few molecules, even in some research system, only observe individual gene and are just judged as the key factor of decision host to the susceptibility of certain pathogenic agent.But due to both sides' complicacy and adaptability to the other side separately, the interaction between pathogenic agent-host is in dynamic variation.At present not fully aware of for the interaction mechanism of pathogenic bacteria and corneal cell.Known pathogenic fungi and bacterial adhesion are the first steps that keratitis occurs in host's corneal epithelial cell, this adhesion that wherein combination between ligands specific-acceptor or nonspecific physical and chemical effect produce all plays an important role, so block these adhesion process, are good strategies preventing and treating keratitis, disease is controlled to bud.
Aspergillus fumigatus (Aspergillus fumigatus) is as one of pathogenic species of modal fungal keratitis, and its toxicity is stronger, and the PD of initiation is rapid.But the concrete mechanism that at present adheres to corneal epithelial cell for aspergillus fumigatus is also and not bery clear, therefore for how in the research of adhesion phase blocking-up aspergillus fumigatus infection corneal cell also in the starting stage.
Summary of the invention
The object of this invention is to provide and suppress the polypeptide that aspergillus fumigatus adheres to cornea, can be more early, block sooner, more by force the action target spot of aspergillus fumigatus infection cornea, for the clinical treatment of infectious keratopathy provides new thinking, to make up the deficiencies in the prior art.
The present invention utilizes people's corneal epithelial cell to carry out three-wheel biocompatible to random 12 peptide libraries of phage display and washes in a pan sieve, after a large amount of amplifications of the clone of gained, carry out extraction and the sequencing of DNA, then 10 screened polypeptide are carried out to synthetic, through ELISA method, further verify the peptide section of synthetic and the avidity of people's corneal epithelial cell.Then utilize cell model and organ model research section of synthesized peptide on aspergillus fumigatus and the interactional impact of corneal epithelial cell, thus the prevention effect of checking polypeptide to infectious keratopathy.
The present invention includes following components:
One aspect of the present invention is to provide and suppresses the polypeptide that aspergillus fumigatus adheres to cornea, includes:
(1) sequence is respectively the polypeptide of SEQ ID NO:1-10,
(2) respectively with (1) in polypeptide have 70% or the polypeptide of above homology, the function of this polypeptide is same or similar with the polypeptide function in (1).
For the polypeptide described in (2), can be to have the polypeptide of 75% homology (3 amino acid whose different), 80% above homology (2 amino acid whose differences) and 90% above homology (1 amino acid whose difference) from arbitrary polypeptide in (1), the function of this polypeptide be same or similar with the polypeptide function in (1).
Another aspect of the present invention is to provide and comprises the fusion polypeptide that sequence is arbitrary polypeptide in SEQ ID NO:1-10.Described fusion polypeptide can be by Chemical bond method, enzyme blanking method or physical rupture method, to process polypeptide fragment to obtain.For example sequence is the protein complexes that in SEQ ID NO:1-10, arbitrary polypeptide and antigen vectors are connected to form, and antigen vectors can be oralbumin (OVA) or bovine serum albumin (BSA).
The present invention also provides a kind of polynucleotide, and described polynucleotide are:
1) encoding sequence is the Nucleotide of arbitrary polypeptide in SEQ ID NO:1-10
2) coding and polypeptide in (1) have 70% or the Nucleotide of the polypeptide of above homology, and the polypeptide function in the function of coded polypeptide and (1) is same or similar.
Described Nucleotide is DNA or RNA, preferably DNA.
The present invention also provides a kind of pharmaceutical composition, comprises that sequence is any one or several in the polypeptide of SEQ ID NO:1-10; Described goods have the function that prevention aspergillus fumigatus adheres to cornea.
Sequence provided by the present invention is to appoint one or several to can be used for Dispersal risk in the polypeptide of SEQ ID NO:1-10.Preferably 10 polypeptide carry out Dispersal risk as common antigen after mixing.
The antibody of preparation is applied in control aspergillus fumigatus and adheres in the medicine of cornea, and the antibody of preparation can also be applied in the product that detects aspergillus fumigatus simultaneously.
10 polypeptide that the present invention screens have competitive inhibition in various degree to the interaction of aspergillus fumigatus and corneal epithelial cell, part that can specific blocking-up aspergillus fumigatus surface and the adhesion of corneal epithelial cell acceptor, become the novel targets of fungal keratitis medicine design, thereby provide new thinking for its clinical treatment.
Accompanying drawing explanation
Fig. 1: the effect histogram that polypeptide of the present invention and people's corneal epithelial cell avidity ELISA detect.
Fig. 2: the effect histogram that polypeptide of the present invention detects the ELISA of the competitive inhibition of phage clone adhesion people corneal epithelial cell.
Fig. 3: polypeptide of the present invention adheres to the restraining effect of people's corneal epithelial cell to the common pathomycete aspergillus fumigatus of ophthalmology, result shows that Pc-C and Pc-E adhere to corneal epithelial cell to aspergillus fumigatus and have stronger restraining effect.
Fig. 4: 2 Pc-C in polypeptide of the present invention and Pc-E different concns adhere to the restraining effect of corneal epithelial cell to aspergillus fumigatus.
Fig. 5: 2 Pc-C in polypeptide of the present invention and Pc-E adhere to the restraining effect of corneal epithelial cell in organ model to aspergillus fumigatus.
Fig. 6: in aspergillus fumigatus with the sequence of 2 polypeptide Pc-A of the present invention and Pc-B homology the deletion mutant of corresponding albumen compare the variation that adheres to corneal epithelial cell at cell levels with wild strain, and Pc-A and the restraining effect of 2 polypeptide of Pc-B to their adhesion process.
Fig. 7: in aspergillus fumigatus with the sequence of 2 polypeptide Pc-A of the present invention and Pc-B homology the deletion mutant of corresponding albumen compare variation and Pc-A and the restraining effect of Pc-B2 bar polypeptide to their adhesion process that adheres to corneal epithelial cell in organ model with wild strain.
Embodiment
Below the operation of polypeptide of the present invention and other side is described in detail.
One, the screening of polypeptide
Display technique of bacteriophage is the DNA sequence dna of foreign protein or polypeptide to be inserted into the appropriate location of bacteriophage coat protein structure gene, foreign gene is expressed with the expression of coat protein, simultaneously foreign protein is with the re-assemblying and be shown to the biotechnology of phage surface of phage, and the albumen of expressing on phage ghost can be folded into native conformation and have corresponding biologic activity.These phages that are loaded with foreign protein can form phage peptide library, and it has huge potentiality in pathogenic agent and the interactional research of host.The polypeptide of application phage-displayed library screening simulation part (cytokine etc.), the little peptide screening can with corresponding receptors bind, the activity of its part of competitive antagonism.Display technique of bacteriophage is a kind of method of studying ligand-receptor interaction very widely of application at present, utilize this method to find the stand-in of the aglucon of known receptor, the biotechnology new drug for disease treatment for development with independent intellectual property rights has important theory significance and potential using value.
Embodiment 1: screening suppresses the polypeptide that aspergillus fumigatus adheres to cornea
1) people's corneal epithelial cell (HCEC) and 10 of individual layer will be cultured to
1012 peptide libraries of the phage display of pfu (New England Biolabs company product, in the pIII of M13 phage albumen, embed the random peptide of 12 amino acid longs) 37 ℃ hatch 1hr, PBS washes away unconjugated phage, under glycine elutriant wash-out, be combined in the phage on HCEC surface, through amplification, carrying out same 2 after titration again and take turns and wash in a pan sieve, can there are with HCEC 14 polypeptide of phage of combination in final acquisition.Picking phage mono-clonal carries out sequencing, the nucleotide sequence of acquisition is translated into polypeptide, to the corresponding polypeptide of positive phage clone, utilize the pBLAST that NCBI provides to carry out homology analysis, find wherein certain DNA homolog of 10 polypeptide and aspergillus fumigatus.
2) synthetic of polypeptide fragment and evaluation
The corneal epithelial cell filtering out 12 peptide libraries from phage display is had to adhesive attraction, and by corresponding gene function analysis, likely participating in pathogenic bacteria and place lives interactional 12 peptide fragment and carries out synthetic, N end biotinylation is modified and is beneficial to subsequent detection, and the amidation of C end modifies to prevent degraded.
People's corneal epithelial cell after a certain amount of section of synthesized peptide and sealing is hatched to 12 peptide fragment that the straight target avidin of employing HR synthesizes by the detection of ELISA method and the avidity (Fig. 1) of people's corneal epithelial cell.10 polypeptide that screen can be combined with people's corneal epithelial cell in various degree.
In addition by a certain amount of synthetic polypeptide and 10
10the phage polypeptide of pfu simultaneously with sealing after HCEC at 37 ℃, hatch 1hr, adopt the anti-M13 antibody of the straight target of HR to detect synthetic polypeptide fragment by ELISA method and phage polypeptide adhered to the competitive inhibition (Fig. 2) of HCEC.10 polypeptide that screen can be in various degree the adhesion of the corresponding phage polypeptide of competitive inhibition and people's corneal epithelial cell, synthesize as seen polypeptide and can substitute phage polypeptide.
3) synthetic polypeptide suppresses the experiment that pathogenic bacteria adheres to corneal epithelial cell
The people's corneal epithelial cell that is cultured to individual layer is first hatched 1hr with the polypeptide of 100uM, then add appropriate Aspergillus fumigatus spores to hatch after 1hr at 37 ℃, PBS washes away unconjugated fungi, and the cell after effect and the pathogenic bacteria of adhesion are detected to the fungi number that adheres to cell surface under the microscope; By the system lysate cracking after effect, the fungi wherein adhering to is counted simultaneously.Then candidate's polypeptide that same method is further studied different concns adheres to the restraining effect of corneal epithelial cell to aspergillus fumigatus, thus calculation of half inhibitory concentration (IC
50).And utilize the organ model research candidate polypeptide of cornea aspergillus fumigatus to be adhered to the restraining effect of cornea, final definite polypeptide that can suppress aspergillus fumigatus adhesion corneal epithelial cell.
Ten polypeptide that filter out adhere to the restraining effect of people's corneal epithelial cell to the common pathomycete aspergillus fumigatus of ophthalmology: by being cultured to people's corneal epithelial cell of 80%, first hatch 1hr with 37 ℃ of the polypeptide of 10 100uM respectively, then respectively add 10
6the aspergillus fumigatus of cfu again 37 ℃ hatch 1hr, PBS washes away unconjugated pathogenic bacteria, direct census under microscope after lysate cracking, do not add that polypeptide processes as negative control (Fig. 3).Compared with the control visible, 10 polypeptide can adhere to corneal epithelial cell by inhibition aspergillus fumigatus in various degree, two peptide sections of Pc-C and Pc-F particularly, and inhibiting rate can reach 90%.
The polypeptide that selection filters out by phage display, carry out the mensuration of half Mlc (IC50), adopt aforesaid method to detect different concns polypeptide (100uM, 10uM, 1uM, 0.1uM and 0.01uM) aspergillus fumigatus is adhered to the restraining effect (Fig. 4) of corneal epithelial cell.Result shows that the inhibition that Pc-C and two polypeptide of Pc-F adhere to corneal epithelial cell to aspergillus fumigatus shows certain dose-dependently, along with the rising of peptide concentration, raises to the inhibiting rate adhering to.
Win the agar surface that BALB/c mouse eyeball organ is put in 96 orifice plates, corneal epithelium is crossed and is damaged, and the polypeptide that drips 100uM is hatched 1hr in advance, adds 10
7the aspergillus fumigatus effect 2hr of cfu, then PBS rinses unconjugated fungal spore, cuts cornea homogenate, gradient dilution bed board counting, do not add that polypeptide processes as negative control (Fig. 5).Result shows that Pc-C and two polypeptide of Pc-F adhere to mouse cornea to aspergillus fumigatus and have certain restraining effect, and the bacterium amount adhering to compared with the control significantly reduces.
4) the polypeptid specificity that sieves checking
Pc-A and Pc-B are higher through two sections of amino acid sequence homologies of the Alb1 of homology analysis discovery and aspergillus fumigatus albumen, so utilize the mutant Δ alb1/B-5233 of aspergillus fumigatus B-5233 and alb1 Gene Partial disappearance (just comprising and Pc-A and Pc-B homologous amino acid region) thereof to carry out the experiment of Pc-A and two polypeptide inhibition aspergillus fumigatuses adhesion corneas of Pc-B, thereby determine that the effect of the inhibition pathogenic bacteria adhesion corneal cell of polypeptide is specific competitive blocking-up.
By being cultured to people's corneal epithelial cell of 80% separately or mixing, add the Pc-A of 100uM and Pc-B polypeptide to hatch in advance 1hr, each polypeptide treatment group divides 2 groups to add respectively 10 again
6the aspergillus fumigatus of cfu and alb1 mutant effect 1hr thereof, PBS washes away unconjugated pathogenic bacteria, gradient dilution bed board counting after lysate cracking, without polypeptide, process as negative control (Fig. 6).Result shows that alb1 gene obviously weakens at the relative wild strain of ability of the aspergillus fumigatus mutant adhesion corneal epithelial cell of Pc-A and Pc-B corresponding zone disappearance, and Pc-A and two polypeptide of Pc-B show certain competitive inhibition to existing the wild strain of alb1 gene to adhere to corneal epithelial cell, to not effect of gene-deleted strain.
Utilize equally BALB/c mouse eyeball organ to cross and damage at corneal epithelium, Pc-A and Pc-B polypeptide independent or mixing dropping 100uM are hatched 1hr in advance, each polypeptide treatment group is divided 2 groups of aspergillus fumigatus and alb1 mutant effect 2hr thereof of adding respectively 107cfu again, then PBS rinses unconjugated fungal spore, cut cornea homogenate, gradient dilution bed board counting, do not add that polypeptide processes as negative control (Fig. 7).Result shows that alb1 gene obviously weakens at the relative wild strain of ability of the aspergillus fumigatus mutant adhesion mouse cornea organ of Pc-A and Pc-B corresponding zone disappearance, and Pc-A and two polypeptide of Pc-B show certain competitive inhibition to existing the wild strain of alb1 gene to adhere to cornea, to not effect of gene-deleted strain.
Found that ten polypeptide (table 1) adhere to corneal epithelial cell to aspergillus fumigatus and all have restraining effect, obvious is Pc-C and two polypeptide of Pc-E (P < 0.05), its half-inhibition concentration (IC50) is respectively 4.79uM and 3.02uM, and by description of test that the deletion mutant of Pc-A and aspergillus fumigatus homologous sequence corresponding to Pc-B is carried out with the polypeptide of screening to some extent the corresponding albumen of sequence of homology be the factor playing a role when aspergillus fumigatus adheres to host cell really, also illustrate that the inhibition that polypeptide adheres to corneal cell to aspergillus fumigatus is that structure is specific simultaneously, part that can competitive blocking-up pathogenic fungi surface adheres to the acceptor of corneal epithelial cell.In addition, these 10 polypeptide have good solvability (can be prepared into easily the stock solution of 1mM in pure water), and do not produce any cytotoxicity (add 37 ℃ of 100uM polypeptide to hatch observe growth conditions under the microcytoscope of the longest 4hr good, do not find cell injury sign).
Table 1: polypeptide of the present invention and corresponding nucleotide sequence information
The present invention also comprises that encoding sequence is arbitrary the polypeptide of SEQ ID NO:1-10 or the Nucleotide of its homeopeptide, and the sequence of Nucleotide is SEQ ID NO:11-20 (table 1).Because amino acid has many codons, each polypeptide may have several coding nucleotides, so Nucleotide of the present invention is not only limited to the Nucleotide that sequence is SEQ ID NO:11-20.
Polypeptide, polypeptide fragment and the fusion rotein thereof two, with the polypeptide of SEQ ID NO:1-10 with homology
The present invention also protect with SEQ ID NO:1-10 in polypeptide have respectively 70% or the polypeptide of above homology, the polypeptide function in the function of this polypeptide and SEQ ID NO:1-10 is same or similar.
Described have a homology polypeptide, can be the polypeptide that arbitrary the polypeptide that be SEQ ID NO:1-10 from sequence has 75% homology (3 are amino acid whose different), 80% above homology (2 amino acid whose differences) and 90% above homology (1 amino acid whose difference), the function of this polypeptide with sequence be that arbitrary the polypeptide function of SEQ ID NO:1-10 is same or similar.
For example, sequence is the polypeptide of SEQ ID NO:21, it is compared with the polypeptide that site name is called Pc-C, is to have added that at N end Ser-Gly-Ser (SGS), the sequence of acquisition are that the polypeptide of SEQ ID NO:21 and Pc-C polypeptide that sequence is SEQ ID NO:3 have 80% homology.And suppress experiment that pathogenic bacteria adheres to corneal epithelial cell, show that sequence is that the polypeptide of SEQ ID NO:21 also has and suppresses the function that aspergillus fumigatus adheres to.
Described homology sequence can also be to replace by amino-acid residue the polypeptide obtaining, for example sequence is that the polypeptide of SEQ ID NO:22 is to be that ALA in the polypeptide of SEQ ID NO:5 replaces with GLY by sequence, both compare the homology having more than 90%, and also have the function that suppresses aspergillus fumigatus adhesion.
Described homology polypeptide can be used Applied biosystem synthesizer or Pioneer
tMit is synthetic that the Peptide synthesizer devices such as peptide synthesizer are pressed solid state chemistry technology.Also can give expression to needed polypeptide by inserting the expression vector of object nucleotide fragments.
Article of the present invention ten, polypeptide can also be connected and make complete antigen with protein carrier, protein carrier can be oralbumin (OVA) or bovine serum albumin (BSA), pass through carbodiimide method, the amino of polypeptide N-terminal is connected with the carboxyl on carrier proteins, and then obtains highly purified complete antigen after adopting gradient method dialysis.
Embodiment 2: the preparation of complete antigen:
(1) of the present invention one or several polypeptide is dissolved in to the polypeptide mother liquor that is mixed with 0.1 μ g/ μ L in dimethyl sulfoxide (DMSO) (DMSO); Water-soluble carbodiimide (EDC), N-hydroxy-succinamide (NHS) and oralbumin (OVA) are mixed with respectively to the working fluid of 1mg/mL with 0.01M phosphate buffered saline buffer (PBS, pH7.5);
(2) get 100 μ L polypeptide mother liquors, add 20 μ L EDC working fluids and 12 μ L NHS working fluids, the mol ratio of STX, EDC, NHS reaction is 1: 2-3: 1-2, and room temperature concussion reaction 3h, obtains initial reaction liquid;
(3) protein carrier is made into the working fluid of 1mg/mL with 0.01M phosphate buffered saline buffer (PBS, pH7.5).
(4) optimum mole ratio of STX and carrier proteins reaction is 10: 1; Reactant in (2) step is joined in the carrier proteins working fluid of 100 μ L, 4 ℃ of concussion reaction 12h, obtain end reaction liquid;
(5) above-mentioned end reaction liquid is transferred in dialysis card, with the PBS damping fluid of 300mL0.01M pH7.5 and the mixed solution of DMSO dialysis 72h, every 12h changes liquid 1 time, dialysis adopts gradient dialysis method, the rear taking-up reactant of having dialysed, finally obtains being connected with the complete antigen of one or several polypeptide of the present invention.
In specific experiment, Pc-A polypeptide, the sequence that is SEQ ID NO:1 by sequence be SEQ ID NO:5 Pc-E polypeptide respectively/is simultaneously connected and makes complete antigen with oralbumin OVA, and prepared by the antibody that can be used for subsequent embodiment.
Three, comprise the pharmaceutical composition that sequence is one or several polypeptide of SEQ ID NO:1-10 polypeptide
Polypeptide of the present invention can be used for pharmaceutical compositions, for preventing the adhesion of aspergillus fumigatus to host cell.Described pharmaceutical composition contains treatment significant quantity, and sequence is one or several polypeptide of SEQ ID NO:1-10, and the auxiliary material such as acceptable carrier or solvent on the pharmaceutical industries of one or more.
Embodiment 3: the preparation of complex polypeptide eye drop and control aspergillus fumigatus infection effect detection thereof
Get a certain amount of Pc-C and Pc-E two peptide species lyophilized powders are dissolved in eye drop common solvent borate buffer solution, be mixed with peptide concentration and be respectively 0.1% eye drop, and utilize sodium bicarbonate adjust pH to 5.5-7.5, utilizing glucose to regulate osmotic pressure is 270-310mOsm/kg, makes it meet the standard of conventional eye drop.
Before infecting, 1hr, with complex polypeptide eye drop to healthy BALB/c mouse eye drip, 15min/ time, continuous 5 times, then utilizes cornea to scratch and coordinates contact lens method to set up aspergillus fumigatus (10
7cfu) mouse model infecting, continues to carry out eye droppings control, 15min/ time with complex polypeptide eye drop after infecting, continuous 5 times, after 8hr, take off cornea, gradient dilution bed board counting after homogenate cracking, only infect do not carry out the control of complex polypeptide eye drop synchronous experiment mice in contrast.Found that this eye drop has good prevention effect to the infection of aspergillus fumigatus, bacteriostasis rate can reach 80%.
Four, sequence of the present invention is that SEQ ID NO:1-10 polypeptide is for the preparation of antibody
Embodiment 4: the preparation of antibody
The female BALB/c mouse in immune 6 week age, 200 μ L Pc-E polypeptide for first immunisation (containing 5 μ g polypeptide) and equal-volume Freund's complete adjuvant, after fully emulsified, make emulsion, intraperitoneal injection of mice, every two weeks, get afterwards same amount Pc-E polypeptide and equal-volume Freund's incomplete adjuvant fully emulsified, abdominal injection.After immune three times, get tail hematometry mice serum and tire, if it is not high to tire, continue immunity.Wait tiring, be greater than after required value, kill mouse and get blood, the blood room temperature of taking-up is placed 30min, and the centrifugal 20min of 8000r/min, gets upper serum, with albumin A antibody centrifugal purification test kit purified blood serum, makes the antibody of anti-Pc-E polypeptide.Prepared antibody is surveyed it by indirect competitive ELISA method and is tired, and it is reference that the OD value of positive serum of take is greater than negative 2 times, and mice serum is tired and reached 2400, experimental results show that and in Mice Body, has produced antibody.
Female BALB/c mouse with 6 week age of complete antigen immunity of the Pc-A polypeptide of embodiment 2 preparation or Pc-E polypeptide, indirect competitive ELISA method is surveyed it and is tired, result shows, the mice serum that has connected the Pc-E polypeptide of protein carrier is tired and reached 3500, far above Pc-E polypeptide is haptenic, tires.
The antibody of preparation can be for the preparation of medicine or detection kit, for detection of aspergillus fumigatus.The specific reaction that utilizes the corresponding antigen of prepared antibody and aspergillus fumigatus surface polypeptide, detects aspergillus fumigatus.First in the fungal keratitis model of the BALB/c mouse of utilizing aspergillus fumigatus to build, test, scraping pathological corneas surface lesion tissue, after paving sheet, utilize the capable immunohistochemistry of antibody of preparation to detect, if the aobvious positive illustrates on pathological corneas, have aspergillus fumigatus to exist.In the lesion tissue scraping blade of the pathological corneas of doubtful fungal keratitis, by ImmunohistochemistryMethods Methods, carry out the validation verification of detection kit clinically in addition, if positive antigen can be detected, explanation is pathology due to aspergillus fumigatus infection cornea.
Industrial applicibility
10 polypeptide that the present invention screens have competitive inhibition in various degree to the interaction of aspergillus fumigatus and corneal epithelial cell, part that can specific blocking-up aspergillus fumigatus surface and the adhesion of corneal epithelial cell acceptor, become the novel targets of fungal keratitis medicine design.The mono-clonal of the polypeptide preparation screening or polyclonal antibody can be used for detecting aspergillus fumigatus, thereby can effectively prevent the infection of aspergillus fumigatus corneal.
Claims (5)
1. suppress the polypeptide that aspergillus fumigatus adheres to cornea, it is characterized in that, the sequence of described polypeptide is SEQ ID NO:2.
2. a fusion rotein, described fusion rotein is that polypeptide claimed in claim 1 and carrier proteins are connected to form.
3. fusion rotein as claimed in claim 2, is characterized in that described carrier proteins is oralbumin or bovine serum albumin.
4. polynucleotide, described polynucleotide encoding polypeptide claimed in claim 1, its sequence is SEQ ID NO:12.
5. suppress the pharmaceutical composition that aspergillus fumigatus adheres to cornea, include polypeptide claimed in claim 1.
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Non-Patent Citations (5)
| Title |
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| Hui Guo et al..Innate responses of corneal epithelial cells against Aspergillus fumigatus challenge.《FEMS Immunology & Medical Microbiology》.2009,第56卷(第1期),88-93. |
| Hui Guo et al..Innate responses of corneal epithelial cells against Aspergillus fumigatus challenge.《FEMS Immunology & * |
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| CN102741272A (en) | 2012-10-17 |
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