CN105859838B - A kind of Escherichia coli O 157: H7 protein I vy polypeptide, anti-Ivy polyclonal antibody and its application - Google Patents
A kind of Escherichia coli O 157: H7 protein I vy polypeptide, anti-Ivy polyclonal antibody and its application Download PDFInfo
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- CN105859838B CN105859838B CN201610219949.XA CN201610219949A CN105859838B CN 105859838 B CN105859838 B CN 105859838B CN 201610219949 A CN201610219949 A CN 201610219949A CN 105859838 B CN105859838 B CN 105859838B
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- C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
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Abstract
The invention discloses a kind of Escherichia coli O 157s: H7 Ivy antigen polypeptide, anti-Escherichia coli O 157: the polyclonal antibody of H7 Ivy and its application.A kind of Escherichia coli O 157 of the present invention: the amino acid sequence of H7 Ivy antigen polypeptide is as shown in SEQ ID NO:1.Using a kind of Escherichia coli O 157: anti-Escherichia coli O 157: the polyclonal antibody of H7 Ivy has been prepared as immunogen immune BALB/c mouse in H7 Ivy antigen polypeptide, qualification result shows that the polyclonal antibody can specific recognition Escherichia coli O 157: H7 Ivy albumen, it can be used for Escherichia coli O 157 in the test such as ELISA, Western blot, immunofluorescence: the detection of H7 Ivy albumen, powerful is provided for the basic research of the protein function, is had a extensive future.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of Ivy antigen polypeptide, anti-Escherichia coli O 157: H7 albumen
The polyclonal antibody of Ivy and its application.
Background technique
Lysozyme is prevalent in animal, is the important composition ingredient of many biological innate immune systems, has and exempt from
Epidemic disease function, digestive function, chitinase activity and isopeptidase activity.Since lysozyme has universal bacteriostasis, bacterium
During same lysozyme interacts for a long time, develops and resist the effect of lysozyme by the method for modifying peptide glycan.
In recent years, research found that bacterium also resisted lysozyme activity by generating bacteriolyze enzyme inhibitor.First bacterium lysozyme suppression
Preparation is found in Escherichia coli, due to its can specificity inhibition vertebrate c type lysozyme activity, be named as
Vertebrate bacteriolyze enzyme inhibitor (Inhibitor of vertebrate lysozyme, Ivy).Ivy can make Escherichia coli
Have the function of resisting lysozyme activity to guarantee that it can survive in the saliva of people and egg white.Ivy is primarily present in carefully
Born of the same parents' pericentral siphon plays the role of inhibiting lysozyme activity, to make the peptide on bacteria cell wall by closing lysozyme activity site
Decomposition of the glycan from exogenous lysozyme.Ivy may also have control other than protecting bacteria from exogenous lysozyme infringement
The work of endogenous bacteria autolytic enzyme (autolytic enzyme is the key enzyme of degradation peptide glycan, and cell is grown and divided most important) processed
With.Ivy is in the mechanism of action in terms of Escherichia coli escape host's lysozyme and the relationship between Ivy and Drug Resistance of E. coli
It needs to be studied.Therefore, preparation, which has, inhibits the active antibody meaning of Ivy very great, not only can be the function of the albumen
It can study and lay the foundation, while provide experimental data for Drug Resistance of E. coli research and supporting.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of to be used for ELISA, western blot, exempt from
The anti-Escherichia coli O 157 of epidemic disease fluorescence detection: the polyclonal antibody of H7 Ivy albumen.
Another object of the present invention is to provide a kind of Escherichia coli O 157: H7 Ivy antigen polypeptide.
It is yet another object of the invention to provide above-mentioned anti-Escherichia coli O 157s: the polyclonal antibody of H7 Ivy albumen is answered
With.
Above-mentioned purpose that the invention is realized by the following technical scheme:
A kind of Escherichia coli O 157: H7 Ivy antigen polypeptide, amino acid sequence is as shown in SEQ ID NO:1.Preferably,
The antigen polypeptide and keyhole blood indigo plant carrier protein KLH is coupled.
A kind of polyclonal antibody of anti-Ivy, is using aforementioned Ivy antigen polypeptide as immunogene, immune animal is prepared.
The preparation method of the polyclonal antibody of above-mentioned anti-Ivy, steps are as follows: Ivy antigen polypeptide and maleic amide activation
Keyhole blood indigo plant carrier protein KLH coupling, as immunogene and adjuvant mixed immunity BALB/c mouse after desalting and purifying, during which
It is immunized more than three times, after ELISA detection serum antibody titer reaches 1:100000, acquires mice serum, by purifying,
After ELISA and western blot identification, the polyclonal antibody of anti-Ivy is obtained.
Further, the present invention also provides the polyclonal antibodies detects Escherichia coli O 157: H7 Ivy in preparation
Application in the reagent of albumen infection, the polyclonal antibody inhibit Escherichia coli O 157: H7 Ivy albumen proliferation in preparation
Reagent in application and polyclonal antibody preparation treat Escherichia coli O 157: H7 Ivy albumen infection reagent in
Using.
Chemically synthesized polypeptide antigen is small molecule, itself is difficult the antigenicity having had, and animal can only be induced to generate very
Weak immune response, thus it is critically important for being crosslinked with carrier protein.Carrier protein contains many epitopes, can stimulate
T helper cell, and then induce B cell reaction.There are many carrier proteins for being crosslinked with polypeptide, wherein the load most generally used
Body is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin(BSA) (bovine
Serumalbumin, BSA), ovalbumin (ovalbumin, OVA) and bovine thyroglobulin (bovine
Thyroglobulin, THY).KLH has higher antigenicity, most with immunogenicity after Ivy antigen polypeptide crosslinking of the invention
By force, therefore the present invention selects KLH as coupling carrier albumen.BSA is also often used as peptide carrier, but due to the frequent quilt of BSA
Be used as detection test blocking agent and make this method produce antibody application on there is certain limitations.
Escherichia coli O 157: the polyclonal antibody of H7 Ivy is in the large intestines bar such as ELISA, western-blot, immunofluorescence
Application in bacterium O157:H7 Ivy protein measurement assays.
Compared with prior art, the invention has the following advantages:
(1) present invention is to Escherichia coli O 157: the secondary structure of H7 Ivy protein amino acid sequence, immunogenicity, close and distant
Aqueous, surface accessibility etc. is analyzed, and it is artificial synthesized to determine that suitable one section of intracellular region peptide sequence carries out；By the polypeptide of synthesis
It is coupled with the carrier KLH of maleimide ammonia activation, this coupled product is subjected to desalting column, BALB/c mouse is immunized after purification；Through
It crosses repeatedly immune mice serum to detect antibody titer with ELISA method, potency collects immune mouse blood after reaching ideal value
Clearly, and with Protein G-protein purification column antibody purification；The identification such as ELISA, Western bot is carried out to purified antibodies.
Qualification result shows that the polyclonal antibody can specific recognition Escherichia coli O 157: H7 Ivy albumen；
(2) this antibody purification is used to carry out ELISA, Western bot, the immunofluorescence dyeing of Escherichia coli by the present invention
Deng experiments have shown that it can identify natural Ivy albumen.The polyclonal antibody of this Ivy is further to study Escherichia coli O 157: H7
The function of Ivy is laid a good foundation.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are only used for explaining this hair
It is bright, rather than limit the scope of the invention.Under the premise of without departing substantially from technical solution of the present invention, made for the present invention
Field those of ordinary skill any change easy to accomplish is fallen within scope of the presently claimed invention.
Embodiment 1 prepares Escherichia coli O 157: H7 Ivy antigen polypeptide
1. Escherichia coli O 157: the design and synthesis of H7 Ivy polypeptide: according to the Escherichia coli O 157 on GenBank:
H7 Ivy sequence (accession number: NP_285937.1) obtains Escherichia coli O 157: H7 Ivy protein sequence, contains 157 amino
Acid.
2. analyzing Ivy protein characteristic with DNAstar software, analysis the results are shown in Table 1, and the molecular weight of the albumen is 16871
Er Dun, isoelectric point 6.59 are acidic protein.
Table 1
| Analysis project (Analysis) | Holoprotein (Whole Protein) |
| Molecular weight (Molecular Weight) | 16.871 kDa |
| Length (Length) | 157 aa |
| 1microgram= | 11.724pMoles |
| Molar extinction coefficient (Molar Extinction coefficient) | 95770 |
| 1A(280)= | 1.10mg/mL |
| Isoelectric point (Isoelectric Point) | 6.59 |
| Charge (Charge at pH 7) | - 0.65 |
3. with IEDB software analysis Ivy protein immunogenic, hydrophilic and hydrophobic and surface accessibility, the results showed that large intestine bar
6 aa -157 aa of bacterium O157:H7 Ivy protein 14 has 12 amino acid antigenicities, hydrophily and surface accessibility stronger.
4. Ivy antigen polypeptide screens: passing through above-mentioned analysis, finally screen polypeptide sequence are as follows: SLENHPDGFNFK(146
Aa-157 aa, SEQ ID NO:1).
The preparation of 2 Ivy antigen polypeptide of embodiment and and carrier protein couplet
1. Ivy antigen polypeptide: SLENHPDGFNFK is synthesized by Shanghai gill biochemical corp.Peptide C end is one and half Guang ammonia
Acid, is convenient for and carrier protein couplet.
2. polypeptide and carrier protein couplet
A. the mcKLH that a pipe maleimide activation is diluted with 200 μ L ultrapure waters, is diluted to the solution of 10 mg/mL.
(note: above-mentioned solution is translucent in blue and white, must not vibrate or heat, and otherwise will lead to mcKLH precipitating.)
B. the haptens containing sulfydryl is dissolved (i.e. with the coupling buffer for being equivalent to 1.0~2.5 times of volumes of solution in step a
Ivy antigen polypeptide).Such as: 200~500 μ L coupling buffers of 2mg haptens are dissolved, mcKLH is added to.If polypeptide
Be it is readily soluble, can be added to solid in mcKLH suspension.(if haptens is less soluble, DMSO increase note: can be added
Dissolution.DMSO concentration in coupling lysate is lower than 30%, otherwise carrier protein mutability).
C. polypeptide, mcKLH solution are mixed immediately, are then reacting at room temperature 2 h.
3. coupled product purifies
Conjugate is purified by gel chromatography desalting column.If conjugate is injected within one week, purified with PBS.Such as
Fruit conjugate freezes, and is purified with purifying buffer salt, if forming precipitating in coupling, supernatant is collected in centrifugation, retains precipitating.Only
Purify supernatant.The conjugate of purifying is combined with precipitating.
A. one bottle of purifying buffer salt is dissolved, the ultrapure water of 60mL degassing, 4 DEG C of preservations are added.
B. take away desalting column top and bottom lid, make store liquid discharge.One desalting column can purify 0.5mL sample.
C. pillar is rinsed with the purification buffer of 3~5 times of column volumes (15~25 mL).
D. the peptide carrier mixture of 0.5mL is directly added into column center.The purification buffer of 0.5mL is added, is dividing
From collecting each peak in pipe.
E. absorbance is measured at 280 nm to determine which partially contains conjugate.It is detected in first absorption peak
It will be hapten conjugation object.Mix all parts containing conjugate.
F. after the part containing conjugate occurs, continue that buffer is added into column, it is anti-to collect half not be coupled
It is former.
G. by conjugate filtration sterilization, -80 DEG C are sterilely maintained within.
As a result: coomassie brilliant blue measures protein concentration and content after coupling, every 250 μ g of pipe packing, -80 DEG C of preservations.
The preparation of the anti-Ivy polypeptide BALB/c mouse polyclonal antibody of embodiment 3 and purifying
1. animal immune
4 ~ 5 week old male BALB/c mouses are immunized in coupled product.Freund's complete adjuvant or incomplete Freund's adjuvant are purchased from
Sigma company.
Immune programme is shown in Table 2, first dilutes 100 μ g antigens with PBS, until 0.3mL, is mixed with isometric adjuvant, vortex vibration
Swing 100 min, diffusion is not dissolved in one minute to be immunized in waterborne as a result, taking drop antigen drop for detection emulsification.It is immune
Preceding tail vein takes blood, and serum is taken after 4 DEG C of standings, and negative control sera is made in -80 DEG C of preservations.
The specific immune programme of table 2
| Immune time | Number of days | Antigen dose | Immunization route |
| For the first time | 1st day | 100 μ g+ Freund's complete adjuvants (totally 0.3 mL) | The subcutaneous multi-point injection in vertebra two sides, 3 points |
| Second | 15th day | 50 μ g+ incomplete Freund's adjuvants (totally 0.3 mL) | The subcutaneous multi-point injection in vertebra two sides, 3 points |
| For the third time | 29th day | 50 μ g+ incomplete Freund's adjuvant (totally 0.3 mL | The subcutaneous multi-point injection in vertebra two sides, 3 points |
Antibody titer ELISA detection
(1) experiment reagent
A. phosphate buffer (PBS) (10 × concentration): sodium chloride (NaCl) 50g, 1.25 g of potassium chloride (KCl), phosphoric acid
Potassium dihydrogen (KH2PO4) 1.25 g, disodium hydrogen phosphate (Na 2HPO 4·12H2O) 18.1 g, distilled water add to 1000 mL, use 1M
HCl tune pH to 7.2.
B. confining liquid: normal calf serum 10mL, 1 × PBS(are free of tween) 90mL.
C. washing buffer: polysorbas20 (Tween20) 0.2mL, 1 × PBS 1000mL.
D. substrate develop the color A liquid: sodium acetate (CH3COONa) 13.6 g, citric acid (C6H8O7·H2O) 1.6 g, hydrogen peroxide
(H2O230%) 0.3mL, distilled water add to 500mL.
E. substrate develop the color B liquid: disodium ethylene diamine tetraacetate (EDTA-Na2) 0.2 g, citric acid (C6H8O7·H2O) 0.95
G, glycerol (C3H8O3) 50mL, 0.2 g of tetramethyl benzidine (TMB), distilled water adds to 500mL.
F. terminate liquid (2M H2SO4): take dense H2SO427.62mL is added slowly in the distilled water of 473mL, is mixed
?.
G. antigen coat liquid (0.1M carbonate buffer solution): pH 9.6, sodium carbonate (Na2CO3) 1.59g, sodium bicarbonate
(NaHCO3) 2.93g, Sodium azide (NaN3) 0.2g, distilled water adds to 1000mL.
H. the goat anti-mouse igg of horseradish peroxidase-labeled (Beijing health is reagent Biotechnology Co., Ltd).
(2) experimental procedure
A. antigen coat: pure polypeptide antigen final concentration is generally 4 μ g/mL, takes 100 μ L to be added after being diluted with coating buffer poly-
In each hole of the enzyme-linked detection plate of styrene, after 4 DEG C are stayed overnight, washing lotion is washed 3 times.
B. close: every hole adds 200 μ L or fills it up with confining liquid, and 4 DEG C overnight or 37 DEG C after two hours, washs 3 times, pats dry.
4 DEG C of refrigerators are set to save backup.
C. ELISA is detected
1. plus sample to be tested: serum is separated after mouse tail vein blood sampling, with PBS doubling dilution, 50~100 holes μ L/ sample-adding
Into the ELISA Plate being coated with, meanwhile, choosing immune preceding mice serum respectively is negative control, 37 DEG C of 60 min of incubation, washing
It 3 times, pats dry.
2. plus secondary antibody: selecting extension rate, 100 holes μ L/, 37 DEG C of 60 min of incubation, washing 3 according to the potency of ELIAS secondary antibody
It is secondary, it pats dry.
3. colour developing: substrate colour developing each hole 50 μ L/ of A, B liquid, 37 DEG C of colour developing 15min are added.
4. terminating: 100 hole μ L/ of terminate liquid is added.
5. reading: each hole OD value is measured with 450nm Single wavelength, to be greater than 2.5 with the ratio (P/N) of negative control hole OD value
It is limited, as the critical point for being judged as positive or determining potency.
Mice serum is collected:
Using eye socket depletion method, in 37 DEG C of incubation 2h after non-anticoagulant Mouse whole blood collection.
2. antibody purification
(1) saturated ammonium sulphate method
A. saturated ammonium sulfate solution (SAS) is prepared: by 767g(NH4)2SO4It is slowly added to 1L distilled water while stirring
In.
Sulfuric acid pH7.0 is transferred to ammonium hydroxide or sulfuric acid.This i.e. saturation degree be 100% ammonium sulfate (4.1mol/L, 25
DEG C).
B. it precipitates
1. sample (serum) 20 000 × g is centrifuged 30min, cell fragment is removed；
2. retaining supernatant and measuring volume；
3. being slowly added into isometric SAS while stirring into supernatant, final concentration of 1:1；
It stirs 6 hours or is stirred overnight (4 DEG C) 4. solution is placed on magnetic stirring apparatus, precipitate protein sufficiently.
C. it dialyses
1. 10 000 × g of protein solution is centrifuged 30min(4 DEG C).It abandons supernatant and retains precipitating；
2. precipitating is dissolved in a small amount of (10~20mL) PBS-0.2g/L Sodium azide.Bag filter pair is put into after precipitating dissolution
PBS-0.2g/L Sodium azide is dialysed 24~48 hours (4 DEG C), and it is primary to change elution buffer every 3~6 hours, thoroughly to remove
Sulfate of ammoniac；
3. dialyzate is centrifuged, protein content in supernatant is measured.
(2) reagent and special installation needed for Protein G affinity purification: 1.0 mol/L Tris(pH8.0)；100mmol/L
Tris(pH8.0)；10mmol/L Tris(pH8.0)；50mmol/L glycine (pH3.0)；Column chromatography instrument.
Operating procedure:
A. the 1.0mol/L Tris(pH8.0 of 1/10 volume is added) adjust (serum, tissue culture supernatant containing antibody samples
Or ascites) pH value to 8.0.
B. antibody-solutions are passed through into albumin A or Protein G microballoon column.In these columns, every milliliter of wet microballoon can be in conjunction with about
10~20mL antibody (1 albumin A or Protein G microballoon molecule combine 2 molecular antibodies).The about volume of record dress column microballoon.Cause
The amount using cleaning and elution buffer is determined for the volume of microballoon column.
C. microballoon is washed with the 100mmol/L Tris(pH8.0 of 10 times of column volumes).
D. microballoon is washed with the 10mmo1/L Tris(pH8.0 of 10 times of column volumes).
E. microballoon column is eluted with 50mmol/L glycine (pH3.0), the buffer of about 1/2 column volume is added every time, by several times
It is added.Eluent is collected with the test tube of the 1 mol/L Tris(pH8.0 for containing 1/10 column volume), each pipe is slowly shaken up, it is made
PH value is restored to neutrality.
F. the collecting pipe containing antibody is mixed, surveys total protein with SDS-PAGE and coomassie brilliant blue staining.
As a result: being identified after antibody purification through SDS-PAGE protein electrophoresis, it is 0.4 that ultraviolet specrophotometer, which measures antibody concentration,
mg/mL.Purified antibodies are stored in -20 DEG C, and in 0.01 M PBS buffer, 40% glycerol and 0.5%BSA are contained in solution.
Embodiment 4: the identification of the polyclonal antibody of anti-Ivy
1. the ELISA of the polyclonal antibody of anti-Ivy is identified
It is detection antigen with the Ivy polypeptide of synthesis, coated elisa plate is made with the diluted not immune mouse rabbit anteserum of 1:2000
For negative control, antibody doubling dilution by mice serum and after purification is detected using ELISA method, with negative serum
Ratio be greater than 2.1 and be judged as positive, calculate how anti-potency.
As a result, the potency of the anti-Ivy antibody of purifying reaches 1:128000 (table 3).
The anti-Ivy antibody titer (k=1000) of table 3
| Extension rate | 1:1000 | 1:2000 | 1:4000 | 1:8000 | 1:16000 | 1:32000 | 1:64000 | 1:128000 | 1:256000 | Negative control | PBS |
| OD value | 2.893 | 2.569 | 2.316 | 2.025 | 1.534 | 0.936 | 0.469 | 0.237 | 0.119 | 0.098 | 0.0125 |
2. the Western Blot of the polyclonal antibody of anti-Ivy is identified
Collect Escherichia coli O 157 respectively: Escherichia coli O 157 is made after being cracked in H7 and Vero cell: H7 cracks egg
White and Vero cytolytic proteins carry out 10%SDS-PAGE electrophoresis, reference with loading after the cracking of 2 × SDS lysis buffer
Western blotting method described in " molecular cloning ", electrotransfer condition are 100V electrophoresis 1h, and confining liquid is containing 5% skimmed milk power
TBST, primary antibody are the polyclonal antibody (0.1 μ g/mL of final concentration) of the anti-Vero in the present invention, and secondary antibody is horseradish peroxidase
The sheep anti-mouse igg antibody of label carries out immunoblotting assay by ECL Color Appearance System.
As a result: the polyclonal antibody of the anti-Ivy in the present invention can be in Escherichia coli O 157: detecting 17KD in H7 lysate
The protein band of size or so is consistent with Escherichia coli O 157: H7 Ivy molecular weight of albumen size.
3. the identified by immunofluorescence of the polyclonal antibody of anti-Ivy.
Escherichia coli O 157: 48 h of H7 vero cells infection, the fixed 15min of 4% paraformaldehyde are added dropwise 0.25%
TritonX100 is incubated at room temperature 10 min, and cell membrane is made to have permeability, so as to antibody entrance.PBS is washed three times, contains 1%
The PBST room temperature of BSA closes 2 h.PBS is washed 3 times, then with polyclonal antibody (the PBST 1:500 of the anti-Ivy in the present invention
Dilution) 1.5 h of incubation at room temperature.PBS wash 3 times, with FITC(green fluorescence) label sheep anti-mouse igg (PBST 1:1000
Dilution) 1 h of incubation at room temperature.DAPI dyes (blue) 10min after PBS washing.After PBS develops a film, fluorescence microscopy is under the microscope.Together
When with Normal Mouse Serum make negative control.
As a result: observing that green is glimmering in the Vero cytoplasm of the polyclonal antibody dyeing through the anti-Ivy in the present invention
Light, it was demonstrated that the anti-Ivy antibody in the present invention can recognize natural Ivy albumen.
Sequence table
SEQ ID NO:1
SLENHPDGFNFK
Claims (5)
1. a kind of Escherichia coli O 157: H7 Ivy antigen polypeptide, which is characterized in that its amino acid sequence such as SEQ ID NO:1 institute
Show.
2. Escherichia coli O 157 according to claim 1: H7 Ivy antigen polypeptide, which is characterized in that the antigen is more
Peptide and keyhole blood indigo plant carrier protein KLH are coupled.
3. a kind of anti-Escherichia coli O 157: the polyclonal antibody of H7 Ivy, which is characterized in that be with as claimed in claim 2 big
Enterobacteria O157:H7 Ivy antigen polypeptide is immunogene, and immune animal is prepared.
4. anti-Escherichia coli O 157 described in claim 3: the preparation method of the polyclonal antibody of H7 Ivy, it is characterised in that step
It is as follows: by Escherichia coli O 157 described in claim 1: the keyhole blood indigo plant carrier egg of H7 Ivy antigen polypeptide and maleic amide activation
During which white KLH coupling is exempted from more than three times as immunogene and adjuvant mixed immunity BALB/c mouse after desalting and purifying
Epidemic disease acquires mice serum, by purifying, ELISA and western after ELISA detection serum antibody titer reaches 1:100000
After blot identification, anti-Escherichia coli O 157: the polyclonal antibody of H7 Ivy is obtained.
5. anti-Escherichia coli O 157 described in claim 3: the polyclonal antibody of H7 Ivy detects Escherichia coli O 157: H7 in preparation
Application in the reagent of Ivy albumen infection.
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| CN104744581A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Toxoplasma gondii TgVP1 extracellular region antigen polypeptide, anti-toxoplasma gondii TgVP1 polyclonal antibody and application of polyclonal antibody |
| CN104744580A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | TgVP1 extracellular region antigen polypeptide, anti-TgVP1 polyclonal antibody and application of polyclonal antibody |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104744581A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | Toxoplasma gondii TgVP1 extracellular region antigen polypeptide, anti-toxoplasma gondii TgVP1 polyclonal antibody and application of polyclonal antibody |
| CN104744580A (en) * | 2015-03-02 | 2015-07-01 | 南方医科大学 | TgVP1 extracellular region antigen polypeptide, anti-TgVP1 polyclonal antibody and application of polyclonal antibody |
Non-Patent Citations (2)
| Title |
|---|
| Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme;Vincent Monchois，等;《The Journal of Biological Chemistry》;20010525;第276卷(第21期);摘要和图7 |
| The vertebrate lysozyme inhibitor Ivy functions to inhibit the activity of lytic transglycosylase;Chelsea A. Clarke，等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20100514;第285卷(第20期);全文 |
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