CN100374460C - Bacteriophage containing short-peptide specifically combined with avian infectious bronchitis virus and use thereof - Google Patents
Bacteriophage containing short-peptide specifically combined with avian infectious bronchitis virus and use thereof Download PDFInfo
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Abstract
The present invention relates to a bacteriophage and a preparation method thereof, which belongs to the technical field of animal virology, wherein the bacteriophage contains short peptides specifically combined with avian infectious bronchitis viruses. The short peptide has the amino acid sequence of GSHHRHVHSPFV, and can be combined with avian infectious bronchitis viruses H52 specifically after passing through the substitution, deletion or addition of one or a plurality of amino acid residues. The present invention also comprises the application of the bacteriophage in preparing IBV identifying and diagnosing kits, and the IBV identifying and diagnosing kits of the present invention are used for preventing and treating the avian infectious bronchitis viruses.
Description
Technical field
The present invention relates to the animal virology technical field, specifically belong to the display technique of bacteriophage field.Utilize display technique of bacteriophage screening at surface display can with the phage of specificity of infectious bronchitis virus of chickens bound short peptide, and the phage that utilizes animal virus to learn a skill screening is obtained is identified and application etc.
Background technology
Chicken infectious bronchitis (Infections Bronchitis of chickens, IB) be a kind of acute height contagious disease of chicken, its cause of disease is avian infectious bronchitis virus (Infections Bronchitis of chickens Virus, IBV hereinafter is referred to as IBV).IBV causes serious chicken respiratory system disease, be positive chain RNA virus, belong to shell type virales (Nidovirales), coronaviridae (Coronavirdae) member, its cyst membrane spike protein (S) is conjugated protein (the Casais R of Virus receptors, Recombinant AvianInfectious Bronchitis Virus Expressing a Heterologous Spike Gene Demonstrates that the SpikeProtein Is a Determinant of Cell Tropism.J Virol.2003,77 (16): 9084-9089; Wong S K, host's preferendum of decision virus A193-amino-acid fragment of the SARS coronavirus S protein efficiently bindsangiotensin-converting enzyme 2.J.Biol.Chem.2003 Dec 11).
The serotype of IBV is numerous, and the serotype of having reported at present is near 30, and new serotype does not have or only have the part cross-immunity also in continuous appearance between the different serotypes.In addition, preparation of IBV diagnostic antigen and immunne response mechanism is more complicated all, and these have brought a lot of difficulties for diagnosis of this disease and anti-system.
(phdge display) is well received in recent years for display technique of bacteriophage, be used for the research of receptors ligand and antigen antibody interaction, and derive the whole bag of tricks, comprise with monoclonal antibody as antigen antibody interaction research and from phage peptide library, to screen epitope (Wang L F, Epitopeidentification and discovery using phage display libraries:applications in vaccine developmentand diagnostics. Curr Drug Targets.2004,5 (1): 1-15), or fixed virus screens monoclonal antibody (Higo-Moriguchi K, Isolation of human monoclonal antibodies that neutralize human rotavirus.J Virol.2004 Apr from phage antibody library; 78 (7): 3325-32) etc., be used for mechanism research, vaccine design and diagnostic method research etc.Acceptor virus interacts and comprises that the intact cell with expressed receptor screens specific receptors in conjunction with epi-position (Cao J from phage random peptide library, Phage displayselection on whole cells yields a small peptide specific for HCV receptor human CD81.CellRes.2003,13 (6): 473-479), or from the conjugated protein phage peptide library of Virus receptors, screen Virus receptors in conjunction with epi-position (Fontana L with the intact cell of expressed receptor, General strategy for broadening adenovirus tropism.J Virol.2003,77 (20): 11094-104.), or from phage random peptide library, screen receptor domain (FerrerM with viral protein, Peptide ligands tohuman immunodeficiency virus type 1 gp120 identified from phage display libraries.J Virol.1999,73 (7): 5795-5802).Related virus comprises hepatitis C virus, HIV (human immunodeficiency virus), adenovirus etc.The PubMed database is not found up to now as yet with the relevant report of display technique of bacteriophage evaluation with coronavirus interaction polypeptide and application thereof by retrieval.
Summary of the invention
One of purpose of the present invention provide a kind of can with IBV specificity bonded small peptide, its aminoacid sequence is shown in sequence table SEQ ID NO:1.
Two of purpose of the present invention provides a kind of specificity small peptide that contains, can with IBV specificity bonded phage.
Three of purpose of the present invention is to confirm that this phage has the blood clotting restraining effect, the effect of blocking-up cell infection, simultaneously can not block antigen antibody reaction, determine that the contained small peptide of this phage is positioned at IBV virus receptor structural domain, thereby provide a kind of quick, effectively, Lian Jia avian infectious bronchitis virus diagnostic kit.
The present invention is achieved through the following technical solutions:
A kind of can with avian infectious bronchitis virus H52 strain specificity bonded small peptide, its aminoacid sequence is shown in sequence table SEQ ID NO.1.
Comprise the M13 phage Ph.D-H52-IV-14 of aminoacid sequence shown in the sequence table SEQ ID NO.1, be deposited in CCTCC, May 8 2004 preservation time, deposit number CCTCC NO:M204034.
Described small peptide is through replacement, disappearance or the interpolation of one or several amino-acid residue and can be with avian infectious bronchitis virus H52 strain specificity bonded and and by aminoacid sequence deutero-small peptide shown in the SEQ ID NO:1.
A kind of can with the preparation method of avian infectious bronchitis virus H52 strain specificity bonded phage, its step comprises:
A. wrap quilt: by 96 orifice plates, 4 ℃ are spent the night with the purified virus bag of avian infectious bronchitis virus;
B. sealing: add 4 ℃ of sealings of confining liquid 1 hour;
C. absorption: add contained prophage storehouse 10 μ L in the phage display peptide library test kit;
D. wash-out: the phage with 0.1%TBST flush away non-specific binding, add elutriant 100 μ L again, jog 10 minutes, with specificity in conjunction with phage with eluting, change the 1.5mL centrifuge tube over to, add 15 μ L 1M Tris-Hcl (PH9.1) at once and neutralize;
E. amplification: the eluted product that step D is obtained joins in the early stage host bacterium of logarithmic growth and increases, the PEG/NaCl precipitation;
F. next round screening: repeating step A-E, and the content of Tween-20 among the TBST brought up to 0.5%;
G. picking mono-clonal: the single clone of four-wheel screening back picking was with 3mL host bacterium amplification 4 hours, and 12000 leave the heart got supernatant in 1 minute, obtained the single clone of four-wheel screening phage;
H. viral binding characteristic is identified: the single clone of phage that step G is obtained wraps respectively by elisa plate, 4 ℃ of bags are spent the night, 4 ℃ of sealings of inferior daily confining liquid are stack virus successively, anti-IBV rabbit anteserum and goat-anti rabbit ELIAS secondary antibody after 1 hour, and measures the light absorption value at 630nm place;
1. the positive bacteriophage that step H is obtained is assembled into the test kit of differential diagnosis chicken infectious bronchitis.
Phage of the present invention or small peptide can be applied to prepare the test kit of chicken infectious bronchitis differential diagnosis.
Detailed technical scheme is as described below:
1, IBV propagation and purifying: with IBV virus H52 strain (Cheng Liqin etc., journal of Zhejiang university .2002,28 (3): 303-306) allantoic fluid inoculation instar chicken embryo (0.2mL/ piece) on the 10th, collect allantoic fluid after 96 hours,-20 ℃ of freeze thawing three times, low-speed centrifugal 3000 changeed 10 minutes, got supernatant, (NaCl 2.33%, PEG600010.00%) stirs precipitation in 4 ℃ and spends the night for PEG6000.Leave the heart 30 minutes next day 10000, get precipitation, with the suitable dilute suspension of PBS, carry out the 30-60% sucrose density gradient centrifugation, 35000 changeed 3 hours, collected the virus band, converted the PBS dialysis and removed sucrose, and PEG6000 concentrates, and divides to install to 1.5mL centrifuge tube ,-20 ℃ of preservations.
2, anti-IBV Antiserum Preparation: the IBV virus immunity rabbit of the purifying that step 1 is obtained, totally 4 immunity, use for the first time Freund's complete adjuvant (available from SIGMA company), use Freund (available from SIGMA company) to the 4th time for the second time, the 4th immunity collected serum in back 14 days, and ELISA titration antiserum titre is 1: 10240.
3, phage is washed in a pan sieve: according to phage display peptide library test kit (Ph.D.-12
TMPhage Display Peptide Library Kit, available from New England Biolabs company) step described in the specification sheets, the purified virus bag that step 1 is obtained is by 96 orifice plates, add contained prophage storehouse 10 μ L in the phage display peptide library test kit, phage with 0.1%TBST (TBS+Tween-20) flush away non-specific binding, add elutriant 100 μ L again, jog 10 minutes, specificity is eluted in conjunction with phage, change the 1.5mL centrifuge tube over to, add 15 μ L 1MTris-HCl (PH9.1) neutralization at once, join the early stage host bacterium (OD of logarithmic growth then
600~ 0.3) ER2738 (Ph.D.-12
TMProvide among the Phage DisplayPeptide Library Kit, available from New England Biolabs) middle amplification, the PEG/NaCl post precipitation carries out the next round screening, and the content of Tween-20 among the TBST is brought up to 0.5%.Four-wheel screening back picking single clone increased 4 hours with 3ml host bacterium ER2738, and 12000 leave the heart got supernatant in 1 minute, obtains the single clone of four-wheel screening phage.
Wherein the agents useful for same prescription is as follows:
Coating buffer: 0.1M NaHCO
3(pH8.6)
Confining liquid: 0.1M NaHCO
3(pH8.6), 5mg/mLBSA, 0.02%NaN
3. filtration sterilization, 4 ℃ of preservations.
TBS:50mM Tris-Hcl (pH7.5), 150mM NaCl.121 ℃ high-temperature sterilization 30 minutes, room temperature (25 ℃) is preserved.
Elutriant: 0.2M glycine-hydrochloric acid (pH2.2)
PEG/NaCl:20% (w/v) polyoxyethylene glycol 8000,2.5M NaCl, 121 ℃ of high-temperature sterilizations 30 minutes, room temperature (25 ℃) is preserved.
4, binding characteristic is identified: the applicant is with phage display peptide library test kit (Ph.D.-12
TMPhage DisplayPeptide Library Kit is available from New England Biolabs) detect the positive bacteriophage method described in the specification sheets and improved, specific practice is: the single clone's bag of phage that step 3 is obtained is by elisa plate, and 4 ℃ are spent the night.4 ℃ of sealings of inferior daily confining liquid are stack virus successively, anti-IBV rabbit anteserum (being obtained by step 2) and goat-anti rabbit ELIAS secondary antibody (available from Southern Biotechnology Associates company) after 1 hour, substrate colour developing, the light absorption value at mensuration 630nm place.
Prepare agents useful for same according to following proportioning:
Coating buffer: 0.1M NaHCO
3(pH8.6)
Confining liquid: 0.1M NaHCO
3(pH8.6), 5mg/mLBSA
Washings: 50mM Tris-Hcl (pH7.5), 150mM NaCl, 0.5%Tween-20
Substrate solution: substrate solution A:0.006%H
2O
2Damping fluid; Substrate solution B: get Na
2HPO
412H
2O14.2g, citric acid 10.5g is settled to 500mL with distilled water and is made into 0.1 phosphoric acid salt citrate buffer solution (pH5.0), adds benzidine (TMB) then.During use A liquid and B liquid equal-volume are mixed, mix in back 5 minutes and use, now with the current
Stop buffer (0.025%HF): HF (40%) 625 μ L, ddH
2O 100mL
5, order-checking is identified: the positive colony of identifying in the step 4 is checked order.
The sequencing primer sequence is provided by the phage display test kit, for: 5 '-
HOCCC TCA TAG TTA GCG TAA CG-3 ' (Ph.D.-12
TMPhageDisplay Peptide Library Kit, New England Biolabs).
Sequencing result is translated as amino acid, obtains 12 small peptides (deduced amino acid as sequence table 1 shown in) of sequence for " G S H H R H V H S P F V ".
Useful achievement of the present invention is summarized as follows:
1, one of useful achievement of the present invention be obtained can with the specificity bonded phage of IBV H52 strain, and the specificity short peptide sequence of surface display.Because it is short, simple to operate that phage is washed in a pan the sieve process cycle, compare conveniently, quick with the making of monoclonal antibody, and do not lose specificity.Have more advantage when therefore, being used for differential diagnosis.
2, two of useful achievement of the present invention is hemagglutinative functions that this phagocytosis physical efficiency suppresses IBV virus, the derived peptide hemagglutination-inhibition test shows the replacement of the contained specific amino acid of this phage through one or several amino-acid residue, and the phage that contains new aminoacid sequence that disappearance or add obtains still can specificity suppresses the hemagglutinative function of IBV.Simultaneously, non-specific inhibition experiment shows that this restraining effect is specific.
Specific practice is:
1) amplification of positive phage clones: get the positive phage clones that obtains in the 5 μ L technical scheme steps 4 and join the early stage (OD of 20mL logarithmic growth
600~ 0.3) host bacterium ER2738 (Ph.D.-12
TMProvide among the Phage Display Peptide Library Kit, available from New EnglandBiolabs company,) in, increased 4 hours, 10000 left the heart 10 minutes, got supernatant, twice of PEG/NaCl precipitation, at last with 200 μ LTBS dissolving, the positive phage clones that obtains increasing (called after Ph.D-H-IV-14).(required agent prescription is with technical scheme steps 3)
2) hemagglutination-inhibition test: the pure virus that obtains in the technical scheme steps 1 is handled the back with 1% pancreatin measure its HAU according to ordinary method, the Ph.D-H-IV-14 that step 1) is obtained does doubling dilution since 2 times, mix with virus (handling) equal-volume of 4 HAUs of 25 μ L through 1% pancreatin, 37 ℃ act on 1 hour, add the fresh chicken red blood cell of 50 μ L0.5%, 37 ℃ act on 1 hour, the observed and recorded result.With the high dilution of the phage that suppresses blood clotting fully is that blood clotting suppresses to tire.The blood clotting inhibition valency of this phage is 1: 16 (see figure 2)
3) derived peptide hemagglutination-inhibition test: the contained specific amino acid of the phage that technical scheme 5 is obtained is through the replacement of one or several amino-acid residue, disappearance or add the phage obtain containing new aminoacid sequence is according to step 1) and step 2) described step carries out hemagglutination-inhibition test.Experimental result shows, with the replacement of the contained specific amino acid of phage of the present invention through one or several amino-acid residue, the phage that contains new aminoacid sequence that disappearance or add obtains still can specificity suppresses the hemagglutinative function of IBV.(see figure 3)
4) non-specific test: measure Avian pneumo-encephalitis virus (Li Hongbin according to ordinary method, the comprehensive anti-system of newcastle disease, agricultural science and technology communication: 1998.6:23-24) avian influenza virus H9N2 strain (Li Zili etc., the diagnosis of Hubei Province's bird flu and the separation of virus strain and preliminary evaluation, China's Preventive Veterinary Medicine newspaper: 2001.23 (2): 146-148) HAU, the Ph.D-H-IV-14 that step 1) is obtained does doubling dilution since 2 times, respectively with the Avian pneumo-encephalitis virus of 4 HAUs of 25 μ L, avian influenza virus H9N2 strain equal-volume mixes, simultaneously, to contain irrelevant peptide phage since 2 times of doubling dilutions, mix with IBV virus (handling) equal-volume of 4 HAUs of 25 μ L through 1% pancreatin, and establish positive bacteriophage, IBV, Avian pneumo-encephalitis virus, avian influenza virus H9N2 strain and the contrast of red corpuscle self-solidifying, 37 ℃ act on 1 hour, add the fresh chicken red blood cell of 50 μ L0.5%, 37 ℃ act on 1 hour, the observed and recorded result.The result shows that Ph.D-H-IV-14 contains irrelevant peptide phage to the equal unrestraint of the blood clotting of IBV to Avian pneumo-encephalitis virus and avian influenza virus H9N2 strain, has only Ph.D-H-IV-14 that the blood clotting of IBV is had restraining effect.(see figure 4)
3, three of the useful achievement of the present invention is to utilize the antigen-antibody blocking test to confirm that this short peptide sequence is not to be positioned at the virus antigen structural domain, but is present in the virus receptor structural domain.
Specific practice is: the pure virus packets that technical scheme steps 1 is obtained is by elisa plate, the Ph.D-H-IV-14 that the step 1) that superposes successively obtains (10 * beginning doubling dilution from top to bottom), anti-IBV rabbit anteserum (obtaining) by step 2, goat-anti rabbit ELIAS secondary antibody (available from Southern BiotechnologyAssociates company) and substrate are measured 630nm place light absorption value.(required agent prescription is with technical scheme steps 4).Utilize the variance analysis of SAS software that each group data is carried out statistical analysis, show and do not have significant difference (P＞0.05) between phage blocking-up group and virus control group, confirm that phage is to the no blocking-up effect of antiviral antibody reaction, contained small peptide is not the antigen site sequence of virus in the prompting phage, may be the Virus receptors sequence.And because the relative conservative property of receptor sequence, this phage is used for viral diagnosis and compares with antigen antibody reaction and may have more broad spectrum.(concrete outcome sees Table 1)
Table 1 phage small peptide of the present invention is to the blocking effect (OD of antigen antibody reaction
630)
| The phage extension rate | Phage number (PFU) | The phage contrast | Virus control | The phage blocking-up |
| 10× 20× 80× 160× | 2.5×10 31 1.25×10 31 6.25×10 30 3.125×10 30 | 0.000 -0.006 -0.008 0.006 | 0.723 0.711 0.731 0.662 | 0.805 0.767 0.675 0.681 |
| 320 * 640 * 1280 * 2560 * mean value ± SD | 1.563×10 30 7.812×10 29 3.906×10 29 1.953×10 29 | 0.108 0.033 0.031 0.062 0.03±0.03 | 0.737 0.671 0.646 0.721 0.69±0.07 | 0.682 0.638 0.626 0.636 0.70±0.03 |
Annotate 1) in the phage contrast row top 4 be disappearance virus and an anti-layer contrast, below 4 holes contrast for disappearance virus layer;
Notes 2) virus control is classified the antiviral antibody normal reaction that does not add phage as, and all values is repeating hole;
Notes 3) barrier effect of the phage of 1: 10 beginning doubling dilution to the antiviral antibody reaction classified in the phage blocking-up as; All values is the average OD of 3 repeating holes in the row
630Value;
4, four of useful achievement of the present invention is to utilize the virus infection blocking test to prove that this phage can the blocking virus cells infected.
Specific practice is: the pure virus infection HeLa cell with technical scheme steps 1 obtains records TCID with ordinary method
50, use 100 TCID then
50Virus is carried out the virus infection blocking test.Specific practice is: the Ph.D-H-IV-14 that step 1) is obtained is since 10 * doubling dilution, then with isopyknic 200 TCID
50Virus mix, 37 ℃ of effects 2 hours are added drop-wise to 96 orifice plates, every hole 100 μ L add 100 μ LHeLa cell suspensions again, and establish the cell contrast, virus control and phage contrast.The result shows, contains the obviously infection of blocking virus pair cell of phage of specificity small peptide, illustrates that this small peptide combines with the virus receptor binding site, has certain antivirus action.(concrete outcome is seen Fig. 5 and table 2)
The blocking effect that table 2 phage small peptide of the present invention pair cell infects
| The |
1 1∶10 | 2 1∶20 | 3 1∶40 | 4 1∶80 | 5 1∶160 | 61∶320 | 7 1∶640 |
| 10 -1 | 10 -1.3 | 10 -1.6 | 10 -1.9 | 10 -2.2 | 10 -2.5 | 10 -2.8 | |
| Phage number (PFU) | 5×10 31 | 2.5×10 31 | 1.25×10 31 | 6.25×10 30 | 3.125×10 30 | 1.563×10 30 | 7.812×10 29 |
| Cytopathy (cpe) |
1 | 1 | 1 | 3 | 3 | 4 | 5 |
| Cytopathy (cpe) hole count does not appear | 4 | 4 | 4 | 2 | 2 | 1 | 0 |
| Cytopathy (cpe) |
1 | 2 | 3 | 6 | 9 | 13 | 18 |
| Cytopathy (cpe) hole sum does not appear | 17 | 13 | 9 | 5 | 3 | 1 | 0 |
| |
1/18 | 2/15 | 3/12 | 6/11 | 9/12 | 13/14 | 18/18 |
| Protection ratio (%) | 94 | 87 | 75 | 45 | 25 | 7 | 0 |
Annotate 1): above data are and connect poison record in back 2.5 days
Annotate 2): the result presses Reed-Muench Liang Shi method and calculates:
Distance proportion=(75-50)/(75-45)=25/30=0.83
Be higher than the logarithm of the logarithm+distance proportion * dilution factor of 50% protection ratio serum dilution=-1.6+0.83* (0.3)=-antilogarithm of 1.849=-1.85-1.85 is 1/71, the phage of dilution in promptly 1: 71 can protect 50% cell cytopathy to occur.
5, five of the beneficial effect of the present invention differential diagnosis kit that provide are easy to use.The present invention is on the basis that the differential diagnosis method is provided, and also that this method is required all ingredients is assembled into test kit, and operation is simple.
Accompanying drawing and explanation
Fig. 1: techniqueflow chart of the present invention
Fig. 2: the phage blood clotting suppresses determination of activity
The 1st row is the hemagglutination inhibition reaction since 1: 2 doubling dilution phage; The 2nd row is the blood clotting contrast of 4 HAU IBV viruses; The 3rd row adds red corpuscle contrast, no nonspecific agglutination for phage; The 4th row is the contrast of 0.5% red corpuscle self-solidifying, does not have from coagulation phenomena.
Fig. 3: derived peptide hemagglutination-inhibition test
The 1st row are classified as to the 8th and are contained one or several amino-acid residue of the contained specificity small peptide of phage of the present invention through replacing, behind phage beginning in 1: 2 doubling dilution of disappearance or other small peptides of adding to the blood clotting restraining effect of IBV; The 9th classifies the blood clotting contrast of 4 HAU IBV viruses as; The 10th classifies phage as adds red corpuscle contrast, no nonspecific agglutination; The 11st classifies the contrast of 0.5% red corpuscle self-solidifying as, does not have from coagulation phenomena.
Illustrate: one or several amino-acid residue process that contains the contained specificity small peptide of phage of the present invention replaces, and the phage of other small peptides of disappearance or interpolation still has the blood clotting restraining effect similar with phage of the present invention
Fig. 4: non-specific detection test effect
The 1st, 3,7 row are respectively the avian influenza virus H9N2 strain of 4 HAUs, Avian pneumo-encephalitis virus, the contrast of IBV (processing of 1% pancreatin) blood clotting; The 2nd, 4 row are respectively Ph.D-H-IV-14 to avian influenza virus H9N2 strain and Avian pneumo-encephalitis virus blood clotting restraining effect; The 6th classifies as and contains irrelevant peptide phage to IBV (processing of 1% pancreatin) blood clotting restraining effect; The 5th classifies Ph.D-H-IV-14 as to IBV (processing of 1% pancreatin) blood clotting restraining effect; The 8th classifies phage as adds red corpuscle contrast, no nonspecific agglutination; The 9th classifies the contrast of 0.5% red corpuscle self-solidifying as, does not have from coagulation phenomena.Illustrate: Ph.D-H-IV-14 contains irrelevant peptide phage to the equal unrestraint of the blood clotting of IBV to Avian pneumo-encephalitis virus and avian influenza virus H9N2 strain, has only Ph.D-H-IV-14 that the blood clotting of IBV is had restraining effect.
Fig. 5: the blocking effect that phage small peptide pair cell infects
Among the figure: A): virus control; B): the cell contrast; C): the phage contrast;
D): the blocking-up that pair cell infects during 10 times of dilutions of phage
E): the blocking-up that pair cell infects during 320 times of dilutions of phage
F): the blocking-up that pair cell infects during 1280 times of dilutions of phage
Illustrate: more than be and connect the back 2.5 days cell of poison.By figure A, B, C as can be seen, the cell of cell contrast and phage contrast is pathology not all, and the virus control shown in the A has pathology, illustrates that cytopathy is to be caused by virus, phage itself can not cause cytopathy; Cell remains intact among the D, and can be by E, and F draws obvious gradient, illustrates that the infection of phage pair cell has blocking effect.
Embodiment
1.1IBV the purifying of virus
With the H52 strain (Cheng Liqin etc., the clone and the sequential analysis of avian infectious bronchitis virus H52 strain S gene, journal of Zhejiang university .2002,28 (3): 303-306) 10 times of after-filtration of allantoic fluid dilution, get 1mL and do steriling test.Specific practice is: the 1mL allantoic fluid is added in the 25mL substratum, and 37 ℃ of vibrations were cultivated 3 hours, and not becoming muddiness then proves it has been aseptic.
With allantoic fluid inoculation instar chicken embryo on the 10th, preserve moisture to cultivate after 96 hours for 37 ℃ and collect allantoic fluid, freeze thawing three times, low-speed centrifugal 3000 changeed 10 minutes, got supernatant, and (Nacl 2.33%, PEG600010.00%) stirs precipitation in 4 ℃ and spends the night for PEG6000.Leave the heart 30 minutes next day 10000, get precipitation,, carry out the 30-60% sucrose density gradient centrifugation with the suitable dilute suspension of PBS, 35000 changeed 3 hours, collected the virus band, converted the PBS dialysis and removed sucrose, and PEG6000 concentrates, obtain the IBV virus of purifying, its branch is installed to the 1.5mL centrifuge tube ,-20 ℃ of preservations.
1.2 the anti-IBV Antiserum Preparation of rabbit
The IBV virus immunity rabbit of the purifying that step 1.1 is obtained, totally 4 immunity, use equal-volume Freund's complete adjuvant (available from SIGMA company) the 1st time, the 2nd time to the 4th use equal-volume Freund (available from SIGMA company), the interval is 14 days between per twice immunity, serum, ELISA titration antiserum titre were collected in the 4th immunity in back 14 days.
1.3 phage is washed in a pan sieve
Add the pure virus of 2 μ L 1.3.1 specific phage is washed in a pan sieve in 150 μ L coating buffers, bag is placed wet environment to spend the night in 4 ℃ by 96 hole enzyme plates.The coating buffer that inclines next day is filled it up with the confining liquid that contains 1%BSA, 4 ℃ of sealings 1-2 hour.The deblocking liquid that inclines with TBST (TBS+0.1%Tween-20) washing 6 times, adds 1.5 * 10
11Individual (among the 100 μ LTBST) phage (Ph.D.-12
TMProvide among the Phage Display Peptide Library Kit, available from New England Biolabs company), room temperature (25 ℃) 60 minutes.The phage that inclines is washed 10 times with TBST, adds elutriant, and jog is eluted to many 10 minutes, and eluate is gone in the 1.5mL centrifuge tube, adds 15 μ L 1M Tris-Hcl (PH9.1) neutralization at once.Eluate is added the early stage (OD of 20mL logarithmic growth
600～0.3) host bacterium ER2738 (Ph.D.-12
TMProvide among the Phage Display Peptide Library Kit, available from New England Biolabs company) in, cultivated 4.5 hours for 37 ℃, 10000 left the heart 10 minutes, get supernatant, once centrifugal again, get 80% supernatant to the 50mL centrifuge tube, the PEG/Nacl that adds 1/6 volume, 4 ℃ of precipitations are spent the night.Leave the heart 15 minutes next day 10000, get precipitation, suspend, forward in the 1.5mL centrifuge tube with 1mLTBS, remove remaining cell debris in centrifugal 5 minutes, supernatant is gone in the new pipe, add 1/6 volume PEG/Nacl precipitation 1 hour, centrifugal 10 minutes, get precipitation, with 200 μ LTBS, 0.02%NaN
3Suspend, removed not molten residue in centrifugal 1 minute, supernatant is gone in the new pipe, be amplified production.
1.3.2 phage titre is measured 10 times of dilutions of phage amplified production (or eluted product) (1.3.1 obtains by step), the phage of getting after 10 μ L dilute joins in the top agar of 3mL preheating, be laid on fast on the LB/IPTG/X-gal plate, 37 ℃ of overnight incubation, locus coeruleus is counted, calculate phage titre.
Calculate 1.5 * 10 1.3.3 utilize phage titre
11The volume of the required amplified production of individual phage carries out the next round screening, and the content of Tween-20 among the TBST is brought up to 0.5%.Positive bacteriophage is detected in four-wheel screening back.
Prepare agents useful for same according to following proportioning:
Coating buffer: 0.1M NaHCO
3(PH8.6)
Confining liquid: 0.1M NaHCO
3(PH8.6), 5mg/mLBSA, 0.02%NaN
3. filtration sterilization, 4 ℃ of preservations.
TBS:50mM Tris-Hcl (PH7.5), 150mM Nacl.121 ℃ high-temperature sterilization 30 minutes, room temperature (25 ℃) is preserved.
Elutriant: 0.2M glycine-hydrochloric acid (PH2.2)
PEG/Nacl:20% (w/v) polyoxyethylene glycol 8000,2.5M Nacl, 121 ℃ of high-temperature sterilizations 30 minutes, room temperature (25 ℃) is preserved.
The LB substratum: the 10g tryptone, the 5g yeast extract, 5gNacl adds water to 1L, 121 ℃ of high-temperature sterilizations 30 minutes, room temperature (25 ℃) is preserved.
The LB/IPTG/x-gal plate: LB substratum+15g/L agar powder, 121 ℃ of high-temperature sterilizations 30 minutes are cooled to＜70 ℃, add IPTG and X-gal, and making the IPTG final concentration is 0.05mg/mL, and the X-gal final concentration is 0.04mg/mL.4 ℃ keep in Dark Place.
Top agar (Agarose Top): 10g tryptone, 5g yeast extract, 5gNacl, 1gMgcl6H
2O, the 7g agarose.121 ℃ of high-temperature sterilizations 30 minutes, room temperature (25 ℃) is preserved.
1.4 the phage small peptide is identified (ELISA) with virus (IBV) binding characteristic
Do not do amplification by the 4th phage of taking turns screening that step 1.3 obtains, directly measure titre, the single plaque that grows on the picking plate is to the early stage (OD of logarithmic growth
600~ 0.3) ER2738 (Ph.D.-12
TMProvide among the Phage Display Peptide Library Kit, available from New EnglandBiolabs company) the middle amplification 4.5 hours, the centrifuging and taking supernatant repeats once.Each clone's supernatant is made the doubling dilution bag respectively by elisa plate, and 4 ℃ are spent the night.The coating buffer that inclines next day is filled it up with confining liquid, 4 ℃ 1-2 hour.TBST washes 3 times, and every hole adds the pure virus of 2 μ L (TBST dilution), 37 ℃ 2 hours, TBST washes plate, adds anti-IBV rabbit anteserum (600 times of dilutions of confining liquid) (being obtained by step 2), 37 ℃ 1 hour, TBST washes plate, add goat-anti rabbit ELIAS secondary antibody (available from Southern Biotechnology Associates company) (4000 times of dilutions of confining liquid), 37 ℃ 1 hour, TBST washes plate, every hole adds 100 μ L substrate solutions, black out left standstill 15 minutes, read 630nm place light absorption value, thereby detected positive bacteriophage.
Prepare agents useful for same according to following proportioning:
Coating buffer: 0.1M NaHCO
3(PH8.6)
Confining liquid: 0.1M NaHCO
3(PH8.6), 5mg/mLBSA
Washings: 50mM Tris-Hcl (PH7.5), 150mM Nacl, 0.5%Tween-20
Substrate solution: substrate solution A:0.006%H
2O
2Damping fluid; Substrate solution B: get Na
2HPO
412H
2O14.2g, citric acid 10.5g is settled to 500mL with distilled water and is made into 0.1 phosphoric acid salt citrate buffer solution (pH5.0), adds benzidine (TMB) then.During use A liquid and B liquid equal-volume are mixed, mix in back 5 minutes and use, now with the current
Stop buffer (0.025%HF): HF (40%) 625 μ L, ddH
2O 100mL
1.5 the order-checking of positive bacteriophage
Get the 500 μ L phage clone supernatants that step 1.4 obtains and add 200 μ L PEG/Nacl, put upside down mixing, room temperature left standstill 10 minutes, centrifugal 10 minutes, remove supernatant, get rid of, with the rifle head remaining supernatant is blotted,, add 250 μ L dehydrated alcohols again with 100 μ L Iodide Buffer dissolution precipitations, incubated at room 10 minutes, centrifugal 10 minutes, remove supernatant, 70% ethanol is washed, with the dissolving of 30 μ L TE damping fluids, promptly get sequencing template after the vacuum-drying.Use automatic sequencer to check order the following (Ph.D.-12 of sequencing primer
TMProvide among the Phage Display Peptide Library Kit, available from New England Biolabs company):
5’-
HOCCC TCA TAG TTA GCG TAA CG-3’
Prepare agents useful for same according to following proportioning:
Iodide Buffer:10mM Tris-HCl (pH8.0), 1mM EDTA, 4M NaI, room temperature (25 ℃) keeps in Dark Place.PEG/Nacl:20% (w/v) polyoxyethylene glycol 8000,2.5M Nacl, 121 ℃ of high-temperature sterilizations 30 minutes, room temperature (25 ℃) is preserved.TE damping fluid: 10mM Tris-HCl (pH8.0), 1mM EDTA, room temperature (25 ℃) is preserved.
The application of embodiment 2 positive colonies
2.1 the amplification of positive phage clones: get the positive phage clones supernatant that obtains in the 5 μ L steps 1.4 and join the early stage (OD of 20mL logarithmic growth
600~ 0.3) host bacterium ER2738 (Ph.D.-12
TMProvide among the Phage Display Peptide Library Kit, available from New EnglandBiolabs company) in, increased 4 hours, 10000 left the heart 10 minutes, get supernatant, PEG/Nacl precipitation twice is dissolved with 200 μ LTBS at last, the positive phage clones that obtains increasing calculates its titre (the same step 3) of required agent prescription again.
2.2 hemagglutination-inhibition test: with the pure virus that obtains in the step 1.1 with 1% pancreatin handle the back on blood-coagulation-board by 100 * begin to do doubling dilution, every hole adds the fresh chicken red blood cell of 50 μ L0.5%, HAU is write down in 37 ℃ of reactions 1 hour.The positive bacteriophage equal-volume of getting the amplification that 25 μ L and step 2.1 obtain mixes, and making viral final concentration is 4 HAUs.37 ℃ act on 1 hour, add the fresh chicken red blood cell of 50 μ L0.5%, and 37 ℃ act on 1 hour, the observed and recorded result.
2.3 derived peptide hemagglutination-inhibition test: the contained specific amino acid of the phage that step 1.5 is obtained is through the replacement of one or several amino-acid residue, the phage that disappearance or interpolation obtain containing new aminoacid sequence carries out hemagglutination-inhibition test according to step 2.1 and the described step of step 2.2.Experimental result shows, with the replacement of the contained specific amino acid of phage of the present invention through one or several amino-acid residue, the phage that contains new aminoacid sequence that disappearance or add obtains still can specificity suppresses the hemagglutinative function of IBV.
2.4 non-specific detection test: measure Avian pneumo-encephalitis virus (Li Hongbin according to ordinary method, the comprehensive anti-system of newcastle disease, agricultural science and technology communication: 1998.6:23-24), avian influenza virus H9N2 strain (Li Zili etc., the diagnosis of Hubei Province's bird flu and the separation of virus strain and preliminary evaluation, China's Preventive Veterinary Medicine newspaper: 2001.23 (2): 146-148) HAU, the Ph.D-H-IV-14 that step 2.1 is obtained does doubling dilution since 2 times, respectively with the Avian pneumo-encephalitis virus of 4 HAUs of 25 μ L, avian influenza virus H9N2 strain equal-volume mixes, simultaneously, to contain irrelevant peptide phage since 2 times of doubling dilutions, mix, and establish positive bacteriophage with IBV virus (handling) equal-volume of 4 HAUs of 25 μ L through 1% pancreatin, IBV, Avian pneumo-encephalitis virus, avian influenza virus H9N2 strain and the contrast of red corpuscle self-solidifying, 37 ℃ act on 1 hour, add the fresh chicken red blood cell of 50 μ L0.5%, 37 ℃ act on 1 hour, the observed and recorded result.
2.5 antigen-antibody blocking test: the pure virus packets that step 1.1 is obtained is by elisa plate, and 4 ℃ are spent the night, the coating buffer that inclines next day, every hole adds 200 μ L confining liquids, 4 ℃ 1 hour.TBST washes 3 times, add the concentrated phage (10 * beginning doubling dilution) that step 2.1 obtains, 37 ℃ 2 hours, TBST washes plate, add anti-IBV rabbit anteserum (600 times of dilutions of confining liquid) (step 2 obtains), 37 ℃ 1 hour, TBST washes plate, add goat-anti rabbit ELIAS secondary antibody (available from Southern Biotechnology Associates company) (4000 times of dilutions of confining liquid), 37 ℃ 1 hour, TBST washes plate, every hole adds 100 μ L substrate solutions, black out left standstill 15 minutes, read 630nm place light absorption value (the agents useful for same same step 4) of filling a prescription.
2.6 virus infection blocking test: the pure virus infection HeLa cell with step 1.1 obtains records TCID with ordinary method
50, use 100 TCID then
50Virus is carried out the virus infection blocking test.Specific practice is: the phage 10 * beginning doubling dilution with step 2.1 obtains, mix with isopyknic virus then, and making the final concentration of virus is 100 TCID
5037 ℃ act on 2 hours, are added drop-wise to 96 orifice plates, and every hole 100 μ L add 100 μ LHeLa cell suspensions again, and establish cell contrast (promptly do not add virus, also do not add phage), virus control (not adding phage) and phage contrast (not adding virus).
2.7 the avian infectious bronchitis virus differential diagnosis kit constitutes and the preparation method
1, test kit comprises:
Positive-virus contrast 1 pipe 0.5mL/ pipe
1 bottle of 50mL/ bottle of 10 * physiological saline
1 bottle of 50mL/ bottle of 10 * PBS damping fluid
1 bottle of 20mL/ bottle of A Shi liquid
2, related solution prescription
10 times of PBS damping fluids: NaCl 80g, KCl 2g, Na
2HPO
412H
2O 29g, KH
2PO
42g, distilled water add to 1000mL (pH7.4).
10 times of physiological saline: NaCl 8.5g, distilled water adds to 100mL
A Shi liquid: glucose 2.05g, structure lemon acid sodium 0.80g, structure lemon acid 0.05g, NaCl 0.42g, distilled water adds to 100ml.Above medicine mixing post-heating dissolves, 115 ℃ of sterilizations in 10 minutes, and 4 ℃ of preservations are standby.
3, the operation steps of test kit and criterion
Operation steps:
1) 0.5% chicken red blood cell preparation: get new freshly-slaughtered poultry blood (A Shi liquid and chicken blood were by 1: 3 mixed) and pour in the centrifuge tube, 3000 rev/mins centrifugal 5 minutes, supernatant liquor is removed in suction, the deposition red corpuscle adds 20-30 times of volume physiological saline, again Red Blood Cells Suspension, again 3000 rev/mins centrifugal 5 minutes, inhale and remove supernatant liquor, repeat again to be made into 0.5% chicken erythrocyte suspension then with physiological salt liquid by the erythrocytic volume of hematocrit for several times.
2) measure viral hemoagglutination unit to be measured according to a conventional method
2) with phage on blood-coagulation-board since 1: 2 times of doubling dilution, add 4 HAU viruses to be measured of 25 μ L equal-volumes, 37 ℃ 1 hour, add the fresh chicken red blood cell of 50 μ L0.5%, 37 ℃ 1 hour, the observed and recorded result.Simultaneously, establish viral hemoagglutination to be measured, positive-virus blood clotting, positive-virus blood clotting suppress and the contrast of red corpuscle self-solidifying.
Criterion:
Result seen in the hole
One deck red corpuscle evenly is laid at the bottom of the hole, i.e. 100% red cell agglutination.++++
Red corpuscle is laid on the pipe end, but the edge is not whole, and great circle is arranged at the bottom of the hole.+++
Red corpuscle is little ring-type at the bottom of the hole, little grumeleuse is arranged all around.++
Red corpuscle is a little group at the bottom of the hole, the edge is rough, and a small amount of aggegation is arranged on every side.+
Red corpuscle is a dot at the bottom of the hole, the edge is smooth neat, and red corpuscle does not have agglutination phenomenon.-
When positive-virus blood clotting and blood clotting suppress normal, viral hemoagglutination to be measured is ++ ++, phage suppress virus to be measured for-time, judge that virus to be measured is positive.
Specificity of infectious bronchitis virus of chickens bound short peptide sequence (SEQUENCE LISTING)
＜110〉Hua Zhong Agriculture University
＜120〉contain phage and application with the specificity of infectious bronchitis virus of chickens bound short peptide
<130>
<141>2004-07-01
<160>1
<170>PatentIn version 3.1
<210>1
<211>12
<212>PRT
＜213〉chicken (Gallus gallus)
<220>
<221>DOMAIN
<222>(1)..(12)
<223>
<400>1
Gly Ser His His Arg His Val His Ser Pro Phe Val
1 5 10
Claims (3)
1. energy and avian infectious bronchitis virus H52 strain specificity bonded small peptide, its aminoacid sequence is shown in sequence table SEQ ID NO.1.
2. the preserving number that comprises aminoacid sequence shown in the sequence table SEQ ID NO.1 is the M13 phage Ph.D-H52-IV-14 of CCTCC NO:M204034.
3. claim 1 or 2 described small peptides or the phage application in preparation chicken infectious bronchitis differential diagnosis kit.
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| GB0911794D0 (en) * | 2009-07-07 | 2009-08-19 | Animal Health Inst | Chimaeric protein |
| CN101805395B (en) * | 2010-03-10 | 2012-02-01 | 中国农业大学 | Polypeptide with inhibiting virus mixed infection activity |
| CN102721812B (en) * | 2012-06-20 | 2014-09-03 | 江苏省农业科学院 | Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof |
| CN108690122B (en) * | 2018-05-29 | 2021-06-25 | 郴州市第一人民医院 | Polypeptide specifically combined with mycoplasma pneumoniae positive serum and diagnostic kit |
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| CN1397648A (en) * | 2001-11-28 | 2003-02-19 | 山东省农业科学院家禽研究所 | Molecular biologic method for fastly diagnosing infections bronchitis virus of chicken |
| CN2578831Y (en) * | 2002-11-21 | 2003-10-08 | 陈福勇 | Antibody inspection reagent box of virus of infectious fowl bronchitis |
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| CN1397648A (en) * | 2001-11-28 | 2003-02-19 | 山东省农业科学院家禽研究所 | Molecular biologic method for fastly diagnosing infections bronchitis virus of chicken |
| CN2578831Y (en) * | 2002-11-21 | 2003-10-08 | 陈福勇 | Antibody inspection reagent box of virus of infectious fowl bronchitis |
Non-Patent Citations (1)
| Title |
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| 鸡传染性支气管炎诊断及免疫的最新研究. 张念华等.山东家禽，第7期. 2003 * |
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