CN1431222A - Monoclone antibody of rice blast germ and detection peridium as well as method for locating antigen surface - Google Patents

Abstract

A monoclonic antibody of rice blast bacterium, its detecting peridium and the locating method on surface of antigen are disclosed, the mixture of conidia, germinal tubes and haptospores for rice blast bacterium are used as antigen to immunize BALB/C mouse, getting its spleen cells and myeloma cells sp2/o, fusing under 50% PEG to obtain hybridoma cells, and indirect ELISA screening to obtain 4 hybridome cells and their monoclonic antibody, which can interfer the formation of appresorium of rice blast bacterium and inhibit the pathogenicity of said rice blast bacterium on rice leaves.

Description

Pyricularia oryzae monoclonal antibody and detect the bag quilt and at the localization method of antigenic surface

Technical field

The present invention relates to a kind of Pyricularia oryzae monoclonal antibody and detect the bag quilt and at the localization method of antigenic surface.

Background technology

Rice blast is the destructive disease of paddy rice, and almost all there is this sick generation in the area of rice-cultivating in the world.Pyricularia oryzae is survived the winter on sick straw and sick paddy with mycelium and conidium, and sick straw and sick paddy are the main sources of disease primary infection in next year.It is that germ is successfully invaded the host and finishes whole one of the round-robin committed step that infects that appressorium forms.Therefore, carrying out the research that the Pyricularia oryzae appressorium forms mechanism necessitates.The influence that does not also utilize monoclonal antibody to analyze the Pyricularia oryzae antigenic structure and the bacterium appressorium is formed in the world.

Summary of the invention

The purpose of this invention is to provide a kind of Pyricularia oryzae monoclonal antibody and detect the bag quilt and at the localization method of antigenic surface.

The monoclonal antibody of 4 strain resisting rice blast bacterias is 4A1,2B4,2H4,1D1, can specific immune response be arranged with rice blast pathogen.

The ELISA of resisting rice blast bacteria monoclonal antibody detects method for coating, with the Millipore aqueous suspension conidium of sterilization, 3-6 * 10 ⁵24h are cultivated for 20 ℃ in individual/mL rice blast pathogen conidiospore suspension 50ul/ hole in the elisa plate in 96 holes, make spore-germination form appressorium, and tight adhesion is at the aperture diapire, again with the fixing 0.5h of the glutaraldehyde of 0.5-1%.

The step at the localization method of antigenic surface of resisting rice blast bacteria monoclonal antibody is as follows:

1) pH7.2 0.02mol/L PBS preparation rice blast pathogen conidiospore suspension is got 100uL on aseptic clean slide glass, cultivates 24h for 20 ℃, make spore-germination form appressorium, and tight adhesion is on slide glass;

2) wash once with above-mentioned PBS, 5min, with normal mice serum as negative control; With the positive contrast of Pyricularia oryzae polyclone mouse antibodies; With 1: 100-200 doubly dilutes monoclonal antibody as testing sample; Above-mentioned each sample adds 300-1000uL on slide glass, repeat 3,1-2h under the room temperature;

3) PBS gives a baby a bath on the third day after its birth time, each 5min, and adding extent of dilution is 1: the sheep anti-mouse igg 300-1000uL that purifies through affinity chromatography that the FITC of 40-100 engages, handle 1-2h under the room temperature;

4) PBS gives a baby a bath on the third day after its birth time, each 5min.9 parts of glycerine add the dropping buffering glycerine of 1 part of PBS, use the cover glass sealing liquid, and are observed and recorded experimental result under the fluorescent microscope of 450-490 in excitation wavelength.

For the monoclonal antibody of attempting to prepare the Pyricularia oryzae surface antigen inquires into that monoclonal antibody forms the effect of mechanism to appressorium and to the analysis of antigenic structure, and further monoclonal antibody is screened conidium cDNA expression library as probe, to obtain forming genes involved with appressorium.Therefore, we are with complete germinating spore immunity BALB/C small white mouse, and with complete germinating spore antigen selection positive hole.This point is most important to the monoclonal antibody that we obtain the Pyricularia oryzae surface antigen, but the difficulty of bag quilt when also having increased indirect ELISA widely and screening positive hole.We find out the method for coating in this paper in this research process, respond well.And in the ELISA process, the antigen quantity in the inspection window on inverted microscope at any time.This method also is because magnaporthe grisea spore can form tight adhesion and determine in germ tube and this characteristic of appressorium of basement membrane.The method for coating of using herein will be accomplished gentleness as far as possible in washing and application of sample process, must guard against beating ELISA reaction and pull, and blots with thieving paper to get final product.Simultaneously, we locate the distribution of monoclonal antibody on the Pyricularia oryzae surface with immunofluorescence technique.

Utilize monoclonal antibody as probe, screen the spore-bearing cDNA expression library of Pyricularia oryzae, to obtain forming relevant gene with appressorium, it is an effective way, we utilize 2B4 monoclonal anti private savings to obtain positive colony from conidium cDNA expression library at present, the gene DNA sequence of this positive colony is finished, and discovery is a new gene, and its function is also among research.Therefore, the monoclonal antibody of Pyricularia oryzae development for the rice blast pathogeny with and the research of antibody gene rice transformation lay the first stone.

Description of drawings

Fig. 1 adopts IiT to show the bonding state figure of various Pyricularia oryzae monoclonal antibodies on the germ surface;

Fig. 2 is the Western-blot analysis chart of monoclonal antibody.

Embodiment

In order to analyze the structure component of Pyricularia oryzae, and inquire into it to the effect that appressorium forms, the present invention utilizes monoclonal antibody technique to prepare the monoclonal antibody of Pyricularia oryzae first.The discovery monoclonal antibody can be sealed the germ surface antigen factor effectively under proper concn, disturb the appressorium of germ to form, and suppresses the infection process host and forms scab.This characteristic of monoclonal antibody provides a new thinking for the control of rice blast.And we utilize monoclonal antibody to be probe, and the cDNA expression library of screening rice blast pathogen conidiospore has obtained one and formed relevant gene with appressorium, for the molecular biology research of Pyricularia oryzae appressorium formation lays the first stone.1. germinating spore and appressorium antigen prepd

Pyricularia oryzae 91-11B1 bacterial classification is cultivated on OMTA.After producing a large amount of spores by mixed culture, use 0.25% gelatin, in the media surface shampooing, collect suspension lightly, after wool paper filters pure conidium.The conidium suspension is through 5, the centrifugal 5min of 000g, and the collecting precipitation spore, suspending with the deionized water (Mil-lipore) of sterilization is diluted to 10 ⁴Individual conidium/mL concentration.Get the conidium suspension 50uL of this concentration, carefully dropwise be added on the glass culture dish (diameter 9cm) of cleaning sterilization with liquid-transfering gun then, 64 in every ware, keep certain distance, note preventing the Colaesce of meeting between the water droplet, behind 23 ℃ of cultivation 10h, this moment, the germinating spore proportion was not 5.4%, it is 61.3% that germinating spore does not form the appressorium ratio, and the ratio that germinating spore has formed appressorium is 33.3%.Scrape gently with the little rubber of cleaning sterilization, collect the Pyricularia oryzae culture on the above-mentioned culture dish, centrifugal then collection contains the mixture of spore, germ tube and appressorium, and 4 ℃ of preservations are standby as antigen.2. MONOCLONAL ANTIBODIES SPECIFIC FOR immune animal

Antigen and isopyknic Fu Shi Freund's complete adjuvant (Sigma) is fully emulsified, the female mouse of BALB/C in age in 4-8 week is made abdominal injection, 3-4 was emulsified in same doses of antigen in isopyknic freund 's incomplete adjuvant after week, made the peritoneal immunity second time.2-3 makes booster immunization once more with the antigen of doubling dose after week, and the aseptic mouse spleen of getting is used for cytogamy after 3 days.Cytogamy

Get above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in 5-10: 1 ratio, mixing in the RPMI-1640 of serum-free (Gibco) substratum, the centrifugal 5min of 1500rpm, remove substratum, with molecular weight is that 3350 50%PEG (Sigma) is as fusogen, under 37 ℃ of following water-baths, add 0.5-0.7ml, make it merge 2min, RPMI-1640 substratum with serum-free stops merging the centrifugal 5min of back 1500rpm, precipitation suspends with the HAT substratum, branch installs to 96 holes and contains in the cell plate of feeder cell, 37 ℃, cultivates in the cell cultures vessel of 5%CO2.The screening in hybridoma and positive hole and clone

After 5 days, change liquid once with the HAT substratum, changed liquid with the HT substratum on the 10th day, by the time at the bottom of the fused cell coverage hole during 10%-50%, adopt the antibody cell hole of indirect ELISA method screening to the secretion Pyricularia oryzae, method is as follows:

1) bag is by rice blast pathogen conidiospore suspension (3 * 10 ⁵Individual conidium 1mL) 24h is cultivated for 20 ℃ in the 50ul/ hole in the elisa plate in 96 holes, makes spore-germination form appressorium, and tight adhesion is at the aperture diapire, as the antigen of bag quilt.As again with the fixing 0.5h of 1% acetaldehyde, better effects if.

2) with the 37 ℃ of sealings in 5% calf serum (diluting) confining liquid 100ul/ hole 30min with PBST.

3) application of sample: the every hole of cells and supernatant to be checked adds 50ul, repeats 2 holes.Establish each 2 hole of polyclonal serum positive control and diluent negative control on every deblocking reaction plate, 37 ℃ of incubation 1h.

4) the PBST washing lotion is given a baby a bath on the third day after its birth time, and each 3min adds the sheep anti-mouse igg binding substances 50ul/ hole of the horseradish peroxidation synthase mark of dilution in 1: 2000,37 ℃ of incubation 30min.

5) the PBST washing lotion is given a baby a bath on the third day after its birth time, each 3min.Add the every hole 50ul of freshly prepared substrate solution (o-phenylenediamine solution), 37 ℃ of incubation 10min.

6) add stop buffer (2mol/L H ₂SO ₄) every hole 25ul, termination reaction.Judge with elisa reading instrument, survey with elisa reading instrument and read the photometric quantity (OD) of each reacting hole at the 490nm place.Be calculated as follows: P/N=treats verify OD value/negative control hole OD value, and P/N value＞2.1 in tested supernatant hole are judged to the positive.Measured the culture supernatant of 850 hybridomas altogether, find that the culture supernatant of 230 cell strains can be positive in the indirect ELISA colour developing, wherein the TCS of 23 cell strains is strong positive.Further clone 4 strain cell strains, its feature sees Table 1.

The character of table 1 Pyricularia oryzae monoclonal antibody

Single gram antibody	Antibody type and subclass	Antibody titer	Antibody is in the distribution of antigenic surface
					4A1	????IgA	?1∶64000	Spore
2B4	????IgA	?1∶64000	Spore, germ tube, appressorium
					2H4	????IgA	?1∶36000	Appressorium
1D1	????IgA	?1∶16000	Germ tube

Three time clonings are carried out continuously with limiting dilution assay in positive cell hole to secretory antibody on 96 well culture plates, obtain the monoclonal antibody hybridoma cell strain, and the cell strain enlarged culturing is used to prepare monoclonal antibody ascites.MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying:

Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5-10 * 10 in 7-10 days ⁵Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm collects supernatant liquor, is monoclonal antibody.Get 1 times of volume ascites and add 2 times of volume 0.06M PH4.8 acetate buffer solution dilutions, add sad (30ul/ml ascites), the following edged of room temperature stirs, and clarifies 1 hour for 4 ℃, the centrifugal 20min of 12000rpm, collect supernatant, use 50% saturated ammonium sulphate immunoglobulin (Ig) again, placed 2 hours for 4 ℃, the centrifugal 20min of 3000rpm, precipitation is dissolved with the PBS solution of 2 times of volumes, promptly obtains the ascites antibody of purifying after 4 ℃ of mobile dialysis 24 hours ,-70 ℃ of preservations.3. determine the distribution of antibody with IiT at antigenic surface

Adopting indirect method and change slightly, mainly is to utilize behind the spore-germination can the characteristic of tight adhesion on slide glass to save to fix this step.Concrete steps are as follows:

1. with pH7.2 0.02mol/L PBS preparation rice blast pathogen conidiospore suspension (10 ⁴Individual conidium/mL). get 100uL on aseptic clean slide glass, cultivate 24h for 20 ℃, make spore-germination form appressorium, and tight adhesion is on slide glass.2. wash once 5min with above-mentioned PBS.3. with normal mice serum (Sigma) as negative control; With the positive contrast of Pyricularia oryzae polyclone mouse antibodies; Monoclonal antibody was as testing sample in 1: 100; Above-mentioned each sample adds 300uL on slide glass, repeat 3, handles 2h under the room temperature.4. PBS gives a baby a bath on the third day after its birth time, each 5min.5. (USA) 300uL handles 2h under the room temperature to the sheep anti-mouse igg of purifying through affinity chromatography that the FITC that added degree of releasing and be 1: 40 engages for full molecule, Saint Lauis.6. PBS gives a baby a bath on the third day after its birth time, each 5min.7. drip buffering glycerine (9 parts of glycerine add 1 part of PBS), use the cover glass sealing liquid, and be observed and recorded experimental result under the fluorescent microscope of 450-490 in excitation wavelength.Found that 4 strain monoclonal antibody antigen binding sites have different distribution situations respectively on conidium, germ tube and the appressorium surface of Pyricularia oryzae.See Fig. 1, A:2B4 can have keying action with conidium, germ tube and appressorium; B:4A1 can have very strong keying action to spore, but very weak to germ tube and appressorium reaction, C:1D1 only has keying action to germ tube, to conidium and appressorium debond.D:2H4 can only play very strong keying action with appressorium, but mitogenetic bundle and germ tube are not acted on; 4 monoclonal antibodies are measured the interference effect that appressorium forms

Each monoclonal antibody mouse ascites IgG is diluted to 5,10 and three concentration gradients of 20ug/mL.Each the extent of dilution 20uL that gets each monoclonal antibody mouse ascites IgG be added on the onion epidermis and the aperture of 24 well culture plates in, add 2 * 10 of 20uL then ⁴The spore suspension of individual conidium/mL makes spore concentration and IgG concentration all release rare one times after the mixing.With normal mouse serum IgG (Sigma) as positive control, triplicate.Measure the rate of formation of appressorium behind 28 ℃ of following 18h.Test repeats secondary.The result shows that 2B4,4A1,1D1 and 2H4 can pull the formation that suppresses the Pyricularia oryzae appressorium effectively at onion epidermis and 24 hole polyethylene cultivations.2B4 action effect the best can also play restraining effect even the concentration of its IgG is 5ug/mL; Particularly evident on the onion epidermis surface, observe behind the 10h and found restraining effect, but the appressorium rate of contrast formation this moment is also not high, therefore, contrast not obviously, and contrast appressorium rate of formation reaches 100% behind the 18h, and about the same behind experimental group appressorium rate of formation and the 10h, it is obvious to suppress effect.5. monoclonal antibody is measured the pathogenic restraining effect of Pyricularia oryzae

Get five leaf phase rice leaves, be cut into the 5.00cm long segment, place the culture dish (diameter is 9cm) that contains wet disinfecting silk or cotton bouquet then, blade back up, and is standby.Simultaneously, adopt the inhibited monoclonal antibody IgG of Pyricularia oryzae appressorium is mixed with the conidium suspension, making conidium concentration is 10 ⁴Individual conidium/mL, monoclonal antibody IgG concentration is corresponding be diluted to original corresponding to the inhibited concentration of appressorium.Get 10uL then altogether and be added to segmental leaf back one side of paddy rice (is the boundary with middle arteries and veins), 3 on every leaf adds the same concentration of 10uL at the corresponding opposite side of leaf back but does not contain the conidium suspension (10 of monoclonal antibody ⁴Individual conidium/mL), same 3,3 blades of every ware repeat 3 wares.Place 28 ℃ of following light dark (13h/11h) to cultivate 5d in above-mentioned culture dish, write down experimental result every day.Experiment repeats 2 times.Test shows that monoclonal antibody 2B4,4A1,1D1 and 2H4 all have effective restraining effect to the scab formation of Pyricularia oryzae.The effect that monoclonal antibody 2B4 performance is the strongest when its IgG concentration only is 5ug/mL, just can have effective restraining effect to scab.Thereby monoclonal antibody mainly is to disturb germ to form the formation that appressorium suppresses or delay scab fully at the rice leaf surface to the pathogenic influence of germ.6. the Western-blot of monoclonal antibody analyzes

1) sample preparation: collect each 0.5g of conidium that conidium and band germinate, add 1%SDS respectively and contain 1mmol/LPMSF and 40mmol/L beta-mercaptoethanol 0.5mL, place the Eppendorf centrifuge tube, in boiling water, boil 30min, then 8, the centrifugal 5min of 000r/min gets supernatant, adds to contain SDS protein electrophoresis sample-loading buffer.Electrophoresis is standby.Other collects each 0.5g of conidium that conidium and band germinate, and adds cold 100% trifluoroacetic acid (TFA) of 0.5mL respectively, abundant mixing 10min, and 8, the centrifugal 10min of 000r/min, low temperature vacuumizes, and after the drying, adds and contains SDS protein electrophoresis buffering and tuck in, and mixing is standby.

2) SDS-PAGE, Western blotting analyze: carry out with reference to ordinary method, the result shows that monoclonal antibody 2B4 can play keying action with the proteantigen of 15KDa.Because this proteantigen is present in the cold 100%TFA extract on conidium and germ tube surface, and is not present in the hot 1%SDS extract, may be certain hydrophobin so infer this albumen.Western blotting analysis revealed, other three strains monoclonal antibody, 4A1,1D1 and 2H4 all can not take up keying action with any albumen one of cold 100%TFA extract on conidium or germ tube surface.4A1 only reaches from molecular weight marker (Promega the conidium extract 31kD albumen that is dissolved in hot 1%SDS, USA) the carboxylase albumen of component 31kD plays keying action, germ tube extract and cold 100%TFA extract are not played keying action, and 1D1 only discerns the 60kD albumen in the germ tube extract that is dissolved in hot 1%SDS; 2H4 does not all find special in conjunction with histogram 2 to above-mentioned various materials (TFA and SDS extract).

Claims (3)

1. the monoclonal antibody of a resisting rice blast bacteria is characterized in that, 4A1,2B4,2H4,1D1 can have specific immune response with rice blast pathogen.

2. the ELISA of a resisting rice blast bacteria monoclonal antibody detects method for coating, it is characterized in that: with the Millipore aqueous suspension conidium of sterilization, 3-6 * 10 ⁵24h are cultivated for 20 ℃ in individual/mL rice blast pathogen conidiospore suspension 50ul/ hole in the elisa plate in 96 holes, make spore-germination form appressorium, and tight adhesion is at the aperture diapire, again with the fixing 0.5h of the glutaraldehyde of 0.5-1%.

3. a resisting rice blast bacteria monoclonal antibody is characterized in that at the localization method of antigenic surface its step is as follows:

1) pH7.2 0.02mol/L PBS preparation rice blast pathogen conidiospore suspension is got 100uL on aseptic clean slide glass, cultivates 24h for 20 ℃, make spore-germination form appressorium, and tight adhesion is on slide glass;

2) wash once with above-mentioned PBS, 5min, with normal mice serum as negative control; With the positive contrast of Pyricularia oryzae polyclone mouse antibodies; With 1: 100-200 doubly dilutes monoclonal antibody as testing sample; Above-mentioned each sample adds 300-1000uL on slide glass, repeat 3,1-2h under the room temperature;

3) PBS gives a baby a bath on the third day after its birth time, each 5min, and adding extent of dilution is 1: the sheep anti-mouse igg 300-1000uL that purifies through affinity chromatography that the FITC of 40-100 engages, handle 1-2h under the room temperature;

4) PBS gives a baby a bath on the third day after its birth time, each 5min, and 9 parts of glycerine add the dropping buffering glycerine of 1 part of PBS, use the cover glass sealing liquid, and are observed and recorded experimental result under the fluorescent microscope of 450-490 in excitation wavelength.

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