CN104610445A - Portunus trituberculatus reovirus receptor, screening method and application thereof - Google Patents
Portunus trituberculatus reovirus receptor, screening method and application thereof Download PDFInfo
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- CN104610445A CN104610445A CN201410833447.7A CN201410833447A CN104610445A CN 104610445 A CN104610445 A CN 104610445A CN 201410833447 A CN201410833447 A CN 201410833447A CN 104610445 A CN104610445 A CN 104610445A
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Abstract
The invention discloses a portunus trituberculatus reovirus receptor, a screening method and application thereof. The portunus trituberculatus reovirus receptor is characterized by being microtubulin. The screening method comprises the following steps: anatomizing healthy trituberculatus on ice, taking branchiae and adding the branchiae to a membrane protein extracting liquid, homogenizing, and performing resuspension on sediments obtained through centrifugation by the membrane protein extracting liquid so as to obtain a branchia membrane protein extracting liquid of portunus trituberculatus; performing separation on the membrane protein by using 8% SDS-PAGE and electrotransferring the membrane protein to a PVDF (polyvinylidene fluoride) membrane, performing overnight incubation by low-fat milk under 4 DEG C, washing the incubated membrane for three times, incubating the washed membrane for an hour by SCRV virus under room temperature, washing the incubated membrane for three times again, incubating the washed membrane by chicken antibody SCRV under room temperature for two hours, washing the incubated membrane for three times, incubating the washed membrane by a goat-anti-chicken second antibody in HRP coupling under room temperature for an hour, washing the incubated membrane for four times, performing color development by OPD so as to obtain a protein band, namely the SCRV receptor. The portunus trituberculatus reovirus receptor has the advantages that a receptor blocking agent has inhibitory activity to SCRV infection cells, and a medicine for preventing and treating SCRV infection can be developed.
Description
Technical field
The invention belongs to field of biology, the control particularly relating to a kind of novel Portunus trituberculatus Miers reovirus (SCRV) acceptor and utilize receptor protein antibody to carry out SCRV virus infection.
Background technology
Portunus trituberculatus Miers (Portunus trituberculatus) is a kind of important marine economic animal, and meat flavour is delicious, nutritious, and the dark favor by consumers in general is also important foreign exchange earning kind.Current Portunus trituberculatus Miers cultivates largest marine economy crab class in the world.In recent years, autumn and winter season often occurs that the epidemic disease caused by swimming crab reovirus makes the phenomenon of swimming crab mortality, carrys out important financial loss to cultivation industrial zone.The strain of described Portunus trituberculatus Miers reovirus is R1015 strain, and preserving number is CGMCC No.5379, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2011.(class) reovirus is the important pathogen of mitten crab (Eriocheir sinensi), Young Crab (Scylla serrata) and Portunus trituberculatus Miers (Portunus trituberculatus), and mammalian reovirus acceptor studies comparatively that system is deeply and the research of crab class reovirus acceptor is not yet in the news.
Because virus receptor and the mutual work between virus and acceptor are the key point of virus infection and are paid attention to widely thus become research and development focus, the cell receptor of existing numerous people, animal, avian viruses is illustrated, and the research of Aquatic animals virus acceptor far lags behind people, animal, avian viruses.Main report has lefteye flonder lymphocystic virus, WSSV and ocean double RNA virus Marine birnavirus (MABV).Hydrobiont reovirus and cell receptor make aspect mutually, and few have several sections reports are mainly about GCRV.By providing available strategy to the research of Portunus trituberculatus Miers reovirus acceptor to this virus of prevention and control.
The research method of virus receptor is many, and the method as started with from gene level has yeast-two hybrid technique, clonal expression technology, display technique of bacteriophage etc.From the method that protein level is started with, as virus paving covers western blotting technique (VOPBA), Immunoprecipitation etc., the advantage of these methods is direct, objective.At present, the correlative study report of any blocking-up inhibitor about Portunus trituberculatus Miers reovirus acceptor and this receptor is not also disclosed both at home and abroad.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Portunus trituberculatus Miers reovirus acceptor, utilize antibody that this receptor protein immunization mouse obtains as receptor blocking agent simultaneously, this blocker has inhibit activities to Portunus trituberculatus Miers reovirus infection cell, can develop the medicine of control Portunus trituberculatus Miers reovirus infection.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of Portunus trituberculatus Miers reovirus acceptor, described acceptor is tubulin.
The screening method of above-mentioned Portunus trituberculatus Miers reovirus acceptor, concrete steps are as follows:
(1) by the Portunus trituberculatus Miers of health on ice solution take its gill, by mass volume ratio 1g:(5 – 10) ml join that ice bath in advance crosses containing homogenate in the Membrane protein extraction liquid of 1mM phenylmethylsulfonyl fluoride, by homogenate in 4 DEG C, the centrifugal 3min of 600g, gets supernatant liquor in 4 DEG C, after the centrifugal 5min of 6000g, get supernatant liquor again in 4 DEG C, the centrifugal 2h of 35000g, by resuspended for the precipitation Membrane protein extraction liquid obtained, namely obtains Portunus trituberculatus Miers gill Membrane protein extraction liquid;
(2) after Portunus trituberculatus Miers gill Membrane protein extraction liquid being carried out 8%SDS-PAGE electrophoresis, turning in liquid at electricity is transferred on pvdf membrane by protein bands all on gel, 300mA, 4 DEG C of transfer 3h, after transfer terminates, pvdf membrane is taken out from electric turn trough, after deionized water and PBST damping fluid a little rinsing, be immersed in 20-25ml confining liquid, the shake of 40r/min room temperature is closed and is spent the night, 3 times are washed by closing the pvdf membrane PBST damping fluid spent the night, each 5min, pvdf membrane after washing is put into plastics bag, add 1mL12.9ug/ml Portunus trituberculatus Miers reovirus, seal with sealing machine, after 1h is hatched in room temperature shake, with PBST buffer solution pvdf membrane 3 times, each 5min, then pvdf membrane is placed in chicken anti-Portunus trituberculatus Miers reovirus primary antibodie diluent, 2h is hatched at room temperature shake, then uses PBST buffer solution 3 times, each 5min, again pvdf membrane is placed in the anti-diluent of HRP-goat-anti chicken two, at room temperature slowly 1h is hatched in shake, use PBST buffer solution again 3 times, after each 10min, after pvdf membrane PBS damping fluid is washed 5min, with O-Phenylene Diamine lucifuge colour developing 15min at 37 DEG C, the protein band that pvdf membrane develops the color is Portunus trituberculatus Miers reovirus acceptor.
The compound method of the Membrane protein extraction liquid described in step (1) is as follows: 1mol/L Tris-HCl 100ml, MgCl of pH 8.0
26H
2o 0.4063g, 0.1mol/L EDTA 10ml, TritonX-100 2ml, supply distilled water to 1L, autoclaving.
Electricity described in step (2) turns liquid making method: Tris 3g, glycine 14.4g, and methyl alcohol 200ml, supplies distilled water to 1L; Described confining liquid is the PBST damping fluid containing 5wt% skimmed milk; Described chicken anti-Portunus trituberculatus Miers reovirus primary antibodie diluent is that the PBST damping fluid of chicken anti-Portunus trituberculatus Miers reovirus primary antibodie containing 0.5wt% skimmed milk dilutes 1000 times of gained; To be the anti-use of HRP-goat-anti chicken two dilute 5000 times of gained containing the PBST damping fluid of 0.5wt% skimmed milk to the described anti-diluent of HRP-goat-anti chicken two.
Described PBST buffer method is as follows: NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.2g, 500 μ L tween 20s, supply distilled water to 1L, regulate pH to 7.4, autoclaving; Described PBS buffer method is as follows: NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.2g, supplies distilled water to 1L, regulates pH to 7.4, autoclaving.
The preparation of the chicken anti-Portunus trituberculatus Miers reovirus primary antibodie described in step (2): get healthy first hen of laying eggs as immunization, by Portunus trituberculatus Miers reovirus antigen and the mixing of Freund's complete adjuvant equal-volume, the fully emulsified rear dipteron to hen, both legs, fundamental immunity is carried out in back and the intramuscular injection of chest muscle position, every hen immunizing dose is 500-800 μ g/mL, after 15-20 days, Portunus trituberculatus Miers reovirus antigen and Freund's incomplete adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to hen intramuscular injection, every hen immunizing dose is 400-600 μ g/mL, booster immunization repeats three times, each interval time is 7-10 days, collect egg mensuration after carrying out three immunity to tire, egg is collected when yolk antibody tires >1:20000, be separated yolk, yolk antibody is purified by saturated ammonium sulphate method.The concrete grammar of the purifying of yolk antibody is as follows: take appropriate yolk, acetic acid-sodium acetate buffer solution (0.05M is added by 1:9, pH5.0), stir rear 4 DEG C of hold over night, the centrifugal 20min of 8000g, getting supernatant liquor, to add saturated ammonium sulphate to saturation ratio be 40%, 4 DEG C are stirred 6h, the centrifugal 20min of 10000g, the bi-distilled water of precipitation 10-20 times of yolk quality is resuspended, adding saturated sodium sulfate to saturation ratio is 40%, 4 DEG C of stirrings are spent the night, the centrifugal 20min of 10000g, get a small amount of PBS solution (0.01M of precipitation, pH7.4) resuspended, dialysed overnight in PBS solution, namely chicken anti-Portunus trituberculatus Miers reovirus primary antibodie is obtained, save backup in-80 DEG C.
The purposes of described tubulin in the receptor blocking agent for the preparation of blocking-up SCRV virus infected cell.
Described receptor blocking agent is Portunus trituberculatus Miers reovirus receptor antibody, and its concrete preparation method is as follows:
Described tubulin 0.9% sodium-chlor suspends, by tubulin and the mixing of Freund's complete adjuvant equal-volume, fully emulsifiedly rear fundamental immunity is carried out to mouse subcutaneous injection, every mouse immune dosage is 10-20 μ g/mL, after 15 days, tubulin and Freund's incomplete adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to mouse subcutaneous injection, every mouse immune dosage is 8-15 μ g/mL, booster immunization repeats 3 times, each interval time is 7-10 days, after last immunity, eyeball is plucked to mouse and gets blood, blood ambient temperatare is put 1h, be transferred to 4 DEG C of standing 3-5h, after gently blood being fiddled with syringe needle, in 4 DEG C, the centrifugal 10min of 3000g, get serum, namely Portunus trituberculatus Miers reovirus receptor antibody is obtained, save backup in-80 DEG C.
Described Portunus trituberculatus Miers reovirus receptor antibody is for being developed to the application of control Portunus trituberculatus Miers reovirus infection aspect.
Compared with prior art, the invention has the advantages that: the present invention makes public for the first time a kind of Portunus trituberculatus Miers reovirus acceptor and screening method thereof and application, utilize antibody that this receptor protein immunization mouse obtains as receptor blocking agent, this blocker has inhibit activities to Portunus trituberculatus Miers reovirus infection cell, the medicine of control Portunus trituberculatus Miers reovirus infection can be developed, thus provide a brand-new thinking for the studying and combating of Portunus trituberculatus Miers reovirus, expand the scope of research, very important meaning is had for actual production.
Accompanying drawing explanation
Fig. 1 is for carry out the mass spectroscopy of Portunus trituberculatus Miers reovirus receptor protein fingerprint with Mascot software, and X-coordinate represents protein fractions, and ordinate zou represents number of clicks;
Fig. 2 is the co-immunoprecipitation result figure of the combination of Portunus trituberculatus Miers reovirus (SCRV) and SCRV acceptor;
Fig. 3 is the negative control in external Portunus trituberculatus Miers reovirus receptor blockade studies, PBS group;
Fig. 4 is that in external Portunus trituberculatus Miers reovirus receptor blockade studies, antireceptor antibody dilutes 10 times of groups;
Fig. 5 is that in external Portunus trituberculatus Miers reovirus receptor blockade studies, antireceptor antibody dilutes 20 times of groups;
Fig. 6 is that in external Portunus trituberculatus Miers reovirus receptor blockade studies, antireceptor antibody dilutes 40 times of groups.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
With western blotting technique screening Portunus trituberculatus Miers reovirus acceptor
1, Portunus trituberculatus Miers reovirus (SCRV) purifying
Get and attack poison and infect dead Portunus trituberculatus Miers, solution takes its internal organ on ice, weighs, by mass volume ratio 1g:(5-20) ml joins the TNE containing 1mM phenylmethylsulfonyl fluoride (PMSF) that ice bath is in advance crossed
2homogenate in buffered soln, by homogenate in 4 DEG C, the centrifugal 20min of 8000g, the TNE of precipitation containing 1mM PMSF
2again in 4 DEG C after the homogenate of damping fluid Eddy diffusion, the centrifugal 20min of 8000g, merges two times centrifugal gained supernatant liquor, by supernatant liquor in 4 DEG C, after the centrifugal 10min of 6000g, gets supernatant liquor in 4 DEG C, the centrifugal 1.5h of 38000g, with the TM of precooling
2damping fluid washes away precipitation upper strata and to loosen part, takes off the TM of the precooling of the solid white precipitate part of layer containing 1mM PMSF
2in 4 DEG C after damping fluid is resuspended, after the centrifugal 5min of 3500g, get supernatant liquor in 4 DEG C, the centrifugal 1.5h of 38000g, uses TM
2damping fluid washes away upper strata and to loosen part, and the solid white precipitate part of lower floor is again with the TM of the precooling containing 1mM PMSF
2in 4 DEG C after damping fluid is resuspended, after the centrifugal 5min of 3500g, get supernatant liquor again in 4 DEG C, the centrifugal 1.5h of 38000g, uses TM
2damping fluid washes away upper strata and to loosen part, and the solid white precipitate of the lower floor finally obtained is Portunus trituberculatus Miers reovirus antigen.
Above-mentioned TNE
2buffer method is as follows: Tris 6.0578g, NaCl 23.4g, disodium ethylene diamine tetraacetate 1.8612g, supplies distilled water to 1L, regulates pH to 8.5, autoclaving;
Above-mentioned TM
2buffer method is as follows: Tris 6.0578g, MgCl
26H
2o 2.0338g, NaCl 15g, supply distilled water to 1L, regulates pH to 7.4, autoclaving.
2, Portunus trituberculatus Miers gill Membrane protein extraction
Get healthy Portunus trituberculatus Miers, solution takes its gill on ice, weigh, by mass volume ratio 1g:(5 – 10) ml join that ice bath in advance crosses containing homogenate in the Membrane protein extraction liquid (Buffer M) of 1mM phenylmethylsulfonyl fluoride (PMSF), by homogenate in 4 DEG C, the centrifugal 3min of 600g, get supernatant liquor in 4 DEG C, after the centrifugal 5min of 6000g, get supernatant liquor again in 4 DEG C, the centrifugal 2h of 35000g, by resuspended for the precipitation Membrane protein extraction liquid obtained, namely obtains Portunus trituberculatus Miers gill Membrane protein extraction liquid.
The compound method of above-mentioned Membrane protein extraction liquid is as follows: 1mol/L Tris-Hcl (pH 8.0) 100ml, MgCl
26H
2o 0.4063g, 0.1mol/L EDTA 10ml, TritonX-1002ml, supply distilled water to 1L, autoclaving; The compound method of above-mentioned 1mol/L Tris-Hcl (pH 8.0) is as follows: Tris 121.1g, dense HCl 42ml, supplies distilled water to 1L, regulates pH to 8.0, autoclaving; The compound method of above-mentioned 0.1mol/L EDTA is as follows: EDTA-Na
237.22g, supplies distilled water to 1L, regulates pH to 8.0, autoclaving.
3, western blotting technique screening
After Portunus trituberculatus Miers gill Membrane protein extraction liquid is carried out 8%SDS-PAGE electrophoresis, (electricity turns liquid formula: Tris3g, glycine 14.4g to turn liquid at electricity, methyl alcohol 200ml, supply distilled water to 1L) in protein bands all on gel are transferred to (0.45um) on pvdf membrane, 300mA, 4 DEG C of transfer 3h, after transfer terminates, pvdf membrane is taken out from electric turn trough, after deionized water and PBST damping fluid a little rinsing, be immersed in confining liquid (the PBST damping fluid containing 5wt% skimmed milk) 20-25ml, the shake of 40r/min room temperature is closed and is spent the night.Wash 3 times, each 5min by closing the pvdf membrane PBST damping fluid spent the night, the pvdf membrane after washing is put into plastics bag, add 1mL12.9ug/ml Portunus trituberculatus Miers reovirus, seal with sealing machine, after 1h is hatched in room temperature shake, with PBST buffer solution pvdf membrane 3 times, each 5min; Then pvdf membrane is placed in chicken anti-Portunus trituberculatus Miers reovirus primary antibodie diluent (this diluent is that the PBST damping fluid of chicken anti-Portunus trituberculatus Miers reovirus primary antibodie containing 0.5wt% skimmed milk dilutes 1000 times of gained), 2h is hatched at room temperature shake, use PBST buffer solution again 3 times, each 5min; (this diluent is that anti-(health is century for HRP-goat-anti chicken two pvdf membrane to be placed in the anti-diluent of HRP-goat-anti chicken two again, CW6162) 5000 times of gained are diluted with the PBST damping fluid containing 0.5wt% skimmed milk) in, at room temperature slowly 1h is hatched in shake, use PBST buffer solution again 3 times, after each 10min, after pvdf membrane PBS damping fluid is washed 5min, with O-Phenylene Diamine (Shanghai Ai Bi chemical reagent company limited, AR100G) lucifuge colour developing 15min at 37 DEG C, the protein band that pvdf membrane develops the color is Portunus trituberculatus Miers reovirus acceptor.
Above-mentioned PBST buffer method is as follows: NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.2g, 500 μ L tween 20s, supply distilled water to 1L, regulate pH to 7.4, autoclaving; Above-mentioned PBS buffer method is as follows: NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.2g, supplies distilled water to 1L, regulates pH to 7.4, autoclaving.
4, Portunus trituberculatus Miers reovirus associated proteins carries out MALDI-TOF-MS-MS analysis
The gel obtain SDS-PAGE electrophoresis puts into centrifuge tube with the protein band cutting on pvdf membrane corresponding to Portunus trituberculatus Miers reovirus acceptor, with ParafilmTM, send it to Hui Jun bio tech ltd, Guangzhou and carry out the high flight time mass spectrum of substance assistant laser desorpted electricity (MALDI-TOF-MS-MS) analysis.Mass spectral results as shown in Figure 1, by Mascot software analysis, when albumen mark is greater than 41 (p<0.05), result is significant, it is 48 that Portunus trituberculatus Miers reovirus acceptor mates mark with the tubulin of Scylla paramamosain, it can thus be appreciated that above-mentioned Portunus trituberculatus Miers reovirus acceptor is tubulin (tubulin).
Specific embodiment two
The preparation of Portunus trituberculatus Miers reovirus (SCRV) receptor antibody
Above-mentioned Portunus trituberculatus Miers reovirus receptor protein is cut off homogenate from 8%SDS-PAGE gel, suspend with 0.9% sodium-chlor, by receptor protein and the mixing of Freund's complete adjuvant equal-volume, fully emulsifiedly rear fundamental immunity is carried out to mouse subcutaneous injection, every mouse immune dosage is 10-20 μ g/mL, after 15 days, Portunus trituberculatus Miers reovirus receptor protein and Freund's incomplete adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to mouse subcutaneous injection, every mouse immune dosage is 8-15 μ g/mL, booster immunization repeats 3 times, each interval time is 7-10 days, after last immunity, eyeball is plucked to mouse and gets blood, blood ambient temperatare is put 1h, be transferred to 4 DEG C of standing 3-5h, after gently blood being fiddled with syringe needle, in 4 DEG C, the centrifugal 10min of 3000g, get serum, namely Portunus trituberculatus Miers reovirus receptor antibody is obtained, save backup in-80 DEG C.
Specific embodiment three
The combination of Immunoprecipitation checking Portunus trituberculatus Miers reovirus (SCRV) and SCRV acceptor
The Portunus trituberculatus Miers gill Membrane protein extraction liquid of fresh for 800ul specific embodiment one preparation and 30ul Portunus trituberculatus Miers reovirus antigen are joined in centrifuge tube, 4 DEG C of slow jolting 8h, add 10ul mouse-anti receptor protein antibody again, anti-soft-shelled turtle systemic septicaemia spherical viruses yolk antibody as negative control, 4 DEG C of overnight incubation.Get 60ul albumin A/G agarose (Santa Cruz biotech company, sc-2003) 4 DEG C of slow jolting 3h in centrifuge tube are added after fully putting upside down mixing, increase salt NETN damping fluid 1ml, in 4 DEG C, 3000rpm is centrifugal, get precipitation repeated centrifugation to operate 4 times and wash, finally add NETN damping fluid 1ml, in 4 DEG C, 3000rpm is centrifugal, get precipitation and add 30ul albumen sample-loading buffer, boiling water boiling 5min, the centrifugal 1min of 12000g, get the cleer and peaceful Portunus trituberculatus Miers reovirus as positive control and carry out 12%SDS-PAGE electrophoresis, electric transferring film 4 DEG C transfer 3h is carried out after electrophoresis terminates, after transfer terminates, pvdf membrane is taken out from electric turn trough, after deionized water and PBST damping fluid a little rinsing, be immersed in confining liquid (the PBST damping fluid containing 5wt% skimmed milk) 20-25ml, the shake of 40r/min room temperature is closed and is spent the night.3 times are washed by closing the pvdf membrane PBST damping fluid spent the night, each 5min, then pvdf membrane is placed in chicken anti-Portunus trituberculatus Miers reovirus primary antibodie diluent (this diluent is that the PBST damping fluid of chicken anti-Portunus trituberculatus Miers reovirus primary antibodie containing 0.5wt% skimmed milk dilutes 1000 times of gained), 2h is hatched at room temperature shake, use PBST buffer solution again 3 times, each 5min; (this diluent is that anti-(health is century for HRP-goat-anti chicken two pvdf membrane to be placed in the anti-diluent of HRP-goat-anti chicken two again, CW6162) 5000 times of gained are diluted with the PBST damping fluid containing 0.5wt% skimmed milk) in, at room temperature slowly 1h is hatched in shake, use PBST buffer solution again 3 times, after each 10min, pvdf membrane is washed 1 time with PBS damping fluid, 5min, with O-Phenylene Diamine (Shanghai Ai Bi chemical reagent company limited, AR100G) lucifuge colour developing 15min at 37 DEG C.As shown in Figure 2, swimming lane 1 is with the Portunus trituberculatus Miers reovirus under the effect of mouse-anti receptor protein antibody to result; Swimming lane 2 is the Portunus trituberculatus Miers reovirus of positive control; Swimming lane 3 is negative control.As seen from the figure, Portunus trituberculatus Miers reovirus can combine with receptor protein.
Above-mentioned high salt NETN buffer method is as follows: NaCl 29.22g, NP-40 5ml, 1.0M Tris-Hcl (pH8.0) 20ml, 0.1M EDTA 10ml, supplies distilled water to 1L, regulates pH to 7.6, autoclaving; Above-mentioned NETN buffer method is as follows: 1.0M Tris-Hcl (pH8.0) 20ml, 0.1M EDTA 10ml, NaCl 5.844g, NP-40 5ml, supplies distilled water to 1L, autoclaving.
The preparation of above-mentioned anti-soft-shelled turtle systemic septicaemia spherical viruses yolk antibody:
Get healthy first hen of laying eggs as immunization, by soft-shelled turtle systemic septicaemia spherical viruses antigen and the mixing of Freund's complete adjuvant equal-volume, the fully emulsified rear dipteron to hen, both legs, fundamental immunity is carried out in back and the intramuscular injection of chest muscle position, every hen immunizing dose is 500-800 μ g/mL, after 15-20 days, soft-shelled turtle systemic septicaemia spherical viruses antigen and Freund's incomplete adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to hen intramuscular injection, every hen immunizing dose is 400-600 μ g/mL, booster immunization repeats three times, each interval time is 7-10 days, collect egg mensuration after carrying out three immunity to tire, egg is collected when yolk antibody tires >1:20000, be separated yolk, yolk antibody is purified by saturated ammonium sulphate method.The concrete grammar of the purifying of yolk antibody is as follows: take appropriate yolk, acetic acid-sodium acetate buffer solution (0.05M is added by 1:9, pH5.0), stir rear 4 DEG C of hold over night, the centrifugal 20min of 8000g, getting supernatant liquor, to add saturated ammonium sulphate to saturation ratio be 40%, 4 DEG C are stirred 6h, the centrifugal 20min of 10000g, the bi-distilled water of precipitation 10-20 times of yolk quality is resuspended, adding saturated sodium sulfate to saturation ratio is 40%, 4 DEG C of stirrings are spent the night, the centrifugal 20min of 10000g, get a small amount of PBS solution (0.01M of precipitation, pH7.4) resuspended, dialysed overnight in PBS solution, namely anti-soft-shelled turtle systemic septicaemia spherical viruses yolk antibody is obtained, above-mentioned acetic acid-sodium acetate buffer solution formula is as follows: sodium acetate 2.604g, glacial acetic acid 1.095g, supplies bi-distilled water to 1L, adjusts pH to 5.0, autoclaving.
Specific embodiment four
External Portunus trituberculatus Miers reovirus (SCRV) receptor blockade studies
1, fluorescein isothiocyanate (FITC) marks the preparation of Portunus trituberculatus Miers reovirus: after by SCRV and FITC, 1h is hatched in the mixing of 1:100 ratio in mass ratio, the centrifugal 10min of 10000g, gets precipitation TM
2damping fluid suspends, then washs three times with this damping fluid at the centrifugal 10min of 10000g, to remove unconjugated FITC, suspends precipitate with 1ml PBS damping fluid;
2, Portunus trituberculatus Miers blood cell suspension preparation: heart extracting blood is carried out to swimming crab with disposable syringe, the ratio of blood and antithrombotics is that 1:1,500g centrifugal 3min, 1ml PBS damping fluid suspends and precipitates;
Described antithrombotics compound method is as follows: monohydrate potassium 0.547g, two citric acid monohydrate sodium 9.116g, glucose 18.7g, NaCl 26g, supplies distilled water to 1L, regulates pH to 5.66, autoclaving;
3, above-mentioned Portunus trituberculatus Miers hemocyte suspension is added to 24 porocyte culture plates, incubated at room 1h, add Portunus trituberculatus Miers reovirus receptor antibody to dilute 10 times (Fig. 4), 20 times (Fig. 5), 40 times (Fig. 6) three gradients, add PBS damping fluid as negative control, incubated at room 1h, the Portunus trituberculatus Miers reovirus that each hole adds marked by fluorescein isothiocyanate continues incubated at room 30min, with PBS buffer solution twice, each 5min, at microscopy results.Result as seen in figures 3-6, when being 10 times by Fig. 3-6 known Portunus trituberculatus Miers reovirus receptor antibody extent of dilution (Fig. 4), Portunus trituberculatus Miers reovirus number obviously reduces relative to negative control group (Fig. 3), when Portunus trituberculatus Miers reovirus receptor antibody extent of dilution is 20,40 times (Fig. 5, Fig. 6), Portunus trituberculatus Miers reovirus number slightly reduces relative to negative control group (Fig. 3).It can thus be appreciated that adding the suppression infection of Portunus trituberculatus Miers reovirus receptor antibody to Portunus trituberculatus Miers reovirus has obvious effect.
Specific embodiment five
Portunus trituberculatus Miers reovirus (SCRV) receptor blockade studies in body
1, experiment crab and grouping
Portunus trituberculatus Miers is provided by prosperous hundred million plants in Xiangshan, Ningbo, and average 20g/ only; Experiment with crab laboratory inflation support temporarily 7d stable after, person divides three groups to select bouncing, A group for PBS be blank group, B group is SCRV infected group, makes negative control group, and C group is SCRV receptor antibody experimental group, 20/group.
2, seed culture of viruses process
Get the lethal Portunus trituberculatus Miers leg muscle of 2g reovirus infection, add 30mlTM
2damping fluid carries out homogenate on ice; Homogenate is at 4 DEG C, and the centrifugal 20min of 6000g, gets supernatant, adds penicillin, Streptomycin sulphate mixed solution that final concentration is 1000 units, 4 DEG C of hold over night.
3, experimental technique
A group (blank group): from Portunus trituberculatus Miers second step injection PBS, 150ul/ only;
B group (negative control group): attack venom from Portunus trituberculatus Miers second step injection, 150ul/ is only;
C group (SCRV receptor antibody experimental group): from Portunus trituberculatus Miers second step injection SCRV receptor antibody 50ul, after half hour, every only injection again attacks venom 100ul.
4, experimental result detects
After infecting, every day carries out changing water, inflation cultivation, observes swimming crab morbidity and death condition, carries out record, and calculate relative protection ratio to dead crab quantity:
Relative protection ratio (%)=(the dead mantissa of 1-positive group/dead mantissa of feminine gender group) × 100%.
5, result
Infection experiment result: by the survival condition of infection experiment crab, the experimental group swimming crab carrying out seed culture of viruses infection after the injection of result display SCRV receptor antibody more comparatively infects control group survival rate and is improved, and its relative protection ratio is 50%.Infectable infection is a kind of very fierce mode of infection, when SCRV adopts and infects swimming crab in this way, its lethal power is extremely strong, and it is larger to attack toxic agent amount, survival rate after SCRV receptor antibody can make virus infection swimming crab in this case improves, prove that the infection activity of this SCRV receptor antibody to Portunus trituberculatus Miers reovirus has obvious restraining effect, can be used for the medicine developing control Portunus trituberculatus Miers reovirus.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (8)
1. a Portunus trituberculatus Miers reovirus acceptor, is characterized in that: described acceptor is tubulin.
2. a screening method for Portunus trituberculatus Miers reovirus acceptor according to claim 1, is characterized in that concrete steps are as follows:
(1) get healthy Portunus trituberculatus Miers on ice solution take its gill, by mass volume ratio 1 g:(5 – 10) ml join that ice bath in advance crosses containing homogenate in the Membrane protein extraction liquid of 1mM phenylmethylsulfonyl fluoride, by homogenate in 4 DEG C, the centrifugal 3min of 600g, gets supernatant liquor in 4 DEG C, after the centrifugal 5min of 6000g, get supernatant liquor again in 4 DEG C, centrifugal 2 h of 35000g, by resuspended for the precipitation Membrane protein extraction liquid obtained, namely obtain Portunus trituberculatus Miers gill Membrane protein extraction liquid;
(2) after Portunus trituberculatus Miers gill Membrane protein extraction liquid being carried out 8%SDS-PAGE electrophoresis, turning in liquid at electricity is transferred on pvdf membrane by protein bands all on gel, 300mA, 4 DEG C of transfer 3h, after transfer terminates, pvdf membrane is taken out from electric turn trough, after deionized water and PBST damping fluid a little rinsing, be immersed in 20-25ml confining liquid, the shake of 40r/min room temperature is closed and is spent the night, 3 times are washed by closing the pvdf membrane PBST damping fluid spent the night, each 5min, pvdf membrane after washing is put into plastics bag, add 1mL12.9ug/ml Portunus trituberculatus Miers reovirus, seal with sealing machine, after 1h is hatched in room temperature shake, with PBST buffer solution pvdf membrane 3 times, each 5min, then pvdf membrane is placed in chicken anti-Portunus trituberculatus Miers reovirus primary antibodie diluent, 2h is hatched at room temperature shake, then uses PBST buffer solution 3 times, each 5min, again pvdf membrane is placed in the anti-diluent of HRP-goat-anti chicken two, at room temperature slowly 1h is hatched in shake, use PBST buffer solution again 3 times, after each 10min, after pvdf membrane PBS damping fluid is washed 5min, with O-Phenylene Diamine lucifuge colour developing 15min at 37 DEG C, the protein band that pvdf membrane develops the color is Portunus trituberculatus Miers reovirus acceptor.
3. the screening method of a kind of Portunus trituberculatus Miers reovirus acceptor according to claim 2, is characterized in that the compound method of the Membrane protein extraction liquid described in step (1) is as follows: 1mol/L Tris-HCl 100ml, MgCl of pH 8.0
26H
2o 0.4063g, 0.1mol/L EDTA 10ml, TritonX-100 2ml, supply distilled water to 1L, autoclaving.
4. the screening method of a kind of Portunus trituberculatus Miers reovirus acceptor according to claim 2, it is characterized in that the electricity described in step (2) turns liquid making method and is: Tris 3g, glycine 14.4g, methyl alcohol 200ml, supplies distilled water to 1L; Described confining liquid is the PBST damping fluid containing 5wt% skimmed milk; Described chicken anti-Portunus trituberculatus Miers reovirus primary antibodie diluent is that the PBST damping fluid of chicken anti-Portunus trituberculatus Miers reovirus primary antibodie containing 0.5wt% skimmed milk dilutes 1000 times of gained; To be the anti-use of HRP-goat-anti chicken two dilute 5000 times of gained containing the PBST damping fluid of 0.5wt% skimmed milk to the described anti-diluent of HRP-goat-anti chicken two.
5. the screening method of a kind of Portunus trituberculatus Miers reovirus acceptor according to claim 4, is characterized in that described PBST buffer method is as follows: NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.2g, 500 μ L tween 20s, supply distilled water to 1L, regulate pH to 7.4, autoclaving; Described PBS buffer method is as follows: NaCl 8g, KCl 0.2g, Na
2hPO
412H
2o 2.9g, KH
2pO
40.2g, supplies distilled water to 1L, regulates pH to 7.4, autoclaving.
6. a purposes for the Portunus trituberculatus Miers reovirus acceptor according to any one of claim 1-5, is characterized in that: the purposes of tubulin according to claim 1 in the receptor blocking agent for the preparation of blocking-up SCRV virus infected cell.
7. the purposes of a kind of Portunus trituberculatus Miers reovirus acceptor according to claim 6, it is characterized in that described receptor blocking agent is Portunus trituberculatus Miers reovirus receptor antibody, its concrete preparation method is as follows: suspended by tubulin according to claim 1 0.9% sodium-chlor, by tubulin and the mixing of Freund's complete adjuvant equal-volume, fully emulsifiedly rear fundamental immunity is carried out to mouse subcutaneous injection, every mouse immune dosage is 10-20 μ g/mL, after 15 days, tubulin and Freund's incomplete adjuvant are carried out equal-volume mixing, fully emulsifiedly rear booster immunization is carried out to mouse subcutaneous injection, every mouse immune dosage is 8-15 μ g/mL, booster immunization repeats 3 times, each interval time is 7-10 days, after last immunity, eyeball is plucked to mouse and gets blood, blood ambient temperatare is put 1h, be transferred to 4 DEG C of standing 3-5h, after gently blood being fiddled with syringe needle, in 4 DEG C, the centrifugal 10min of 3000g, get serum, namely Portunus trituberculatus Miers reovirus receptor antibody is obtained, save backup in-80 DEG C.
8. the purposes of a kind of Portunus trituberculatus Miers reovirus acceptor according to claim 7, is characterized in that: described Portunus trituberculatus Miers reovirus receptor antibody is for being developed to the application of control Portunus trituberculatus Miers reovirus infection aspect.
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