CN102532312A - Anti-Vibrio anguillarum yolk antibody, and preparation method, titer detection method and application thereof - Google Patents
Anti-Vibrio anguillarum yolk antibody, and preparation method, titer detection method and application thereof Download PDFInfo
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- CN102532312A CN102532312A CN2012100191527A CN201210019152A CN102532312A CN 102532312 A CN102532312 A CN 102532312A CN 2012100191527 A CN2012100191527 A CN 2012100191527A CN 201210019152 A CN201210019152 A CN 201210019152A CN 102532312 A CN102532312 A CN 102532312A
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Abstract
The invention discloses an anti-Vibrio anguillarum yolk antibody, and the preparation method, the titer detection method and the application thereof. The invention is characterized in that the anti-Vibrio anguillarum yolk antibody is obtained by immunizing laying hens with Vibrio anguillarum as antigen, collecting highly-immunized eggs and performing separation and purification of yolk of the immunized eggs. The method comprises a step of preparing a Vibrio anguillarum antigen and inactivating the antigen, a step of performing basic immunization once in laying hens and secondary immunization for two to three times and collecting eggs, and a step of performing centrifugal separation, salting-out and dialysis to obtain the final product. The titer of the anti-Vibrio anguillarum yolk antibody is determined by indirect enzyme-linked immunosorbent assay. The anti-Vibrio anguillarum yolk antibody can be used for prevention and treatment of diseases caused by Vibrio anguillarum infection and can also be used for development of Vibrio anguillarum test kits and immunoreaction and diagnosis in immunology. The invention has the advantages that the yolk antibody is higher in purity and has a significant inhibitory effect on Vibrio anguillarum; the extraction of highly-immunized yolk is cost-efficient and convenient, and is adapted to low-cost and mass production; and the preparation method is simple and feasible.
Description
Technical field
The present invention relates to biological products technology of preparing and Application Areas, especially relate to a kind of anti-vibrio anguillarum yolk antibody.
Background technology
Vibrio anguillarum (
Vibrio anguillarum) be the very wide aquatic pathogenic bacteria of a kind of popularity, all can cause a disease to seawater, freshwater animals, brought tremendous loss to the raiser throughout the year.It is reported that Vibrio anguillarum can infect lefteye flounder, perch, salmon, yellow croaker, rainbow trout, common eel, sweetfish, cod, turbot etc.; It is the pathogenic bacterium of multiple prawn, and the immunity system of shrimps such as Macrobrachium rosenbergii, chlamys farreri, shellfish is had the strong impulse effect, is the higher bacterium of harm in the vibrios.
The means of prevention of the disease that causes for Vibrio anguillarum at present will have following several kinds: microbiotic, herbal medicine, inactivated vaccine, monoclonal antibody etc.Microbiotic and chemicals are prone to cause stress response, and biology is had toxicity in various degree, and use is easy to generate resistance repeatedly, has drug residue, will step down from the stage of history in the today of advocating green agriculture; The lasting medicine of herbal medicine, but be difficult to rapid onset, and each department herbal medicine property of medicine difference is bigger is used waywardly, is used for disease prevention more; Vaccine inoculation is the most directly effective means of control bacteriosis; There has been multiple commercialized vaccine to sell abroad; But China's aquatic products vaccine development starting is late; Up to the present only have the grass carp hemorrhage disease vaccine to obtain commercialization and produce approval, the research and development of vaccine, producing and selling reach to be widely used all needs significant period of time.
Yolk antibody (IgY) is isolatingly from the bird through antigen immune lays eggs to have specific albumen; It has advantages such as productive rate height, good stability, high specificity; Be a kind ofly be easy to produce, cheap antibody, the amount (about 200mg) of the specific IgY that contains in collected only 1 egg of 1 chicken of immunity is higher than the antibody amount in the collected whole serum of immune 1 rabbit far away.IgY has stronger acidproof, alkaline-resisting, temperature capacity.Efficient yolk antibody can play protective material disease treatment effect to aquatic animal through injection, the modes such as bait is thrown something and fed, immersion of mixing, and also can be used for fields such as immunology detection.At present; Yolk antibody has obtained paying close attention to more widely and using in livestock industry; But the research and development at marine industry also are in basic stage; At present application patent open and that authorize has anti-prawn virus disease aspect aquatic products, sick, the anti-Vibrio parahaemolyticus of anti-Cryptosporidium baileyi is sick and the yolk antibody of anti-Aeromonas hydrophila, still, at present also not about the anti-vibrio anguillarum yolk antibody and its production and application the correlative study report.
Summary of the invention
Technical problem to be solved by this invention provides provides a kind of anti-vibrio anguillarum yolk antibody and preparation method thereof, the detection method of tiring and application; This yolk antibody purity is higher; And Vibrio anguillarum there is stronger restraining effect; The obtaining cheapness, conveniently be convenient to give birth to low-costly and in high volume of high-immunity yolk simultaneously, its preparation method is simple and easy to do, on the prevention and treatment of diseases that can be used for being caused by Vibrio anguillarum and immunoreation and diagnosis on exploitation Vibrio anguillarum detection kit and the immunology.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of anti-vibrio anguillarum yolk antibody, adopting Vibrio anguillarum is the antigen immune laying hen, collects high immune egg, immune egg yolk is separated with purifying obtaining the anti-vibrio anguillarum yolk antibody.
A kind of preparation method of anti-vibrio anguillarum yolk antibody may further comprise the steps:
(1) preparation Vibrio anguillarum antigen and deactivation
The inoculation Vibrio anguillarum is in LB solid nutrient agar, and after 18h was cultivated in 28 ℃ of inversions, picking list colony inoculation was in the LB liquid nutrient medium, and at 28 ℃, under the condition of 160rpm, behind the shaking table shaking culture 10h, the Vibrio anguillarum bacterium amount of regulating in the LB liquid nutrient medium is 2 * 10
9CFU/ml, spinning obtains containing throw out and the supernatant of Vibrio anguillarum then, throw out is mixed with SPSS to obtain Vibrio anguillarum bacterium amount be 2 * 10
9The bacteria suspension of CFU/ml adds formaldehyde and makes formaldehyde volume final concentration reach 0.5%, and 37 ℃ of water bath with thermostatic control deactivation 24h then promptly obtain the Vibrio anguillarum antigen of deactivation, and it is subsequent use to put 4 ℃ of refrigerators;
(2) immune laying hen and collect egg
Laying hen is carried out 1 fundamental immunity and 2～3 booster immunizations, and described fundamental immunity is for adding the Freund's complete adjuvant of equivalent in the Vibrio anguillarum antigen that step (1) obtains, and injecting immune is carried out to laying hen in fully emulsified back; Described booster immunization is for adding the Freund's incomplete adjuvant of equivalent in the Vibrio anguillarum antigen that step (1) obtains; Injecting immune is carried out to laying hen in fully emulsified back, and back 10 days of immunity is accomplished in each immunity 15 days at interval; Begin to collect egg, preserve in 4 ℃ of refrigerators;
(3) yolk antibody separates and purifying
The immune egg that step (2) is obtained is with the alcohol wipe eggshell and separate yolk; Acetate-the sodium acetate buffer that in yolk, adds 10 times of yolk volumes then; Leave standstill 10h in 4 ℃ of refrigerators behind the mixing, then at 4 ℃, centrifugal 20min under the 10000g condition; Obtain purification liquid just; First purification liquid is saltoutd, the deposition that obtains saltouing use with the isopyknic concentration of yolk be that 7.2 PBS damping fluid is resuspended as the pH of 0.01mol/L, use interception to obtain the anti-vibrio anguillarum yolk antibody at last as the dialysis tubing dialysis desalination of 100KD.
Salting-out process described in the step (3) is: at first in first purification liquid, add whole saturation ratio and be 40% saturated (NH
4)
2SO
4, after 4 ℃ of refrigerators leave standstill 10h, 4 ℃, 10000g, centrifugal 20min obtains primary sedimentation; Then primary sedimentation is used 140g/LNa
2SO
4Resuspension, after 4 ℃ of refrigerators leave standstill 10h, 4 ℃, 10000g, centrifugal 20min obtains secondary sedimentation, and described secondary sedimentation is the deposition of saltouing and obtaining.
A kind of detection method of tiring of anti-vibrio anguillarum yolk antibody adopts indirect enzyme-linked immunosorbent assay to detect tiring of anti-vibrio anguillarum yolk antibody, specifically comprises the steps: with 10
9CFU/ml Vibrio anguillarum coated elisa plate, 4 ℃ of refrigerator hold over night, liquid in the hole of inclining next day adds the confining liquid that contains 1%BSA with the hole with after the PBST washings washing 3 times; Room temperature sealing 1h wash the anti-vibrio anguillarum yolk antibody of adding doubling dilution after 3 times, and it is 1 parallel that every gradient is established, and sets up blank and negative control simultaneously; Behind 37 ℃ of water bath with thermostatic control 2h, wash 3 times, the goat-anti chicken two that adds the HRPO mark is anti-; 37 ℃ of water bath with thermostatic control 1h add OPD colour developing liquid colour developing 15min, H after washing 5 times
2SO
4Read wavelength 492nm light absorption value with ELIASA after the termination reaction, testing sample and negative control light absorption value are judged to be the positive greater than 2.1, and maximum positive extent of dilution is an antibody titer.
The PBST washings be tween 20 00 with the PBS damping fluid by volume the mixed of 1:2000 obtain.
A kind of application of anti-vibrio anguillarum yolk antibody on the prevention and treatment of diseases that this Vibrio anguillarum yolk antibody can be used for being caused by Vibrio anguillarum, and can be used for developing immunoreation and the diagnosis on Vibrio anguillarum detection kit and the immunology.
Compared with prior art; The invention has the advantages that: the invention discloses a kind of anti-vibrio anguillarum yolk antibody and preparation method thereof, the detection method of tiring and application, the yolk antibody purity that this method prepares is higher, and Vibrio anguillarum is had stronger restraining effect; Simultaneously high-immunity yolk obtains cheapness, convenience; Be convenient to give birth to low-costly and in high volume, its preparation method is simple and easy to do, reaches immunoreation and the diagnosis on exploitation Vibrio anguillarum detection kit and the immunology on the prevention and treatment of diseases that the final yolk antibody highly efficient and productive, higher degree that obtains can be used for being caused by Vibrio anguillarum; This detection method detection efficiency of tiring is high, and operating process is simple and easy to do.The present invention has proposed anti-vibrio anguillarum yolk antibody and preparation method thereof first, the detection method of tiring and application, has filled up the blank of anti-vibrio anguillarum yolk antibody correlative study, and lays the foundation for later research and application.
Description of drawings
Fig. 1 is that anti-vibrio anguillarum yolk antibody purification effect detects figure, swimming lane 1: homemade albumen Marker, swimming lane 2: the anti-vibrio anguillarum yolk antibody IgY of purifying;
Fig. 2 is the external fungistatic effect figure of anti-vibrio anguillarum yolk antibody, and a left side is yolk antibody and bacterium liquid mixture growing way, and the right side is common yolk liquid and bacterium liquid mixture growing way.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
One, specific embodiment
A kind of anti-vibrio anguillarum yolk antibody of the present invention; Through preparation Vibrio anguillarum inactivation antigen immune egg, collect high immune egg and separate and purifying, and carry out determination of protein concentration and SDS-PAGE detection protein band judgement purity; Indirect ELISA detects antibody titer; External bacteriostatic experiment is confirmed effect, finally obtains yolk antibody highly efficient and productive, higher degree, and its preparation process is specific as follows:
(1) preparation Vibrio anguillarum antigen and deactivation
The inoculation Vibrio anguillarum is in LB solid nutrient agar, and after 18h was cultivated in 28 ℃ of inversions, picking list colony inoculation was in the LB liquid nutrient medium, and at 28 ℃, under the condition of 160rpm, behind the shaking table shaking culture 10h, the Vibrio anguillarum bacterium amount of regulating in the LB liquid nutrient medium is 2 * 10
9CFU/ml, spinning obtains containing throw out and the supernatant of Vibrio anguillarum then, throw out is mixed with SPSS to obtain Vibrio anguillarum bacterium amount be 2 * 10
9The bacteria suspension of CFU/ml adds formaldehyde and makes formaldehyde volume final concentration reach 0.5%, and 37 ℃ of water bath with thermostatic control deactivation 24h then promptly obtain the Vibrio anguillarum antigen of deactivation, and it is subsequent use to put 4 ℃ of refrigerators;
(2) immune laying hen and collect egg
Laying hen is carried out 1 fundamental immunity and 2～3 booster immunizations, and described fundamental immunity is for adding the Freund's complete adjuvant of equivalent in the Vibrio anguillarum antigen that step (1) obtains, and injecting immune is carried out to laying hen in fully emulsified back; Described booster immunization is for adding the Freund's incomplete adjuvant of equivalent in the Vibrio anguillarum antigen that step (1) obtains; Injecting immune is carried out to laying hen in fully emulsified back, and back 10 days of immunity is accomplished in each immunity 15 days at interval; Begin to collect egg, preserve in 4 ℃ of refrigerators;
(3) yolk antibody separates and purifying
Get height and exempt from egg,, remove egg white with 75% alcohol wipe eggshell; Separate yolk; Draw acetate-sodium acetate buffer of yolk liquid 10ml and the pH 5.0 of 1:10 adding by volume, fully leave standstill 10h in 4 ℃ of refrigerators, 4 ℃ behind the mixing; Centrifugal 20min under the 10000g condition, obtaining just, purification liquid carries out saltouing for twice.At first, in first purification liquid, add saturated (NH
4)
2SO
4Making its whole saturation ratio is that 40%, 4 ℃ of refrigerator leaves standstill 10h, takes out back 4 ℃, 10000g, and centrifugal 20min, deposition is used 100ml140g/LNa then
2SO
4Resuspension; 4 ℃ of refrigerators leave standstill the centrifugal 20min of 10000g behind the 10h, precipitate resuspended with 10mlPBS (0.01mol/L, pH 7.2); Obtain the anti-vibrio anguillarum yolk antibody with interception 100KD dialysis tubing dialysis desalination at last; Dialyzate is 0.01mol/L, the PBS of pH 7.2, during change dialyzate 3-5 time.
Two, the evaluation of anti-vibrio anguillarum yolk antibody
(1) concentration determination and purity detecting
Dialysis back protein liquid amounts to 14ml, dilute 10 times after, light absorption value is respectively 2.684 and 2.148 under mensuration 260nm and the 280nm wavelength, according to formula (1.45 * OD
280-0.74 * OD
260It is 11.284mg/ml that) * extension rate calculates protein concentration, and the about 160mg of total protein concentration boils 10min after getting protein liquid 15 μ l and equivalent 2 * loading Buffer mixing in normal range; Carry out 12% reductibility SDS-PAGE electrophoresis; Result such as Fig. 1, heavy chain, light chain lay respectively at 66KD, 26KD place band is brighter, the about 180KD of total molecular weight; Assorted band less, a little less than, purity is higher.
(2) indirect enzyme-linked immunosorbent assay (ELISA) detects and tires
With 10
9CFU/ml Vibrio anguillarum coated elisa plate, every hole 100 μ l, 4 ℃ of refrigerator hold over night, liquid in the hole of inclining next day; After the hole removed the antigen impurity on not coated for 3 times with PBST (PBST is a tween 20 00 and the PBS damping fluid mixed of 1:2000 by volume) washings washing, add the confining liquid 100 μ l that contain 1%BSA, room temperature sealing 1h adds by 1:100 after washing 3 times; 1:200,1:400,1:800,1:1600; 1:3200,1:6400,1:12800, the IgY100 μ l of 1:25600 dilution; It is 1 parallel that every gradient is established, and sets up blank and negative control simultaneously, and 37 ℃ of water bath with thermostatic control 2h after scouring 3 times add the goat-anti chicken two anti-100 μ l of the HRPO mark of 1:10000 dilution; 37 ℃ of temperature are bathed 1h, add the OPD colour developing liquid 15min that develops the color after wash 5 times, with the H of 50 μ l, 2M
2SO
4Termination reaction, ELIASA read wavelength 492nm light absorption value, and testing sample and negative sample light absorption value are judged to be the positive greater than 2.1, and maximum positive extent of dilution is an antibody titer.The anti-vibrio anguillarum yolk antibody of this preparation is tired and is 1:12800.
(3) external bacteriostatic experiment
The Vibrio anguillarum of pure culture is diluted to 10
2CFU/ml gets 150 μ l anti-vibrio anguillarum yolk antibodies and common vitellin(Vt) liquid respectively, and 28 ℃ of incubators of adding equivalent bacterium liquid mixing postposition combine 15min, get 100 μ l coating LB agar plate, respectively establish 1 and repeat, the counting of taking pictures behind 28 ℃ of inversion cultivation 24h.Bacteriostatic experiment shows: add one group of growth that can obviously suppress Vibrio anguillarum of yolk antibody, its bacterium amount is the 1/3-1/4 that adds common this group of vitellin(Vt) liquid.
Three, the application of anti-vibrio anguillarum yolk antibody
Select 30 of healthy and strong Procambius clarkiis and be divided into 3 groups at random, 10 every group, inject every 0.15ml in crayfish the 3rd uromere by following the grouping.Group 1:2 * 10
8The CFU/ml Vibrio anguillarum mixes with 0.9% SPSS 1:1; Group 2:2 * 10
8The CFU/ml Vibrio anguillarum mixes with common vitellin(Vt) liquid 1:1; Group 3:2 * 10
8The CFU/ml Vibrio anguillarum mixes with anti-vibrio anguillarum yolk antibody 1:1.The conventional raising observed death condition.The result shows: injects group 1 and all death of group 2 crayfish in back 2 days, and organizes 3 in the 3rd talent's beginning death, and survival 6 tails in 7 days, protection ratio is 66.7%.Show anti-vibrio anguillarum yolk antibody, the crayfish that attacked by the high density Vibrio anguillarum is had the significant protection effect and postpones dead effect, can apply to the disease that aquatic animal prevention and treatment are caused by Vibrio anguillarum by the present invention's preparation.
Claims (6)
1. anti-vibrio anguillarum yolk antibody is characterized in that: adopting Vibrio anguillarum is the antigen immune laying hen, collects high immune egg, immune egg yolk is separated with purifying obtaining the anti-vibrio anguillarum yolk antibody.
2. the preparation method of an anti-vibrio anguillarum yolk antibody according to claim 1 is characterized in that may further comprise the steps:
(1) preparation Vibrio anguillarum antigen and deactivation
The inoculation Vibrio anguillarum is in LB solid nutrient agar, and after 18h was cultivated in 28 ℃ of inversions, picking list colony inoculation was in the LB liquid nutrient medium, and at 28 ℃, under the condition of 160rpm, behind the shaking table shaking culture 10h, the Vibrio anguillarum bacterium amount of regulating in the LB liquid nutrient medium is 2 * 10
9CFU/ml, spinning obtains containing throw out and the supernatant of Vibrio anguillarum then, throw out is mixed with SPSS to obtain Vibrio anguillarum bacterium amount be 2 * 10
9The bacteria suspension of CFU/ml adds formaldehyde and makes formaldehyde volume final concentration reach 0.5%, and 37 ℃ of water bath with thermostatic control deactivation 24h then promptly obtain the Vibrio anguillarum antigen of deactivation, and it is subsequent use to put 4 ℃ of refrigerators;
(2) immune laying hen and collect egg
Laying hen is carried out 1 fundamental immunity and 2～3 booster immunizations, and described fundamental immunity is for adding the Freund's complete adjuvant of equivalent in the Vibrio anguillarum antigen that step (1) obtains, and injecting immune is carried out to laying hen in fully emulsified back; Described booster immunization is for adding the Freund's incomplete adjuvant of equivalent in the Vibrio anguillarum antigen that step (1) obtains; Injecting immune is carried out to laying hen in fully emulsified back, and back 10 days of immunity is accomplished in each immunity 15 days at interval; Begin to collect egg, preserve in 4 ℃ of refrigerators;
(3) yolk antibody separates and purifying
The immune egg that step (2) is obtained is with the alcohol wipe eggshell and separate yolk; Acetate-the sodium acetate buffer that in yolk, adds 10 times of yolk volumes then; Leave standstill 10h in 4 ℃ of refrigerators behind the mixing, then at 4 ℃, centrifugal 20min under the 10000g condition; Obtain purification liquid just; First purification liquid is saltoutd, the deposition that obtains saltouing use with the isopyknic concentration of yolk be that 7.2 PBS damping fluid is resuspended as the pH of 0.01mol/L, use interception to obtain the anti-vibrio anguillarum yolk antibody at last as the dialysis tubing dialysis desalination of 100KD.
3. the preparation method of anti-vibrio anguillarum yolk antibody according to claim 1 is characterized in that the salting-out process described in the step (3) is: at first in first purification liquid, add whole saturation ratio and be 40% saturated (NH
4)
2SO
4, after 4 ℃ of refrigerators leave standstill 10h, 4 ℃, 10000g, centrifugal 20min obtains primary sedimentation; Then primary sedimentation is used 140g/LNa
2SO
4Resuspension, after 4 ℃ of refrigerators leave standstill 10h, 4 ℃, 10000g, centrifugal 20min obtains secondary sedimentation, and described secondary sedimentation is the deposition of saltouing and obtaining.
4. the detection method of tiring of an anti-vibrio anguillarum yolk antibody according to claim 1 is characterized in that adopting indirect enzyme-linked immunosorbent assay to detect tiring of anti-vibrio anguillarum yolk antibody, specifically comprises the steps: with 10
9CFU/ml Vibrio anguillarum coated elisa plate, 4 ℃ of refrigerator hold over night, liquid in the hole of inclining next day adds the confining liquid that contains 1%BSA with the hole with after the PBST washings washing 3 times; Room temperature sealing 1h wash the anti-vibrio anguillarum yolk antibody of adding doubling dilution after 3 times, and it is 1 parallel that every gradient is established, and sets up blank and negative control simultaneously; Behind 37 ℃ of water bath with thermostatic control 2h, wash 3 times, the goat-anti chicken two that adds the HRPO mark is anti-; 37 ℃ of water bath with thermostatic control 1h add OPD colour developing liquid colour developing 15min, H after washing 5 times
2SO
4Read wavelength 492nm light absorption value with ELIASA after the termination reaction, testing sample and negative control light absorption value are judged to be the positive greater than 2.1, and maximum positive extent of dilution is an antibody titer.
5. the detection method of tiring of anti-vibrio anguillarum yolk antibody according to claim 1 is characterized in that: the PBST washings be tween 20 00 with the PBS damping fluid by volume the mixed of 1:2000 obtain.
6. the application of an anti-vibrio anguillarum yolk antibody according to claim 1; It is characterized in that: on the prevention and treatment of diseases that this Vibrio anguillarum yolk antibody can be used for being caused by Vibrio anguillarum, and can be used for developing immunoreation and the diagnosis on Vibrio anguillarum detection kit and the immunology.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628921A (en) * | 2014-11-07 | 2016-06-01 | 华东理工大学 | Preparation and applications of rapid Vibrio anguillarum detection system |
| CN105759036A (en) * | 2016-04-11 | 2016-07-13 | 新疆农业大学 | Detection method and detection kit used for detecting immune antibody of micro-encapsulation escherichia coli oral vaccine |
| CN105891412A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Test strip for detecting citrobacter freundii and preparation method thereof |
| CN105884890A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Citrobacter freundii resisting yolk antibody, preparation method therefor and application of Citrobacter freundii resisting yolk antibody |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
-
2012
- 2012-01-20 CN CN201210019152.7A patent/CN102532312B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
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| 朱香萍等: "对虾鳗弧菌卵黄抗体（IgY）的制备及其对人工感染的保护研究", 《海洋科学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628921A (en) * | 2014-11-07 | 2016-06-01 | 华东理工大学 | Preparation and applications of rapid Vibrio anguillarum detection system |
| CN105891412A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Test strip for detecting citrobacter freundii and preparation method thereof |
| CN105884890A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Citrobacter freundii resisting yolk antibody, preparation method therefor and application of Citrobacter freundii resisting yolk antibody |
| CN105891412B (en) * | 2014-11-14 | 2018-08-21 | 宁波大学 | A kind of citrobacter freundii test strip and preparation method thereof |
| CN105759036A (en) * | 2016-04-11 | 2016-07-13 | 新疆农业大学 | Detection method and detection kit used for detecting immune antibody of micro-encapsulation escherichia coli oral vaccine |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
| CN108003240B (en) * | 2017-12-04 | 2020-06-05 | 西安德轩驰生物科技有限公司 | Multi-connected anti-idiotype egg yolk antibody vaccine for marine cultured fish and preparation method thereof |
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| CN102532312B (en) | 2014-03-12 |
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