CN113376381A - Breeding method of high-antibody-level parent strain for nervous necrosis of grouper - Google Patents
Breeding method of high-antibody-level parent strain for nervous necrosis of grouper Download PDFInfo
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Abstract
The invention relates to a method for breeding a parent with high antibody level for nervous necrosis of grouper, belonging to the technical field of disease-resistant breeding of grouper. The antibody level determines the strength of disease resistance, and grouper with high natural antibody level is selected as a parent for breeding disease-resistant varieties. The method can quickly and simply select the disease-resistant individuals, simplify the breeding process of the disease-resistant varieties and save the breeding cost.
Description
The technical field is as follows:
the invention belongs to the technical field of disease-resistant breeding of groupers, and particularly relates to a breeding method of groupers nervous necrosis disease high-antibody level parents.
Background art:
the grouper is a rare fish in China coastal, with the breakthrough of grouper fry breeding technology, the breeding industry develops rapidly, the breeding scale is enlarged continuously, the disease problem in the breeding process is increasingly obvious, particularly in the last two years, the disease toxic nervous necrosis disease caused by Nervous Necrosis Virus (NNV) infection in the breeding process is very harmful to larval fish and juvenile fish, the death rate of severe people in one week can reach 100%, adult fish is infected continuously, clinical symptoms can not be displayed under some conditions, and the virus vertical transmission is carried out through gonads, so that the control difficulty of the disease is increased.
The realization of the anti-nervous necrosis disease breeding of the grouper is a problem which needs to be solved urgently by the industry, no good strategy is available in the industry at present, and the disease-resistant breeding of the grouper has more important significance for solving the technical problem.
The invention content is as follows:
the invention aims to solve the technical problem of providing a method for breeding a grouper nervous necrosis disease high-antibody-level parent, wherein the method detects the natural antibody level in the grouper body by an ELISA method, the antibody level determines the strength of disease resistance, and the grouper with high natural antibody level is selected as the parent for breeding disease-resistant varieties.
The method provided by the invention is used for analyzing the level of the anti-NNV virus antibody in the sample by constructing a recombinant NNV capsid protein-target-HRP enzyme-labeled rabbit anti-grouper secondary antibody indirect method detection system and utilizing the change of the substrate chromogenic detection light absorption value. The principle is as follows: coating the recombinant NNV capsid protein on a 96-pore plate, adding the serum of the grouper to be detected, capturing an anti-NNV antibody in a sample by using the fixed recombinant capsid protein, and combining the anti-NNV antibody of the grouper by using a rabbit anti-grouper secondary antibody marked by HRP. And (4) developing with a horseradish peroxidase developing kit, and analyzing the level of the anti-NNV virus antibody according to the change of a light absorption value.
The specific technical scheme of the invention is as follows:
a method for breeding a parent with high antibody level for the nervous necrosis disease of grouper comprises the steps of recombining NNV capsid protein, establishing an indirect ELISA method based on the detection of the recombined NNV capsid protein and a rabbit anti-grouper secondary antibody marked by HRP, carrying out antibody detection in an asymptomatic breeding population of natural grouper by using the method, and selecting the grouper with high antibody level to form a disease-resistant variety breeding parent;
the method of the recombinant NNV capsid protein is as follows: NNV capsid protein sequence and GenBank Accession: the sequences in ATJ33902 are consistent, the expression vector is pGEX-6P-1, the competent cell is Escherichia coli BL21(DE3), the induction condition is that IPTG is added when OD reaches 0.6 under the conditions of 37 ℃ and 250rpm until the final concentration of IPTG is 0.5mM, the temperature is reduced to 25 ℃, and the induction expression is carried out for 6 hours; the recombinantly expressed GST fusion protein sequence is: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSPMVRKGEKKLAKPATTKAANPQPRRRANNRRRSNRTDAPVSKASTVTGFGRGTNDVHLAGMSRISQAVLPAGTGTDGYVVVDATIVPDLLPRLGHAARIFQRYAVETLEFEIQPMCPANTGGGYVAGFLPDPTDNDHTFDALQATRGAVVAKWWESRTVRPQYTRTLLWTSSGKEQRLTSPGRLILLCVGNNTDVVNVSVLCRWSVRLSVPSLETPEETTAPIMTQGSLYNDSLSTNDFKSILLGSTPLDIAPDGAVFQLDRPLSIDYSLGTGDVDRAVYWHLKKFAGNAGTPAGWFRWGIWDNFNKTFTDGVAYYSDEQPRQILLPVGTVCTRVDSEN
The indirect ELISA method (1) comprises the following steps: embedding the recombinant protein into a 96-well plate by using an embedding solution, and cleaning;
(2) and (3) sealing: adding sealing liquid for sealing and cleaning;
(3) capture of anti-NNV antibody: adding the grouper sample into a pore plate, incubating and combining to ensure that the recombinant protein fully captures the anti-NNV antibody in the sample to be detected, and cleaning;
(4) and (3) detection: adding a rabbit anti-grouper secondary antibody labeled with horseradish peroxidase (HRP) into the pore plate, and cleaning;
(5) color development of the TMB color development kit: adding a TMB substrate developing solution into each hole, reacting at room temperature in a dark place, adding a stop solution to stop the reaction, carrying out colorimetric observation, measuring the absorbance value at 450nm by using an enzyme-linked immunosorbent assay (ELISA), and analyzing the anti-NNV antibody level according to the absorbance value.
Furthermore, the washing is carried out by adopting TBST.
Further, 0.1. mu.g of recombinant capsid protein of the nervous necrosis virus of grouper was added in said step (1) and embedded overnight at 4 ℃.
Further, the confining liquid added in the step (2) is skimmed milk powder with the mass fraction of 5%, and the culture box is incubated for 2 hours at 37 ℃.
Further, the sample in the step (3) is a serum sample, the dilution factor is 400 times, and the sample is incubated for 1 hour in an incubator at 37 ℃.
Further, in the step (4), 100. mu.l of a secondary antibody was added at 1.6. mu.g/ml, and the mixture was incubated at 37 ℃ for 45 minutes in an incubator. The preparation method of the secondary antibody comprises purifying antibody in the serum of Epinephelus coioides with ProteinA affinity chromatography column to immunize rabbit, collecting serum after four times of immunization, and purifying antibody with ProteinG affinity chromatography column. The HRP labeling method is to label with a Glue activated horseradish peroxidase (HRP) type A kit according to the instruction method.
Compared with the prior art, the invention has the beneficial effects that:
the recombinant NNV capsid proteins of the invention were immobilized in 96-well plates while the rabbit anti-grouper antibodies were linked to horseradish peroxidase. The invention uses a recombinant NNV capsid protein-target-HRP enzyme-labeled rabbit anti-grouper secondary antibody indirect detection system, and the recombinant NNV capsid protein and the HRP enzyme-labeled rabbit anti-grouper secondary antibody are respectively used for capturing a target and detecting the target.
Aiming at the problems of common asymptomatic NNV infection phenomenon, difficult disease-resistant breeding work and the like in the grouper breeding industry, the invention establishes an indirect ELISA detection method based on the detection of recombinant NNV capsid protein and HRP-labeled rabbit anti-grouper secondary antibody by utilizing the characteristics of high affinity and high specificity of antigen antibody, can be used for detecting anti-NNV antibody in grouper breeding, guides the preferential selection of parent fish with high antibody level for breeding in production, and provides technical support for the disease-resistant breeding work of grouper.
The traditional disease-resistant breeding method mainly comprises the steps of detecting NNV antigen and selecting healthy fish without virus infection for breeding, but the detection of the antigen alone cannot effectively breed parent fish because of the universal existence of asymptomatic infection of NNV, and the antigen mainly infects central nervous tissue and is not easy to carry out in vivo detection. The recombinant NNV capsid protein recombinant expression system can efficiently express in vitro in a soluble way, is easy to prepare and has low cost; the recombinant NNV capsid protein has target specificity, the rabbit anti-grouper secondary antibody can be specifically combined with the grouper antibody specifically combined with the recombinant capsid protein, the detection has high sensitivity and specificity, the operation is simple and convenient, the number of detection samples is large, high-throughput detection can be carried out, and the time consumption is short; the detection method can detect the obvious difference of the anti-nervous necrosis virus antibody level in the asymptomatic grouper culture population of the grouper, the detected OD450 value of a sample is at least 3 times that of individuals of a control group, which accounts for about 20 percent of the population, and in the aspects of quick detection and disease-resistant breeding of the anti-NNV antibody of the grouper, the disease-resistant individuals can be quickly and simply selected, the breeding process of disease-resistant varieties is simplified, and the breeding cost is saved.
Description of the drawings:
FIG. 1 is a SDS-PAGE electrophoretic test of prokaryotic expression and purification of NNV recombinant CP protein in an embodiment of the invention; m: marker, 1. intracellular soluble component of bacteria before induction, 2: intracellular soluble fraction of bacteria after induction, 3: a purified recombinant NNV capsid protein;
FIG. 2 shows the result of SDS-PAGE electrophoresis of the antibody in the serum of grouper; m: marker, 1: purified Epinephelus coioides antibody;
FIG. 3 shows the result of SDS-PAGE electrophoresis of rabbit anti-grouper antibody; m: marker, 1: a purified rabbit anti-grouper secondary antibody;
FIG. 4 is a graph showing the results of measuring serum anti-NNV antibody levels in cultured populations of Epinephelus coioides from 2 cultures in Hainan province using an indirect ELISA constructed in accordance with the present invention.
FIG. 5 is a graph showing the results of measuring the levels of serum anti-NNV antibodies in a cultured population of Epinephelus contorti, a certain culture company, Shandong, using an indirect ELISA constructed in the examples of the present invention.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: preparation of grouper nervous necrosis virus recombinant capsid protein
Step 1: the primer sequence of the recombinant protein coding region clone is as follows: f: gaattcATGGTACGCAAAGGTGAGAAGAAAT, respectively; r: ctcgagGTTTTCCGAGTCAACCCTGGTG, restriction enzyme sites of EcoRI and XhoI, expression vector pGEX-6P-1, and recombinant expression strain BL21(DE 3).
Step 2: positive clones were inoculated into LB medium containing 50. mu.g/mL kanamycin sulfate and grown overnight at 37 ℃ 200rpm on a shaker. The next day, the above-mentioned bacterial solution was inoculated into LB medium containing 50. mu.g/mL kanamycin sulfate resistance at a ratio of 1:100, placed in a shaker at 37 ℃ and 200rpm until OD600 became 0.6-0.8, and then added with 0.5mM IPTG at the final concentration, and induced to grow for 6 hours at 25 ℃ and 200rpm in the shaker. The induced bacterial solution was centrifuged at 4 ℃ 10000rpm for 10min to discard the supernatant, the cells were collected, resuspended in 1/10 culture volume of phosphate buffered saline PBS (pH8.0), and the cell walls were disrupted by ultrasonication.
And step 3: centrifuging the sonicated solution at 4 deg.C and 10000rpm for 10min, collecting supernatant, purifying recombinant protein with GST affinity chromatography column, dialyzing with 3000Da dialysis bag in 10mM PBS (pH8.0) in ice bath overnight, and collecting dialyzed protein, and storing at-20 deg.C. The purity of the recombinant protein was checked by SDS-PAGE electrophoresis (FIG. 1), and the protein concentration was checked by BCA. The protein concentration is 0.1mg/ml, and about 150mg of recombinant protein can be obtained per liter of LB culture solution.
Example 2: preparation of garrupa serum antibody
Diluting 40ml of brown spot grouper serum with 0.02M PB, filtering with a 0.22 mu M filter membrane, loading on a ProteinA column, eluting with 0.1M PH3.0 glycine, adjusting the pH of an elution product to be neutral by saturated sodium carbonate, carrying out ultrafiltration concentration to about 5ml by a 10kDa ultrafiltration tube, dialyzing 5L of 0.01M PH 7.4PBS overnight, changing the solution for 1 time the next day, and collecting the antibody after dialysis, and storing at-20 ℃. The antibody was checked for purity by SDS-PAGE electrophoresis (FIG. 2) and protein concentration by BCA. The total amount of the obtained antibody is 3mg, the concentration of the antibody is 0.3mg/ml, and the purity is about 80%.
Example 3: preparation of rabbit anti-grouper secondary antibody and HRP labeling
8ml antiserum, after diluting with 0.02M PB, 0.22 μ M filter membrane filtration and column, 0.1MPH3.0 glycine elution, saturated sodium carbonate to adjust the pH of the elution product to neutral, 10kDa ultrafiltration tube, ultrafiltration concentration to about 1-3ml, 5L 0.01M pH 7.4PBS overnight, next day fluid change 1 time, collection after dialysis antibody-20 ℃ storage. The antibody was checked for purity by SDS-PAGE electrophoresis (FIG. 3) and protein concentration by BCA. The total amount of the obtained antibody is 20mg, the concentration of the antibody is 10mg/ml, and the purity is more than 90%.
The antibody was diluted to 1mg/ml with 0.01M PH 7.4PBS, secondary antibodies were HRP-labeled with Glue-activated horseradish peroxidase (HRP) type a kit according to the instructions, and the labeled antibody was stored at-20 ℃. The antibody titers before and after labeling were measured by ELISA. The immunogens measured by rabbit antiserum are all over 1:50K, rabbit polyclonal antibody detects garrupa antibody, the OD450 detection value is 1.0153 when the rabbit polyclonal antibody is diluted by 78ng/ml, the OD450 detection value is 1.225 when the rabbit polyclonal antibody is marked by HRP when the rabbit polyclonal antibody is diluted by 625 ng/ml.
Example 4: construction of Indirect ELISA for detection of anti-NNV antibody levels
Antigen coating: antigen was diluted to 1. mu.g/ml with coating solution (50mM sodium carbonate/sodium bicarbonate, pH 9.6), 100. mu.L/well was added to a 96-well reaction plate, and left overnight at 4 ℃.
And (3) sealing: discarding the coating liquid on the next day, washing with 200 μ L/hole of washing solution for 1 time, throwing off and completely drying the liquid in the plate, sealing with 200 μ L/hole of sealing solution in a constant temperature incubator at 37 ℃ for 2h, taking out the ELISA plate, throwing off the liquid, washing the plate with the washing solution for 3-4 times, and completely drying the liquid in the plate. The confining liquid is skimmed milk powder with the mass fraction of 5%; and the washing liquid is washed by TBST.
Antibody incubation: diluting the grouper serum by different times (80 times, 400 times and 2000 times), adding 100 μ l into 96-well plate, incubating at 37 deg.C for 1h, and washing the plate with washing solution for 3-4 times; adding HRP-labeled rabbit anti-grouper secondary antibody 100 microliters with different dilution times (0.8 mug/ml, 1.6 mug/ml, 3.2 mug/ml), incubating for 45min at 37 ℃, and washing the plate for 3-4 times by using a washing solution; at this time, a solid-phase antigen-test antibody-enzyme-labeled secondary antibody complex is formed on the solid phase.
Color development: immediately after washing, a single-component color reagent TMB (Trimethoprim corporation) was added thereto in an amount of 90. mu.l per well, covered with a film, and incubated in a 37 ℃ incubator for 20 minutes.
And (4) terminating: after the development was complete, 50. mu.l of stop solution (Solebao) was added to each well in turn, and the plate reader was used to read the plate as soon as possible (wavelength 450 nm).
The result shows that when the serum of the grouper is diluted by 400 times, the detection result is better when the enzyme-labeled secondary antibody is 1.6 mu g/ml.
Example 5: detecting the serum antibody level in grouper culture populations of 2 culture companies in Hainan province
About 0.5ml of serum of the epinephelus fuscoguttatus in prophase of asymptomatic cultured population of the epinephelus fuscoguttatus of 2 culturing companies in Hainan province is respectively collected at 22-25 days 4 months in 2021, 71 tails and 88 tails are respectively sampled and marked with electrons, and the average weights are 2510.3 +/-541.6 g and 3674.7 +/-369.5 g respectively. The constructed indirect ELISA was used to measure anti-NNV antibody levels in the samples. The result shows that the serum antibody level in the grouper culture population has larger individual difference, 28 percent of individual sample detection values OD450 in the culture population 1 reach more than 0.6 and are at least 3 times of the detection value of a control group, 20 percent of individual sample detection values OD450 in the culture population 2 reach more than 0.6 and are at least 3 times of the detection value of the control group, and the parent fish 38 tails with high detection values are preferentially selected for breeding disease-resistant excellent varieties (figure 4). The method can well detect parent fish with high antibody level, and provides technical support for disease-resistant breeding of grouper in the future.
Example 6: detecting the serum antibody level in a grouper culture group of a certain culture company in Shandong province
Collecting about 0.5ml of serum of the epinephelus malabaricus in prophase of asymptomatic culture population of the epinephelus malabaricus of a certain culture company in Shandong province on 14 days 5 and 14 months in 2021, sampling 390 tails and marking with an electronic mark, wherein the average weight is 5846.9 +/-1675.6 g. The constructed indirect ELISA was used to measure anti-NNV antibody levels in the samples. The results show that the serum antibody level in the grouper culture population has larger individual difference, 20 percent of individual sample detection values OD450 in the culture population reach more than 0.6, and 100 tails of parent fishes with higher detection values are selected and reserved for breeding disease-resistant fine varieties (figure 5). The method provided by the invention can be used for detecting not only Epinephelus coioides, but also parent fishes with high Epinephelus coioides antibody level, and provides technical support for the disease-resistant breeding work of Epinephelus coioides in the future.
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> method for breeding grouper nervous necrosis disease high-antibody level parent strain
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Trp Glu Ser Arg Thr Val Arg Pro Gln Tyr Thr Arg Thr Leu Leu Trp
385 390 395 400
Thr Ser Ser Gly Lys Glu Gln Arg Leu Thr Ser Pro Gly Arg Leu Ile
405 410 415
Leu Leu Cys Val Gly Asn Asn Thr Asp Val Val Asn Val Ser Val Leu
420 425 430
Cys Arg Trp Ser Val Arg Leu Ser Val Pro Ser Leu Glu Thr Pro Glu
435 440 445
Glu Thr Thr Ala Pro Ile Met Thr Gln Gly Ser Leu Tyr Asn Asp Ser
450 455 460
Leu Ser Thr Asn Asp Phe Lys Ser Ile Leu Leu Gly Ser Thr Pro Leu
465 470 475 480
Asp Ile Ala Pro Asp Gly Ala Val Phe Gln Leu Asp Arg Pro Leu Ser
485 490 495
Ile Asp Tyr Ser Leu Gly Thr Gly Asp Val Asp Arg Ala Val Tyr Trp
500 505 510
His Leu Lys Lys Phe Ala Gly Asn Ala Gly Thr Pro Ala Gly Trp Phe
515 520 525
Arg Trp Gly Ile Trp Asp Asn Phe Asn Lys Thr Phe Thr Asp Gly Val
530 535 540
Ala Tyr Tyr Ser Asp Glu Gln Pro Arg Gln Ile Leu Leu Pro Val Gly
545 550 555 560
Thr Val Cys Thr Arg Val Asp Ser Glu Asn
565 570
<210> 2
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaattcatgg tacgcaaagg tgagaagaaa t 31
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctcgaggttt tccgagtcaa ccctggtg 28
Claims (6)
1. A method for breeding a parent with high antibody level for the nervous necrosis disease of grouper is characterized by comprising the steps of recombining NNV capsid protein, establishing an indirect ELISA method based on the recombined NNV capsid protein and HRP marked rabbit anti-grouper secondary antibody detection, carrying out antibody detection in an asymptomatic breeding population of natural grouper by using the method, and selecting the grouper with high antibody level to form a disease-resistant variety breeding parent;
the method of the recombinant NNV capsid protein is as follows: NNV capsid protein sequence and GenBank Accession: the sequences in ATJ33902 are consistent, the expression vector is pGEX-6P-1, the competent cell is Escherichia coli BL21(DE3), the induction condition is that IPTG is added when OD reaches 0.6 under the conditions of 37 ℃ and 250rpm until the final concentration of IPTG is 0.5mM, the temperature is reduced to 25 ℃, and the induction expression is carried out for 6 hours; the sequence of the recombinant expressed GST fusion protein is shown as SEQ ID NO. 1;
the indirect ELISA method comprises the following specific steps:
(1) embedding: embedding the recombinant protein into a 96-well plate by using an embedding solution, and cleaning;
(2) and (3) sealing: adding sealing liquid for sealing and cleaning;
(3) capture of anti-NNV antibody: adding the grouper sample into a pore plate, incubating and combining to ensure that the recombinant protein fully captures the anti-NNV antibody in the sample to be detected, and cleaning;
(4) and (3) detection: adding a rabbit anti-grouper secondary antibody with horseradish peroxidase markers into the pore plate, and cleaning;
(5) color development of the TMB color development kit: adding a TMB substrate developing solution into each hole, reacting at room temperature in a dark place, adding a stop solution to stop the reaction, carrying out colorimetric observation, measuring the absorbance value at 450nm by using an enzyme-linked immunosorbent assay (ELISA), and analyzing the anti-NNV antibody level according to the absorbance value.
2. The method of claim 1, wherein the washing is performed using TBST.
3. The method according to claim 1, wherein 0.1 μ g of recombinant capsid protein of the nervous necrosis virus of Epinephelus is added in step (1) and embedded overnight at 4 ℃.
4. The method according to claim 1, wherein the confining liquid added in step (2) is skimmed milk powder with a mass fraction of 5%.
5. The method according to claim 1, wherein the grouper sample in step (3) is a serum sample, diluted 400-fold, and incubated in an incubator at 37 ℃ for 1 hour.
6. The method according to claim 3, wherein the secondary antibody is added in step (4) at a concentration of 1.6. mu.g/ml of 100. mu.l, and the mixture is incubated at 37 ℃ in an incubator for 45 minutes.
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| CN105785023A (en) * | 2016-04-22 | 2016-07-20 | 中国科学院南海海洋研究所 | Aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers |
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| CN107389921A (en) * | 2017-07-18 | 2017-11-24 | 福建省水产研究所 | A kind of preparation method and application of rabbit-anti grouper serum immune globulin HRP labelled antibodies |
| CN111363758A (en) * | 2020-03-26 | 2020-07-03 | 华南农业大学 | Preparation and application of polyclonal antibody of major capsid protein of epinephelus neocalifornicus iridovirus |
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2021
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| CN105785023A (en) * | 2016-04-22 | 2016-07-20 | 中国科学院南海海洋研究所 | Aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers |
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