CN115474564A - Method for breeding epinephelus coioides - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The invention aims to solve the problem of high mortality rate after infecting cryptocaryon irritans in the grouper culture process, and injects a recombinant protein vaccine with good immune protection effect to the grouper culture process, thereby greatly improving the disease-resistant survival rate of the grouper. The invention also aims to solve the technical problems of high preparation cost and poor immune protection effect of the cryptocaryon irritans vaccine, and provides a preparation method of a subunit vaccine based on cryptocaryon irritans membrane protein CiIA1, which is used for screening and identifying the CiIA1 membrane protein of the cryptocaryon irritans for the first time, carrying out optimized recombinant expression on the CiIA1 membrane protein, and preparing the vaccine by using the CiIA1 membrane protein as an antigen, thereby greatly improving the capacity of the grouper for resisting cryptocaryon irritans infection.
Description
Technical Field
The invention belongs to the field of aquaculture, and particularly relates to a method for culturing epinephelus coioides.
Background
Epinephelus coioides is commonly called as Qingban and belongs to the genus of Epinephelus, is an important and valuable marine fish cultured in southern areas of China, but parasitic diseases of the Epinephelus coioides are more and more frequently outbreak along with continuous expansion of culture scale, deterioration of culture environment and the like. Cryptocaryon irritans is a most serious parasite for infecting marine culture fishes in south China, mainly infects marine teleost fishes to cause lethal and fulminant infectious diseases, and causes huge economic loss to marine culture industries in Guangdong, guangxi, fujian and other places. Epinephelus coioides is an important marine culture fish in coastal areas of China, has delicious meat quality and rich nutrition, and has very high economic value. The cryptocaryon irritans is a common disease in grouper breeding, and the frequent occurrence of the disease also causes huge economic loss to the grouper breeding industry. How to efficiently and safely prevent and treat the cryptocaryon irritans of grouper has been a hot problem of domestic and foreign research. The life history of cryptocaryon irritans includes four stages, of which the most harmful to farmed fish is the trophozoite stage, which parasitizes the host body surface, and the most vulnerable stage is the free larval stage. The cryptocaryon irritans in the cyst life history stage have a plurality of layers of cyst walls, dense substances are arranged between layers, the outer layer of the cyst is difficult to break, common medicaments are difficult to enter the inner part of the cyst, so that the traditional prevention and control method mainly aims at killing cryptocaryon irritans larvae. The current prevention and control methods for diseases and insect pests of the groupers cultured in seawater mainly comprise a chemical agent prevention and control method, a physical prevention and control method, a Chinese herbal medicine prevention and control method, an immune prevention and control method, an ecological prevention and control method and the like. The disease control effect can be achieved by killing pathogenic organisms in the aquaculture water body, but the traditional control method has the problems of high cost, great harm to the aquaculture fish, poor control effect and the like.
Cryptocaryon irritans (Cryptocaryon irritans) belong to the phylum Protozoa, the subdivision ciliata, the class Oligocilia, the class Mitocida, the order Futocida, the family Hitaceae, the genus Cryptocaryon, which are often parasitic on the body surface and gills of marine teleostomia species, cause infectious diseases in fish. The parasite has wide host range, and can infect almost all marine teleosts. Although the damage is serious, the disease is extremely difficult to prevent and control due to the characteristics of unique parasitic parts, complex life history, openness of a mariculture environment and the like.
The method is characterized in that the larvae in three life history stages of cryptocaryon irritans are respectively used as antigens to carry out intraperitoneal injection immunization on the grouper, the larvae are found to have the best immune effect, and the larvae with high dose have better immune effect than those with low dose, so that the cryptocaryon irritans can be prevented and controlled through immunization (Bai et al, 2008 Dan et al, 2013. However, cryptocaryon irritans cannot be cultured and propagated in vitro like bacteria, and can only be passaged with live fish (yoshinagaga et al, 2007); although stable cryptocaryon irritans living passage technology is established, it is still difficult to obtain enough whole insect antigen in a short time for scale production of vaccines, and the cryptocaryon irritans antigen produced by living passage is very expensive, which hinders the industrialized application of vaccines.
The use of genetically engineered vaccines to prevent and treat this disease has been reported in succession. The truncated Cryptocaryon irritans CiSA32.6 gene is connected to a pcDNA expression vector to prepare a recombinant expression plasmid, and the recombinant expression plasmid is used as a DNA vaccine to immunize large yellow croakers and can give the large yellow croakers a relative protection rate of 36.26%; the full length or the truncated gene sequence of the papain-like enzyme stimulating the cryptocaryon irritans is connected to a pcDNA3.1 expression vector, or the truncated papain-like enzyme gene and a profilein gene are connected in series and then connected to the pcDNA3.1 expression vector, and the recombinant expression vector is extracted to carry out intramuscular injection on the immune large yellow croaker, so that the relative protection rate of about 40 percent can be provided; the cryptocaryon irritans profilin gene, actin gene or the genes of profilin and actin are connected in series through PCR and then connected to a pcDNA expression vector, so that the large yellow croaker is immunized by the cryptocaryon irritans profilin gene, actin gene or the genes of profilin and actin, and immune protection rates of different degrees can be provided for the large yellow croaker; an i-antigen gene stimulating cryptocaryon irritans is connected to a pcDNA3.1 expression vector, and the i-antigen gene alone or in combination with recombinant expressed i-antigen protein can provide 40-46% of relative protection rate, and when the i-antigen gene and an hsp 70C-terminal sequence stimulating cryptocaryon irritans are connected in series to the pcDNA3.1 vector, the immune protection rate to fish bodies can be obviously improved; the full-length or truncated sequence of the cryptocaryon irritans cysteine protease is connected to a pcDNA3.1 expression vector, and the cryptocaryon irritans cysteine protease can be used as a DNA vaccine to immunize paralichthys olivaceus and can provide certain immune protection (Zhang Yinan, 2019; litting, 2019; jiangshuiya, 2019, josepray et al, 2012, 2015. Although DNA vaccines can provide immune protection to marine fish against cryptocaryon irritans, there is a risk that DNA vaccines integrate into host DNA or induce autoimmunity.
The cryptocaryon irritans actin depolymerizing factor is connected to a pGEX-4T-1 expression vector, is transformed into escherichia coli, and is induced by IPTG and purified to immunize large yellow croaker, so that 59% of relative protection rate can be provided for the large yellow croaker; amplifying to obtain actin, enolase and 26S proteosome genes stimulating the cryptocaryon, optimizing and modifying codons of the genes, connecting the genes to a pET-28a expression vector, and immunizing the groupers after nickel column purification, wherein immune protection is 23% -46%; connecting the cryptocaryon irritans cysteine protease gene to pET-26b or pET-28b expression vector, transforming into BL21 for induction expression, preparing vaccine by using the recombinant expressed protein as antigen, able to give significant immune protection rate to puffer fish; the truncated Cryptocaryon irritans CiSA32.6 gene is connected to a pET28a expression vector and is transformed into escherichia coli BL21 for recombinant expression, and subunit vaccine prepared by taking purified recombinant CiSA32.6 protein as antigen can give 50.1% of relative protection rate to large yellow croaker; we immunized Epinephelus with recombinant Cryptocaryon irritans surface protein C1 or i-antigen, tetrahymena tululin, both provided their ability to resist Cryptocaryon irritans (Zhang Yinan, 2019; jianshuqing, 2019, watanabe et al, 2021, jiang et al, 2022, mo et al, 2019, 2022; nevertheless school et al, 2021). The recombinant expression of the actin depolymerizing factor for stimulating the cryptocaryon volvatus, actin, enolase, 26S proteosome, cysteine protease and the tubulin of the tetrahymena are intracellular proteins, and the antibodies generated by the induced fishes enter the cryptocaryon volvatus to play the role of resisting the insects. However, there are two problems associated with having a sufficiently large number of functional antibodies present in the cryptocaryon irritans: on the one hand, the total amount of antibodies that can enter the worm is limited, and on the other hand, part of the antibodies that enter the worm are also digested by the worm. Therefore, intracellular proteins are not ideal candidates as vaccine candidate antigens.
The cryptocaryon irritans surface protein is used as an antibody generated by antigen induction, can effectively identify the proteins which irritate the cryptocaryon surface, directly plays a role in resisting insects, and is a more ideal candidate antigen. CiSA32.6 is a surface protein, but whether the protein exists in different Cryptocaryon irritans strains or is conserved is not clear, which can seriously affect the protective effect of the protein as a vaccine on different strains of Cryptocaryon irritans; the immunoprotection rate of the surface antigen C1 or i-antigen screened earlier in the subject group was low. The CiIA1 protein used in the patent is a surface membrane protein, and subject group analysis finds that the protein is conservative among different geographical isolates stimulating cryptocaryon and the vaccine prepared from the protein can provide 25% -50% of immune protection rate. Therefore, the vaccine prepared by using the CiIA1 protein as the antigen has wider application value, and can reduce the economic loss of the marine culture fish caused by the cryptocaryon irritans disease.
Disclosure of Invention
The invention aims to solve the problem of high mortality rate after infecting cryptocaryon irritans in the grouper culture process, and injects a recombinant protein vaccine with good immune protection effect to the grouper culture process, thereby greatly improving the disease-resistant survival rate of the grouper. The invention also aims to solve the technical problems of high preparation cost and poor immune protection effect of the cryptocaryon irritans vaccine, and provides a preparation method of a subunit vaccine based on the cryptocaryon irritans membrane protein CiIA1, which is used for screening and identifying the cryptocaryon irritans membrane protein CiIA1 for the first time, optimizing, recombining and expressing the cryptocaryon irritans membrane protein CiIA1, and preparing the vaccine by using the cryptocaryon irritans membrane protein CiIA1 as an antigen, so that the cryptocaryon irritans infection resistance of the grouper is greatly improved.
The invention is realized by the following technical scheme:
a method for breeding epinephelus coioides is characterized by comprising the following steps: putting epinephelus coioides fry into a culture pond, reserving 50-100 fries when 7 months of the epinephelus coioides fry grow to 10-15cm, putting the fries into a new culture pond as a control group, taking all the remaining fries as an immune group, injecting 0.1ml of CiIA1 subunit vaccine per tail fish into the abdominal cavity of the fry of the immune group, and simultaneously injecting 0.1ml of PBS per tail fish into the abdominal cavity of the fry of the control group; after two weeks, injecting CiIA1 subunit vaccine two 0.1 ml/tail fish into the abdominal cavity of the immunized group fry, and simultaneously injecting PBS 0.1 ml/tail fish into the abdominal cavity of the control group fry; after the first immunization injection is carried out for four weeks, part of groupers in the immune group and the control group are attacked by cryptocaryon irritans larvae of 4000 larvae/tail fish; the next day after the toxin is attacked, transferring the attacked fish to a special cyst collecting device, collecting cryptocaryon irritans cysts falling off from each fish body, counting, and calculating the insect reduction rate of the immune group; compared with a PBS control group, the insect reduction rate of the immune group reaches 25-50%;
the preparation method of the CiIA1 subunit vaccine I comprises the following steps: mixing 1.0mg/ml of recombinant CiIA1 protein solution with Freund's complete adjuvant at a ratio of 1: 1, and emulsifying by a homogenizer; the preparation method of the CiIA1 subunit vaccine II comprises the following steps: mixing 1.0mg/ml of recombinant CiIA1 protein solution with Freund's incomplete adjuvant according to the ratio of 1: 1, and emulsifying by a homogenizer; the recombinant CiIA1 protein solutions in both vaccines were prepared as follows:
1. construction of expression vectors
The cryptocaryon irritans membrane protein CiIA1 gene sequence encodes 320 amino acids in total, wherein the first 19 amino acids are signal peptide regions, and the last 65 amino acids are transmembrane structural domains; according to the analysis of hydrophilic and hydrophobic properties, a hydrophobic part is deleted, 232 amino acids are selected as target proteins, and the sequence is as follows: <xnotran> KTTAADWKGTFVVTSSSCLATCGWKIGTTVVIADKTGDATKVTWQGTVHTTDTTNVDVATGSCKYVSIVATEGQVGTATEVLNNDDTCEFGNGACTVMGKKQKTPGVVAFKRDMDLDTKPFQILYKQVEMVKKTSTTQKAAATDQANDCDTQASLVDTTTDAKAIVGTLKLSKATCDKCSWDTSKDLKITQDATKKYMVTLAGTIKETTAGDCANKLTASETCYVTKKDDKT; </xnotran>
The preference of cryptocaryon irritans codon and the type of coded amino acid are greatly different from those of escherichia coli, in order to realize good expression of cryptocaryon irritans in escherichia coli, the nucleic acid sequences for coding the protein sequences are recoded one by one according to the characteristics of escherichia coli codons, and the redesigned nucleic acid coding sequences are as follows: <xnotran> AAAACCACCGCAGCAGATTGGAAAGGGACGTTTGTGGTGACCTCAAGTAGCTGTTTAGCAACCTGTGGCTGGAAAATTGGCACCACCGTGGTTATTGCAGATAAAACCGGCGATGCAACCAAAGTGACCTGGCAGGGCACCGTTCATACCACCGATACCACCAATGTTGATGTTGCAACCGGCTCTTGTAAATATGTGAGTATTGTTGCAACCGAAGGTCAGGTGGGCACCGCAACCGAAGTGCTGAATAATGATGATACCTGTGAATTTGGTAATGGCGCATGTACCGTTATGGGCAAAAAACAGAAAACACCAGGCGTGGTTGCCTTTAAACGCGATATGGATCTGGATACCAAACCGTTTCAGATTCTGTATAAACAGGTTGAAATGGTTAAAAAAACCTCTACCACCCAGAAAGCAGCCGCAACCGATCAGGCCAATGATTGTGATACCCAGGCCTCATTAGTTGATACCACCACCGATGCCAAAGCCATTGTGGGTACGCTGAAATTAAGTAAAGCAACCTGTGATAAATGTAGCTGGGATACCTCTAAGGACCTGAAAATTACCCAGGATGCAACCAAAAAATATATGGTGACCTTAGCCGGCACCATTAAAGAAACCACCGCCGGCGATTGTGCCAATAAACTGACCGCATCAGAAACCTGTTATGTTACCAAAAAAGATGATAAAACC; </xnotran> Artificially synthesizing the gene segments, connecting the gene segments to a pMD-18T plasmid vector, and transforming the pMD-18T-CiIA1 plasmid into escherichia coli DH5 alpha; after amplification culture, extracting pMD-18T-CiIA1 plasmid from Escherichia coli DH5 alpha;
designing primers according to a CiIA1 gene sequence after codon optimization, and respectively adding homologous arm sequences of pET32 a-delta TRX vectors at the 5' ends of a forward primer and a reverse primer, wherein the forward primer sequence is as follows: CACCACCACCACGGTACCAAACCACCGCAGATT, and the reverse primer sequence is as follows: TTGTCGACGGAGCTCGAATTCTTAGGTTTTTTATCATTTTTTTGGTAACA; the PCR reaction system for amplifying the CiIA1 gene expression segment is as follows: pMD-18T-CiIA1 plasmid vector 4. Mu.l, 10. Mu.M forward primer 4. Mu.l, 10. Mu.M reverse primer 4. Mu.l, primeSTAR MAX mix 50. Mu.l, water 38. Mu.l; the PCR reaction program is: pre-denaturation at 98 ℃ for 1min, (98 ℃,15s, 55 ℃,15s, 72 ℃,2 min) x 35 cycles, and finally extension at 72 ℃ for 5min; carrying out agarose gel electrophoresis on the PCR product, cutting gel and recovering an amplified CiIA1 gene segment;
extracting pET32 a-delta TRX plasmid from Escherichia coli DH5 alpha containing pET32 a-delta TRX plasmid, and using Kpn I and EcoR I two restriction enzymes to cut pET32 a-delta TRX plasmid, wherein the cutting reaction system is as follows: pET32 a-. DELTA.TRX plasmid 1. Mu.g, quickcut TM Kpn I 1μl、QuickCut TM EcoR I1. Mu.l, 10X Quickcut Green Buffer 5. Mu.l, water to total volume of 50. Mu.l; after 30min at 37 ℃, agarose gel electrophoresis is carried out, and the mixture is separated from the gelCollecting pET32 a-delta TRX plasmid after double enzyme digestion;
the CiIA1 gene is connected with pET32 a-delta TRX plasmid recovered after enzyme digestion, and the connection reaction system is as follows: pET32 a-DeltaTRX plasmid 118ng, ciIA1 gene 28ng, 5 XCE II Buffer 4. Mu.l and Exnase II 2. Mu.l, adding water to total volume of 20. Mu.l; connecting at 37 deg.C for 30min, immediately cooling on ice; and transforming the ligation product into a new Escherichia coli DH5 alpha, selecting a positive clone for sequencing, and obtaining the positive clone containing pET32 a-delta TRX-CiIA1 plasmid with correct sequencing.
2. Screening of recombinant CiIA1 protein expression bacteria
Extracting pET32 a-delta TRX-CiIA1 plasmid from positive escherichia coli DH5 alpha obtained by sequencing in the last step, and then transforming the plasmid into BL21 expression bacteria; selecting BL21 bacteria containing pET32 a-delta TRX-CiIA1 plasmid to 1ml LB liquid culture medium added with ampicillin, culturing at 37 ℃ and 180rpm for 5h; adding 0.5 mul IPTG into 500 mul of bacterial liquid, and continuously culturing for 2h to obtain a CiIA1 protein induced expression group; taking another 500 mu l of bacterial liquid without adding any reagent, and continuously culturing for 2h to serve as an uninduced control group; collecting the bacterial liquid of the CiIA1 protein induced expression group and the non-induced control group, and performing SDS-PAGE gel electrophoresis; the results show that recombinant CiIA1 protein is successfully expressed in the CiIA1 protein inducible expression group, and the corresponding protein is not expressed in the uninduced control group.
3. Purification of recombinant CiIA1 protein
(1) Inoculating the bacteria which can express the recombinant CiIA1 protein and are confirmed in the previous step into 5ml of LB liquid culture medium, and carrying out shaking culture at 37 ℃ overnight;
(2) Inoculating 5ml of overnight-cultured bacterial liquid into 500ml of LB liquid medium, and adding 250 mul of ampicillin at the same time;
(3) Carrying out shaking culture at 37 ℃ and 180rpm for 4h until the OD600 of the bacterial liquid reaches 0.5;
(4) Adding 350 μ l of 1M IPTG into the bacterial liquid, and continuously culturing at 37 deg.C and 180rpm for 3h;
(5) Putting the bacterial liquid at 4 ℃ to precipitate bacteria for 24 hours;
(6) Removing the supernatant, and reserving 100-150ml of bacterial liquid;
(7) Centrifuging the bacterial solution at 10000 Xg for 15min;
(8) Removing the supernatant, retaining the bacterial pellet, and then resuspending the bacteria with 30ml of PBS;
(9) Ultrasonically breaking the bacterial suspension, wherein the ultrasonic treatment lasts for 250w, 9s and 9s for 30min;
(10) Centrifuging the crushed bacteria liquid at 10000 Xg for 5min, discarding the supernatant, and keeping the precipitate;
(11) Adding 20ml 1M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(12) Adding 20ml 2M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(13) Adding 20ml 3M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(14) Adding 20ml 4M urea solution into the precipitate, re-suspending by a refiner, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging 15min by 10000 Xg of re-suspending solution, sucking supernatant, storing in another tube, and retaining the precipitate;
(15) Adding 20ml 6M urea solution into the precipitate, resuspending by a refiner, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging a heavy suspension solution at 10000 Xg for 15min, sucking supernatant, storing in another tube, and retaining the precipitate;
(16) Adding 20ml 8M urea solution into the precipitate, re-suspending by a refiner, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and discarding the precipitate;
(17) Taking part of the supernatant collected in the steps (11) - (16) to perform SDS-PAGE gel electrophoresis, and then performing Coomassie brilliant blue staining to find that the recombinant CiIA1 protein is mainly dissolved in 8M urea;
(18) And (3) centrifuging the supernatant collected in the step (16) at 10000 Xg for 10min, removing the precipitate, taking the supernatant, adding the supernatant into a dialysis bag, sequentially putting the dialysis bag into a solution containing 6M, 4M, 2.5M, 1.5M, 1M and 0.5M urea for dialysis, and finally transferring the dialysis bag into a PBS solution. The dialysis bags were placed in each solution for 12h.
(19) And collecting the solution in the dialysis bag dialyzed by PBS, namely the solution of the recombinant CiIA1 protein, wherein the concentration of the recombinant CiIA1 protein is 1.18mg/ml.
The invention has the beneficial effects that: the membrane protein CiIA1 of the cryptocaryon irritans is screened, identified, optimized and recombined for the first time, and is applied to the preparation of subunit vaccines for the first time, and is used in the culture process of the epinephelus coioides, so that the disease resistance of the epinephelus coioides to the cryptocaryon irritans is greatly improved, and the culture survival rate of the epinephelus coioides is improved. In 7 months of the year, the high incidence stage of cryptocaryon irritans in marine fish is observed, and in general, the protective effect of the vaccine is gradually reduced after a long injection period, so that the duration of the vaccine injection is selected to be 7 months to optimize the resistance against cryptocaryon irritans.
Drawings
FIG. 1 shows the expression of recombinant CiIA1 protein.
Wherein, lane 1 is bacterial liquid before induction, lane 2 is bacterial liquid after induction, lane 3 is purified recombinant CiIA1 protein.
Detailed Description
1. Passage of cryptocaryon irritans
Cryptocaryon irritans are separated from trachinotus ovatus cultured in the white region of the Maine city power in Guangdong province, the trophozoites on infected gills are scraped off by a glass slide, cleaned and transferred to a beaker containing 100ml of seawater for hatching; infecting trachinotus ovatus with newly hatched larvae of cryptocaryon irritans, and temporarily culturing the infected trachinotus ovatus in a 700L plastic bucket after 2 h; transferring the infected trachinotus ovatus to a 200L plastic barrel at night 2 days after infection to allow the cysts to naturally fall off from the fish body, and collecting the cysts falling off from the bottom of the plastic barrel 2 days later; and (3) placing the cysts in sterilized seawater, culturing by inflating, hatching a large number of larvae after 48-72h, re-infecting the trachinotus ovatus with the larvae, and carrying out next passage.
2. Screening of CiIA1 Gene
Cryptocaryon irritans are collected according to the method in the step 1, total RNA of insect bodies is extracted, transcriptome sequencing is carried out after a cDNA library is constructed, surface protein genes highly expressed in various life history stages are analyzed and identified, and CiIA1 is a main target gene. The CiIA1 gene is amplified, optimized and modified by codon according to the method of the following step 3-5, then connected to a pET32 a-delta TRX expression vector, and transformed into escherichia coli BL21 for induced expression. And carrying out SDS-PAGE gel electrophoresis on the purified CiIA1 protein, carrying out Western blot by taking the rockfish anti-cryptocaryon irritans whole-worm antiserum as a primary antibody after membrane conversion, and finding that the rockfish antiserum can effectively identify the CiIA1 with prokaryotic expression, thereby indicating that the recombinant CiIA1 has the same or similar epitope of natural protein thereof and can be used as a good antigen for preparing vaccines.
3. Construction of expression vector
The gene sequence of the cryptocaryon irritans membrane protein CiIA1 totally encodes 320 amino acids, wherein the first 19 amino acids are signal peptide regions, and the last 65 amino acids are transmembrane domains. According to the analysis of hydrophilic and hydrophobic properties, a hydrophobic part is deleted, 232 amino acids are selected as target proteins, and the sequence is as follows: <xnotran> KTTAADWKGTFVVTSSSCLATCGWKIGTTVVIADKTGDATKVTWQGTVHTTDTTNVDVATGSCKYVSIVATEGQVGTATEVLNNDDTCEFGNGACTVMGKKQKTPGVVAFKRDMDLDTKPFQILYKQVEMVKKTSTTQKAAATDQANDCDTQASLVDTTTDAKAIVGTLKLSKATCDKCSWDTSKDLKITQDATKKYMVTLAGTIKETTAGDCANKLTASETCYVTKKDDKT. </xnotran>
The preference of cryptocaryon irritans codon and the type of coded amino acid are greatly different from those of escherichia coli, in order to realize good expression of cryptocaryon irritans in escherichia coli, the nucleic acid sequences for coding the protein sequences are recoded one by one according to the characteristics of escherichia coli codons, and the redesigned nucleic acid coding sequences are as follows: <xnotran> AAAACCACCGCAGCAGATTGGAAAGGGACGTTTGTGGTGACCTCAAGTAGCTGTTTAGCAACCTGTGGCTGGAAAATTGGCACCACCGTGGTTATTGCAGATAAAACCGGCGATGCAACCAAAGTGACCTGGCAGGGCACCGTTCATACCACCGATACCACCAATGTTGATGTTGCAACCGGCTCTTGTAAATATGTGAGTATTGTTGCAACCGAAGGTCAGGTGGGCACCGCAACCGAAGTGCTGAATAATGATGATACCTGTGAATTTGGTAATGGCGCATGTACCGTTATGGGCAAAAAACAGAAAACACCAGGCGTGGTTGCCTTTAAACGCGATATGGATCTGGATACCAAACCGTTTCAGATTCTGTATAAACAGGTTGAAATGGTTAAAAAAACCTCTACCACCCAGAAAGCAGCCGCAACCGATCAGGCCAATGATTGTGATACCCAGGCCTCATTAGTTGATACCACCACCGATGCCAAAGCCATTGTGGGTACGCTGAAATTAAGTAAAGCAACCTGTGATAAATGTAGCTGGGATACCTCTAAGGACCTGAAAATTACCCAGGATGCAACCAAAAAATATATGGTGACCTTAGCCGGCACCATTAAAGAAACCACCGCCGGCGATTGTGCCAATAAACTGACCGCATCAGAAACCTGTTATGTTACCAAAAAAGATGATAAAACC. </xnotran> The gene segments are artificially synthesized, connected to a pMD-18T vector and transformed into DH5 alpha by a pMD-18T-CiIA1 plasmid. After amplification culture, extracting pMD-18T-CiIA1 plasmid from Escherichia coli DH5 alpha;
designing a primer according to the CiIA1 gene sequence after codon optimization, and respectively adding a homologous arm sequence of a pET32 a-delta TRX vector at the 5' ends of a forward primer and a reverse primer, wherein the forward primer sequence is as follows: CACCACCACCACGGTACCAAACCACCGCAGATT, and the reverse primer sequence is as follows: TTGTCGACGGAGCTCGAATTCTTAGGTTTTTTATCATTTTTGGTAACA. The PCR reaction system for amplifying the CiIA1 gene expression segment is as follows: pMD-18T-CiIA1 plasmid vector 4. Mu.l, 10. Mu.M forward primer 4. Mu.l, 10. Mu.M reverse primer 4. Mu.l, primeSTAR MAX mix 50. Mu.l, water 38. Mu.l. The PCR reaction program is: pre-denaturation at 98 ℃ for 1min, (98 ℃,15s, 55 ℃,15s, 72 ℃,2 min) x 35 cycles, and finally extension at 72 ℃ for 5min. And carrying out agarose gel electrophoresis on the PCR product, cutting the gel and recovering the amplified CiIA1 gene segment.
Extracting pET32 a-delta TRX plasmid from Escherichia coli DH5 alpha containing pET32 a-delta TRX plasmid, and using Kpn I and EcoR I two restriction enzymes to cut pET32 a-delta TRX plasmid, wherein the cutting reaction system is as follows: pET32 a-. DELTA.TRX plasmid 1. Mu.g, quickcut TM Kpn I 1μl、QuickCut TM EcoR I1. Mu.l, 10X Quickcut Green Buffer 5. Mu.l, water to a total volume of 50. Mu.l. After digestion at 37 ℃ for 30min, agarose gel electrophoresis was carried out, and the pET32 a-. DELTA.TRX plasmid after double digestion was recovered from the gel.
The CiIA1 gene is connected with pET32 a-delta TRX plasmid recovered after enzyme digestion, and the connection reaction system is as follows: pET32 a-. DELTA.TRX plasmid 118ng, ciIA1 gene 28ng, 5 XCE II Buffer 4. Mu.l, and Exnase II 2. Mu.l were added with water to a total volume of 20. Mu.l. The ligation was carried out at 37 ℃ for 30min and immediately cooled on ice. And transforming the ligation product into a new Escherichia coli DH5 alpha, selecting a positive clone for sequencing, and obtaining the positive clone containing pET32 a-delta TRX-CiIA1 plasmid with correct sequencing.
4. Screening of recombinant CiIA1 protein expression bacteria
pET32 a-delta TRX-CiIA1 plasmid is extracted from positive colibacillus DH5 alpha obtained by sequencing in the step 3, and then is transformed into BL21 expression bacteria. BL21 bacteria containing pET32 a-Delta TRX-CiIA1 plasmid are picked up to 1ml LB liquid culture medium added with ampicillin, and cultured for 5h at 37 ℃ and 180 rpm. Adding 0.5 mul IPTG into 500 mul of bacterial liquid, and continuously culturing for 2h to obtain a CiIA1 protein induced expression group; another 500. Mu.l of the bacterial solution was taken without adding any reagent, and cultured for 2 hours to serve as an uninduced control group. And collecting bacterial liquid of the CiIA1 protein induced expression group and the non-induced control group, and performing SDS-PAGE gel electrophoresis. The results are shown in FIG. 1, where recombinant CiIA1 protein was successfully expressed in CiIA1 protein-induced expression group (FIG. 1, lane 2), while no expression of the corresponding protein was observed in uninduced control group (FIG. 1, lane 1).
5. Purification of recombinant CiIA1 protein
(1) Inoculating the bacteria which can express the recombinant CiIA1 protein and are confirmed in the step 4 into 5ml of LB liquid culture medium, and carrying out shaking culture at 37 ℃ overnight;
(2) Inoculating 5ml of overnight-cultured bacterial liquid into 500ml of LB liquid medium, and adding 250 mul of ampicillin at the same time;
(3) Carrying out shaking culture at 37 ℃ and 180rpm for 4h until the OD600 of the bacterial liquid reaches 0.5;
(4) Adding 350 μ l of 1M IPTG into the bacterial liquid, and continuously culturing at 37 deg.C and 180rpm for 3h;
(5) Putting the bacterial liquid at 4 ℃ to precipitate bacteria for 24 hours;
(6) Removing the supernatant, and reserving 100-150ml of bacterial liquid;
(7) Centrifuging the bacterial liquid at 10000 Xg for 15min;
(8) Removing the supernatant, retaining the bacterial pellet, and then resuspending the bacteria with 30ml of PBS;
(9) Ultrasonically breaking the bacterial suspension, wherein the ultrasonic treatment lasts for 250w, 9s and 9s for 30min;
(10) Centrifuging the crushed bacteria liquid at 10000 Xg for 5min, discarding the supernatant, and keeping the precipitate;
(11) Adding 20ml 1M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(12) Adding 20ml 2M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(13) Adding 20ml 3M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(14) Adding 20ml 4M urea solution into the precipitate, re-suspending by a refiner, shaking for 10min on a shaking table to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(15) Adding 20ml 6M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(16) Adding 20ml 8M urea solution into the precipitate, re-suspending by a refiner, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and discarding the precipitate;
(17) Taking part of the supernatant collected in the steps (11) - (16) to perform SDS-PAGE gel electrophoresis, and then performing Coomassie brilliant blue staining to find that the recombinant CiIA1 protein is mainly dissolved in 8M urea;
(18) And (4) centrifuging the supernatant collected in the step (16) at 10000 Xg for 10min, removing the precipitate, taking the supernatant, adding the supernatant into a dialysis bag, sequentially putting the dialysis bag into a solution containing 6M, 4M, 2.5M, 1.5M, 1M and 0.5M urea for dialysis, and finally transferring the dialysis bag into a PBS solution. The dialysis bags were placed in each solution for 12h.
(19) And collecting the solution in the dialysis bag dialyzed by PBS, namely the solution of the recombinant CiIA1 protein, wherein the concentration of the recombinant CiIA1 protein is 1.18mg/ml.
6. Preparation of vaccines
Recombinant CiIA1 protein dialyzed into PBS was diluted to a final concentration of 1.0mg/ml with PBS, and the recombinant CiIA1 protein solution was mixed with Freund's adjuvant at 1: 1 and emulsified in a homogenizer. And (3) dropping the prepared vaccine on the water surface for observation, determining that the vaccine drops cannot be scattered, and storing the vaccine at 4 ℃.
7. Immunoprotection of recombinant CiIA1 protein against grouper
And (3) immunizing the grouper by using the vaccine prepared in the step (6), immunizing the vaccine prepared by using the Freund complete adjuvant for the first time, and reinforcing the vaccine prepared by using the Freund incomplete adjuvant for the reinforcing immunization once after two weeks. Grouper was injected with PBS as a control group. At 4 weeks after the first immunization, the groupers of the immunized and control groups were challenged with larvae of cryptocaryon irritans of 4000 larvae/tail fish. And 2d, transferring the attacked fish to a special cyst collecting device, collecting the cysts of the cryptocaryon irritans falling off from each fish body, counting the cysts, and calculating the insect reduction rate of the vaccine immune group. As a result, it was found that: compared with a PBS control group, the recombinant CiIA1 protein immune group has a pest reduction rate of 25-50%.
Claims (1)
1. A method for breeding epinephelus coioides is characterized by comprising the following steps: putting the epinephelus coioides fry into a culture pond, reserving 50-100 fries when 7 months fish fry grow to 10-15cm, putting the fries into a new culture pond as a control group, taking all the remaining fries as an immune group, injecting 0.1ml of CiIA1 subunit vaccine/tail fish into the abdominal cavity of the immune group fry, and simultaneously injecting 0.1ml of PBS/tail fish into the abdominal cavity of the control group fry; injecting CiIA1 subunit vaccine two 0.1 ml/tail fish into the abdominal cavity of the immunized group of fries two weeks later, and injecting PBS 0.1 ml/tail fish into the abdominal cavity of the control group of fries at the same time; after the first immunization injection is carried out for four weeks, part of groupers in the immune group and the control group are detoxified by the larvae of the cryptocaryon irritans of 4000 larvae/tail fish; the next day after toxin attacking, transferring the attacked fish to a special cyst collecting device, collecting cryptocaryon irritans cysts dropped from each fish body, counting, and calculating the insect reduction rate of the immune group; compared with a PBS control group, the insect reduction rate of the immune group reaches 25-50%;
the preparation method of the CiIA1 subunit vaccine I comprises the following steps: mixing 1.0mg/ml of recombinant CiIA1 protein solution with Freund's complete adjuvant at a ratio of 1: 1, and emulsifying by a homogenizer; the preparation method of the CiIA1 subunit vaccine II comprises the following steps: mixing 1.0mg/ml of recombinant CiIA1 protein solution with Freund's incomplete adjuvant at a ratio of 1: 1, and emulsifying by a homogenizer; the recombinant CiIA1 protein solutions in both vaccines were prepared as follows:
1. construction of expression vectors
The cryptocaryon irritans membrane protein CiIA1 gene sequence encodes 320 amino acids in total, wherein the first 19 amino acids are signal peptide regions, and the last 65 amino acids are transmembrane domains; according to the analysis of hydrophilic and hydrophobic properties, a hydrophobic part is deleted, 232 amino acids are selected as target proteins, and the sequence is as follows: <xnotran> KTTAADWKGTFVVTSSSCLATCGWKIGTTVVIADKTGDATKVTWQGTVHTTDTTNVDVATGSCKYVSIVATEGQVGTATEVLNNDDTCEFGNGACTVMGKKQKTPGVVAFKRDMDLDTKPFQILYKQVEMVKKTSTTQKAAATDQANDCDTQASLVDTTTDAKAIVGTLKLSKATCDKCSWDTSKDLKITQDATKKYMVTLAGTIKETTAGDCANKLTASETCYVTKKDDKT: </xnotran>
The preference of cryptocaryon irritans codon and the type of coded amino acid are greatly different from those of escherichia coli, in order to realize good expression of the cryptocaryon irritans in the escherichia coli, the nucleic acid sequences coding the protein sequences are recoded one by one according to the characteristics of the escherichia coli codons, and the redesigned nucleic acid coding sequences are as follows: <xnotran> AAAACCACCGCAGCAGATTGGAAAGGGACGTTTGTGGTGACCTCAAGTAGCTGTTTAGCAACCTGTGGCTGGAAAATTGGCACCACCGTGGTTATTGCAGATAAAACCGGCGATGCAACCAAAGTGACCTGGCAGGGCACCGTTCATACCACCGATACCACCAATGTTGATGTTGCAACCGGCTCTTGTAAATATGTGAGTATTGTTGCAACCGAAGGTCAGGTGGGCACCGCAACCGAAGTGCTGAATAATGATGATACCTGTGAATTTGGTAATGGCGCATGTACCGTTATGGGCAAAAAACAGAAAACACCAGGCGTGGTTGCCTTTAAACGCGATATGGATCTGGATACCAAACCGTTTCAGATTCTGTATAAACAGGTTGAAATGGTTAAAAAAACCTCTACCACCCAGAAAGCAGCCGCAACCGATCAGGCCAATGATTGTGATACCCAGGCCTCATTAGTTGATACCACCACCGATGCCAAAGCCATTGTGGGTACGCTGAAATTAAGTAAAGCAACCTGTGATAAATGTAGCTGGGATACCTCTAAGGACCTGAAAATTACCCAGGATGCAACCAAAAAATATATGGTGACCTTAGCCGGCACCATTAAAGAAACCACCGCCGGCGATTGTGCCAATAAACTGACCGCATCAGAAACCTGTTATGTTACCAAAAAAGATGATAAAACC; </xnotran> Artificially synthesizing the gene segments, connecting the gene segments to a pMD-18T plasmid vector, and transforming the pMD-18T-CiIA1 plasmid into escherichia coli DH5 alpha; after amplification culture, extracting pMD-18T-CiIA1 plasmid from Escherichia coli DH5 alpha;
designing a primer according to the CiIA1 gene sequence after codon optimization, and respectively adding a homologous arm sequence of a pET32 a-delta TRX vector at the 5' ends of a forward primer and a reverse primer, wherein the forward primer sequence is as follows: CACCACCACCACGGTACCAAACCACCGCAGATT, and the reverse primer sequence is as follows: TTGTCGACGGAGCTCGAATTCTTAGGTTTTTTATCATTTTTTTGGTAACA; the PCR reaction system for amplifying the CiIA1 gene expression segment is as follows: pMD-18T-CiIA1 plasmid vector 4. Mu.l, 10. Mu.M forward primer 4. Mu.l, 10. Mu.M reverse primer 4. Mu.l, primeSTAR MAX mix 50. Mu.l, water 38. Mu.l; the PCR reaction program is: pre-denaturation at 98 ℃ for 1min, (98 ℃,15s, 55 ℃,15s, 72 ℃,2 min) for 35 cycles, and finally extension at 72 ℃ for 5min; carrying out agarose gel electrophoresis on the PCR product, cutting gel and recovering an amplified CiIA1 gene segment;
extracting pET32 a-delta TRX plasmid from Escherichia coli DH5 alpha containing pET32 a-delta TRX plasmid, and using Kpn I and EcoR I two restriction enzymes to cut pET32 a-delta TRX plasmid, wherein the cutting reaction system is as follows: pET32 a-. DELTA.TRX plasmid 1. Mu.g, quickcut TM Kpn I 1μl、QuickCut TM EcoR I1. Mu.l, 10X Quickcut Green Buffer 5. Mu.l, water to total volume of 50. Mu.l; carrying out enzyme digestion at 37 ℃ for 30min, carrying out agarose gel electrophoresis, and recovering pET32 a-delta TRX plasmid subjected to double enzyme digestion from the gel;
the CiIA1 gene is connected with pET32 a-delta TRX plasmid recovered after enzyme digestion, and the connection reaction system is as follows: pET32 a-DeltaTRX plasmid 118ng, ciIA1 gene 28ng, 5 XCE II Buffer 4. Mu.l and Exnase II 2. Mu.l, adding water to total volume of 20. Mu.l; connecting at 37 deg.C for 30min, immediately cooling on ice; and transforming the ligation product into a new Escherichia coli DH5 alpha, selecting a positive clone for sequencing, and obtaining the positive clone containing pET32 a-delta TRX-CiIA1 plasmid with correct sequencing.
2. Screening of recombinant CiIA1 protein expression bacteria
Extracting pET32 a-delta TRX-CiIA1 plasmid from positive escherichia coli DH5 alpha obtained by sequencing in the previous step, and then transforming the plasmid into BL21 expression bacteria; selecting BL21 bacteria containing pET32 a-delta TRX-CiIA1 plasmid to 1ml LB liquid culture medium added with ampicillin, culturing at 37 ℃ and 180rpm for 5h; adding 0.5 mul IPTG into 500 mul of bacterial liquid, and continuously culturing for 2h to obtain a CiIA1 protein induced expression group; taking 500 mu l of bacterial liquid without adding any reagent, and continuously culturing for 2h to serve as an uninduced control group; collecting the bacterial liquid of the CiIA1 protein induced expression group and the non-induced control group, and performing SDS-PAGE gel electrophoresis; the results show that recombinant CiIA1 protein is successfully expressed in the CiIA1 protein inducible expression group, and the corresponding protein is not expressed in the uninduced control group.
3. Purification of recombinant CiIA1 protein
(1) Inoculating the bacteria which can express the recombinant CiIA1 protein and are confirmed in the previous step into 5ml of LB liquid culture medium, and carrying out shaking culture at 37 ℃ overnight;
(2) Inoculating 5ml of overnight-cultured bacterial liquid into 500ml of LB liquid medium, and adding 250 mul of ampicillin at the same time;
(3) Carrying out shaking culture at 37 ℃ and 180rpm for 4h until the OD600 of the bacterial liquid reaches 0.5;
(4) Adding 350 μ l of 1M IPTG into the bacterial liquid, and continuously culturing at 37 deg.C and 180rpm for 3h;
(5) Putting the bacterial liquid at 4 ℃ to precipitate bacteria for 24 hours;
(6) Removing supernatant, and reserving 100-150ml of bacterial liquid;
(7) Centrifuging the bacterial liquid at 10000 Xg for 15min;
(8) Removing the supernatant, retaining the bacterial pellet, and then resuspending the bacteria with 30ml of PBS;
(9) Ultrasonically breaking the bacterial suspension, wherein the ultrasonic treatment lasts for 250w, 9s and 9s for 30min;
(10) Centrifuging the crushed bacteria liquid at 10000 Xg for 5min, discarding the supernatant, and keeping the precipitate;
(11) Adding 20ml 1M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(12) Adding 20ml 2M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(13) Adding 20ml 3M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(14) Adding 20ml 4M urea solution into the precipitate, re-suspending by a refiner, shaking for 10min on a shaking table to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(15) Adding 20ml 6M urea solution into the precipitate, re-suspending by a homogenizer, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and retaining the precipitate;
(16) Adding 20ml 8M urea solution into the precipitate, re-suspending by a refiner, shaking on a shaking table for 10min to fully dissolve the precipitate, centrifuging the re-suspending solution for 15min at 10000 Xg, sucking the supernatant, storing in another tube, and discarding the precipitate;
(17) Taking part of the supernatant collected in the steps (11) - (16) to perform SDS-PAGE gel electrophoresis, and then performing Coomassie brilliant blue staining to find that the recombinant CiIA1 protein is mainly dissolved in 8M urea;
(18) And (3) centrifuging the supernatant collected in the step (16) at 10000 Xg for 10min, removing the precipitate, taking the supernatant, adding the supernatant into a dialysis bag, sequentially putting the dialysis bag into a solution containing 6M, 4M, 2.5M, 1.5M, 1M and 0.5M urea for dialysis, and finally transferring the dialysis bag into a PBS solution. The dialysis bag was placed in each solution for 12h
(19) And collecting the solution in the dialysis bag dialyzed by PBS, namely the solution of the recombinant CiIA1 protein, wherein the concentration of the recombinant CiIA1 protein is 1.18mg/ml.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105746410A (en) * | 2016-04-08 | 2016-07-13 | 华南农业大学 | Production method of cryptocaryon irritus-resistant epinephelus fuscoguttatus and epinephelus lanceolatus fry |
| CN105941194A (en) * | 2016-04-08 | 2016-09-21 | 华南农业大学 | Cryptocaryon irritans Brown prevention Epinephelus tauvina fry production method |
| CN105941196A (en) * | 2016-04-08 | 2016-09-21 | 华南农业大学 | Cryptocaryon irritans Brown prevention large yellow croaker fry production method |
| CN107349422A (en) * | 2017-06-20 | 2017-11-17 | 华南农业大学 | A kind of preparation method and applications for stimulating cryptonucleus insect subunit vaccine |
| JP2020000050A (en) * | 2018-06-26 | 2020-01-09 | 日本水産株式会社 | Breeding method of yellowtail cultured fish and yellowtail cultured fish |
| TW202135656A (en) * | 2020-03-26 | 2021-10-01 | 劉冠宏 | Fish breeding assembly and fish breeding system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105746410A (en) * | 2016-04-08 | 2016-07-13 | 华南农业大学 | Production method of cryptocaryon irritus-resistant epinephelus fuscoguttatus and epinephelus lanceolatus fry |
| CN105941194A (en) * | 2016-04-08 | 2016-09-21 | 华南农业大学 | Cryptocaryon irritans Brown prevention Epinephelus tauvina fry production method |
| CN105941196A (en) * | 2016-04-08 | 2016-09-21 | 华南农业大学 | Cryptocaryon irritans Brown prevention large yellow croaker fry production method |
| CN107349422A (en) * | 2017-06-20 | 2017-11-17 | 华南农业大学 | A kind of preparation method and applications for stimulating cryptonucleus insect subunit vaccine |
| JP2020000050A (en) * | 2018-06-26 | 2020-01-09 | 日本水産株式会社 | Breeding method of yellowtail cultured fish and yellowtail cultured fish |
| JP2021177781A (en) * | 2018-06-26 | 2021-11-18 | 日本水産株式会社 | Breeding method of yellowtail cultured fish and yellowtail cultured fish |
| TW202135656A (en) * | 2020-03-26 | 2021-10-01 | 劉冠宏 | Fish breeding assembly and fish breeding system |
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