A yellow catfish calicivirus subunit protein, its preparation method and application

Abstract

The present invention belongs to the field of prevention and control of aquatic pathogenic microorganisms, and specifically relates to a yellow catfish calicivirus subunit protein, a preparation method thereof and an application. This protein has strong immunogenicity, can effectively stimulate the immune response of yellow catfish, effectively reduce the incidence of yellow catfish calicivirus disease, and this protein is expressed by Escherichia coli, with low production cost and easy for large-scale production. In addition, the vaccine prepared with this protein as the active substance can be immunized by oral administration through mixing with fish feed, avoiding the cumbersome operation of catching fish for injection immunization.

Description

Technical Field

[0001] The invention belongs to the field of aquatic pathogenic microorganism prevention and control, and specifically relates to a yellow catfish calicivirus subunit protein and a preparation method and application thereof. Background Art

[0002] The yellow catfish (Petrospora fulvidraco), a member of the family Cyprinidae, order Siluriformes, is a highly prized freshwater aquaculture species in my country, with a rapidly developing aquaculture industry. In recent years, outbreaks of mortality have occurred in key yellow catfish farming areas, including Guangdong, Guangxi, Zhejiang, Hubei, Sichuan, and Jiangsu, causing significant economic losses to the industry. Researchers at the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, have confirmed that the cause of the recent outbreak of yellow catfish mortality is calicivirus, a disease that primarily occurs in the spring and carries a high mortality rate and serious consequences.

[0003] Pelteobagrus calicivirus belongs to the Caliciviridae family within the order Picornaviridae. Its virions are spherical, approximately 30–40 nm in diameter. The virus is highly contagious, infecting fish of all sizes. Pond mortality in severe cases can reach as high as 90%, with acute onset lasting 3–5 days and chronic disease lasting a month or longer. In intensive aquaculture, the disease resistance of yellow catfish is susceptible to various adverse factors, leading to the need for regular vaccination to boost immunity and disease resistance. However, conventional vaccines suffer from weak immunogenicity and high production costs. Therefore, developing a highly immunogenic and low-cost vaccine for yellow catfish calicivirus is of great significance for aquaculture. Summary of the Invention

[0004] To address the problems of the prior art, the present invention provides a subunit protein of calicivirus from yellow catfish. This protein has strong immunogenicity, can effectively stimulate the immune response of yellow catfish, and effectively reduce the incidence of calicivirus disease in yellow catfish. Furthermore, the protein is expressed in Escherichia coli, which has low production costs and is easy to mass-produce. Furthermore, a vaccine made with this protein as the active ingredient can be administered orally by mixing it with fish feed, avoiding the tedious operation of catching fish for injection.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A first aspect of the present invention provides a yellow catfish calicivirus subunit protein, the amino acid sequence of the yellow catfish calicivirus subunit protein is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides a gene encoding the above-mentioned Pelteobagrus fulvidraco calicivirus subunit protein, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] The third aspect of the present invention provides a method for preparing the above-mentioned yellow catfish calicivirus subunit protein, comprising: connecting the coding gene to the vector Pet-30a (+) using BamHI and HindIII as restriction sites to obtain a recombinant expression plasmid containing the target gene fragment; transforming the recombinant expression plasmid containing the target gene fragment into Escherichia coli BL21-DE3, inducing the expression of the target protein by IPTG, and obtaining the yellow catfish calicivirus subunit protein after purification.

[0009] The fourth aspect of the present invention provides a yellow catfish calicivirus subunit vaccine, which includes, by mass, 1 to 3 parts of the above-mentioned yellow catfish calicivirus subunit protein, 40 to 45 parts of an immunopotentiator, 10 to 15 parts of mannitol, 10 to 15 parts of polyvinyl pyrrolidone, and 340 to 350 parts of basic excipients.

[0010] Furthermore, the immunopotentiator comprises the following raw materials, calculated by weight: 10-20 parts of ammonium sulfate, 10-20 parts of astragalus polysaccharide, and 1-5 parts of honeysuckle pollen; and the particle size of the immunopotentiator is above 200 mesh.

[0011] Furthermore, the basic auxiliary materials include the following raw materials, calculated by mass: 2-5 parts of garlic powder, 2-10 parts of broken yeast powder, 5-20 parts of wheat bran, 2-5 parts of mixed oils, 0.5-1 part of salt, 5-20 parts of fish meal, 5-15 parts of soluble dry distiller's grains, and 5-15 parts of peanut residue; the particle fineness of the basic auxiliary materials is above 200 mesh.

[0012] Furthermore, the basic auxiliary materials include the following raw materials, calculated by mass: 3 parts of garlic powder, 5 parts of broken yeast powder, 10 parts of wheat bran, 2 parts of mixed oil, 1 part of salt, 10 parts of fish meal, 10 parts of soluble dry distiller's grains, and 10 parts of peanut residue.

[0013] Among them, garlic powder has antibacterial and immune-enhancing effects.

[0014] Cracked yeast powder, as a source of protein and B vitamins.

[0015] Wheat bran, as a source of fiber.

[0016] Mixed with fats and oils as a source of energy.

[0017] Salt, as an electrolyte and flavoring.

[0018] Fish meal, soluble dried distiller's grains, and peanut residue as protein sources.

[0019] The fifth aspect of the present invention provides a method for preparing the above-mentioned yellow catfish calicivirus subunit vaccine: spray-dried yellow catfish calicivirus subunit protein, immune enhancer, mannitol, and polyvinyl pyrrolidone are added to a blender in sequence, fully stirred, and then basic excipients are added and continued to be stirred to form a yellow catfish calicivirus subunit oral vaccine.

[0020] A sixth aspect of the present invention provides a use of the above-mentioned Pelteobagrus calicivirus subunit protein or the above-mentioned Pelteobagrus calicivirus subunit vaccine in the preparation of feed for preventing Pelteobagrus calicivirus.

[0021] Furthermore, the yellow catfish calicivirus subunit vaccine is added to yellow catfish calicivirus prevention feed at a mass percentage of 0.5% to 1%.

[0022] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0023] (1) The yellow catfish calicivirus subunit protein prepared by the present invention has strong immunogenicity and can effectively stimulate the immune response of yellow catfish. The protein preparation method is simple and can be prepared by expression in Escherichia coli. The production cost is low and it is easy to produce on a large scale.

[0024] (2) The yellow catfish calicivirus subunit protein prepared by the present invention is a dry powder, which has good stability compared to conventional liquid dosage forms and is easy to store and transport;

[0025] (3) The vaccine made with this protein as the active substance can be immunized by feeding it orally by mixing it with fish feed, avoiding the tedious operation of catching fish and injecting them for immunization. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an SDS-PAGE image for verifying the induced expression of the target protein of the yellow catfish calicivirus subunit protein of the present invention;

[0027] Figure 2 This is an SDS-PAGE diagram for the purification verification of the yellow catfish calicivirus subunit protein of the present invention. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] Unless otherwise specified, the materials, reagents, and experimental equipment involved in the embodiments of the present invention are all commercially available products.

[0030] Example 1

[0031] A yellow catfish calicivirus subunit protein, its amino acid sequence (SEQ ID NO.1):

[0032] SYSLVNRFKTVLMYFFPKMKCFRGKRTDLVEPITVVSMGTSFNGFLTPQNEFYCPDFYGESITLSDPPRPRLLELRGDTGGCSITCRACGVKIKKSNLLKHLQVCPKRTQPPRPPCPPKMTYKKTPAGQLKVKQAGPKQIAPIPMRDDPKGKGRWWWISSNLAAYSPAHADQHKGLACGDRGKSLKWTDVVEK SYTFNEVGDLLVLGTGLVESPYAVVATKDGLTSTGSTLPDIFHPVMCVGVNDRDEDDLDVTGSKDDSKLVITKMFDTPESHEKHRMVGSSITITVSGEAGRVNIAELTSGPEKDDLKTVHMTRLHQTDGHTAHNGKEGLYIISKPAHDHDPTNWMDNGDVIPALTSLVSMEDGGADKEHNITGGGGSRKDVY

[0033] Gene sequence codon-optimized for E. coli expression (SEQ ID NO. 2):

[0034]

[0035] The preparation method of the yellow catfish calicivirus subunit protein is as follows:

[0036] Based on the above gene sequence, restriction enzyme sites BamHI and HindⅢ were introduced, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the corresponding restriction enzyme sites of the pET-30a (+) vector through the restriction enzyme sites BamHI and HindⅢ at both ends of the gene sequence. The transfected strain was transformed into the Escherichia coli Top10 clone strain by the heat shock method, and grown on LB solid medium containing 50 μg / ml Kan+. Transformants containing the recombinant plasmid were screened by colony PCR, inoculated into LB liquid medium containing 50 μg / ml Kan+, and cultured at 37°C with shaking at 200 rpm for 14 h. The recombinant plasmid was extracted using a common plasmid extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0037] The obtained recombinant plasmid containing the target gene fragment was transformed into Escherichia coli BL21-DE3 competent cells by heat shock method. The transformed cells were spread on LB solid medium containing 50 μg / ml Kan+ for growth, and BL21-DE3 transformants containing the recombinant plasmid were screened.

[0038] Use a sterile toothpick to pick up a single BL21-DE3 colony containing the recombinant plasmid grown on the Kan+ resistance plate and inoculate it into 10 ml of LB liquid medium containing 50 μg / ml Kan+. Incubate the culture in a constant temperature shaker at 37°C and 200 rpm overnight. The next day, transfer the bacterial liquid to LB liquid medium containing 50 μg / ml Kan+ at a ratio of 1:100. When the OD600 value is about 0.6, add IPTG storage solution (100 m mol / L) at a ratio of 1:200 and continue culturing for 5 hours to induce the expression of the target protein. The SDS-PAGE image of the induced expression of the yellow catfish subunit target protein is shown below. Figure 1 As shown (M: 180 kDa color pre-stained protein molecular weight standard; lane 1: IPTG-induced bacterial solution; lane 2: uninduced bacterial solution).

[0039] Purification of Pelteobagrus fulvidraco Calicivirus Subunit Protein:

[0040] The E. coli fermentation broth expressing the recombinant protein was centrifuged at 5000 rpm for 8 minutes to collect the cells, resuspended in PBS with 1 / 20 volume of the fermentation broth, and homogenized using a high-pressure homogenizer. Appropriate protease inhibitors were added during the disruption process to prevent protein degradation. The disrupted liquid mixture was centrifuged again at 13000 rpm and 4°C for 10 minutes to collect the precipitate. The target protein (precipitate) was resuspended in PBS containing 0.1% Triton-X100 and 0.5M urea, shaken at room temperature for 20 minutes, and then centrifuged at 10000 rpm to collect the precipitate. This was repeated twice to remove most of the soluble impurities. The washed precipitated protein was dissolved in PBS solution containing 8M urea and shaken at room temperature for 30 minutes until the precipitated protein was fully dissolved and free of white particles. Cell debris and unbroken cells were removed by centrifugation at 13000 rpm and 4°C, and the supernatant was collected to obtain the purified protein. The SDS-PAGE image of the purified yellow catfish subunit protein purification verification is shown below. Figure 2 As shown (wherein, M: 180 kDa color pre-stained protein molecular weight standard; lane 1: purified protein solution collected from tube 1; lane 2: purified protein solution collected from tube 2; lane 3: purified protein solution collected from tube 3), the purified protein obtained was spray-dried and stored at 5°C for future use.

[0041] Example 2

[0042] This embodiment provides a yellow catfish calicivirus subunit vaccine, which includes, by mass, 1 part of the yellow catfish calicivirus subunit protein prepared in Example 1, 40 parts of an immunopotentiator, 10 parts of mannitol, 10 parts of polyvinyl pyrrolidone, and 340 parts of basic excipients.

[0043] The immunopotentiator includes the following raw materials, calculated by weight: 15 parts of ammonium sulfate, 15 parts of astragalus polysaccharide, and 3 parts of honeysuckle pollen; the particle size of the immunopotentiator is 200 mesh.

[0044] Calculated by mass, the basic auxiliary materials include the following raw materials: 3 parts of garlic powder, 5 parts of broken yeast powder, 10 parts of wheat bran, 2 parts of mixed oil, 1 part of salt, 10 parts of fish meal, 10 parts of soluble dry distiller's grains, and 10 parts of peanut residue; the particle fineness of the basic auxiliary materials is 200 mesh.

[0045] The preparation method of the yellow catfish calicivirus subunit vaccine comprises the following steps: adding spray-dried yellow catfish calicivirus subunit protein, an immunopotentiator, mannitol, and polyvinyl pyrrolidone to a blender in sequence, stirring them thoroughly, then adding basic auxiliary materials, and continuing to stir them to form the yellow catfish calicivirus subunit oral vaccine.

[0046] Example 3

[0047] Performance evaluation:

[0048] Case 1:

[0049] Method for using the yellow catfish calicivirus subunit vaccine: take 100g of the prepared yellow catfish calicivirus subunit vaccine, add it to about 600ml of water, stir and mix it thoroughly until a suspension is completely formed, then stir and mix it thoroughly with 20kg of feed, and air dry it for more than 10 minutes to obtain feed evenly coated with the yellow catfish calicivirus subunit vaccine.

[0050] Preparation method of yellow catfish calicivirus subunit protein feed: take 0.25g catfish calicivirus subunit protein, add it to about 600ml water, stir it thoroughly, then stir it thoroughly with 20kg feed, and air dry it for more than 10 minutes to obtain fish rhabdovirus binding protein feed.

[0051] 1. Test methods

[0052] 720 healthy yellow catfish fry weighing 8-10g and tested for calicivirus infection were selected. The experiment was randomly divided into four groups, with three replicates per group and 60 fish per replicate. Group 1 was a blank control group, fed a blank diet throughout the experiment. Group 2 was fed a yellow catfish calicivirus subunit vaccine mixed with feed. The feed was fed once daily for 7 consecutive days after the first immunization, and once daily for 5 consecutive days after a 3-week interval. The blank diet was fed at other times. Group 3 was fed a yellow catfish calicivirus subunit protein mixed with feed. The feed was fed once daily for 7 consecutive days after the first immunization, and once daily for 5 consecutive days after a 3-week interval. The blank diet was fed at other times. Group 4 was an injection-immunized control group, immunized by injection.

[0053] The vaccine antigen for injection is prepared by emulsifying the purified inactivated subunit protein antigen of yellow catfish calicivirus with mineral oil adjuvant in a ratio of 1:2. The antigen content is 100 μg / mL, the injection dose is 100 μL / tail, and the second vaccination with the same dose of vaccine is given 28 days after the first vaccination.

[0054] Each group was fed 4% of their body weight twice daily, and fish mortality was observed and recorded. Fourteen days after the end of the feeding immunization (19 days after the second immunization of Group 4, the control group), 0.1 g of visceral tissue from yellow catfish infected with Calicivirus was thoroughly ground, resuspended in an appropriate amount of PBS, and centrifuged at 10,000 rpm at 4°C for 5 minutes. The supernatant was sterilized by filtration through a 0.22 μm syringe filter and diluted to 100 ml with PBS as the virus solution. Fifty fish were sampled from each replicate group, with a challenge dose of 100 μL / fish. Observation was continued for 14 days, and statistical results were recorded.

[0055] According to the formula RPS=( )×100% to calculate the relative immune protection rate.

[0056] 3. Test results

[0057] Fourteen days after the end of feeding immunization, 50 fish from each group and each parallel were challenged with poison. The number of deaths was counted and the relative protection rate was calculated for 14 days after the challenge. The results are shown in Table 1.

[0058] Table 1 Comparison of relative immune protection rates

[0059]

[0060] As shown in Table 1, the relative immune protection rate of the group vaccinated with the calicivirus subunit vaccine for yellow catfish was 69.53%, the relative immune protection rate of the group vaccinated with the calicivirus subunit protein for yellow catfish was 54.67%, and the relative immune protection rate of the injection immunization group was 77.35%. From the above comparison results, it can be seen that the relative immune protection rate of the calicivirus subunit vaccine for yellow catfish can be close to 70%. Although it is slightly lower than the injection immunization, the operation is relatively simple and can meet the clinical immune effect requirements of yellow catfish farming.

[0061] Case 2:

[0062] Three yellow catfish farms in different regions, known for their frequent outbreaks of calicivirus, were selected: Farms A, B, and C. Six ponds were selected from each farm, with three replicates each for the blank control group and the experimental group. The blank control group was fed a blank diet throughout the entire culture period; the experimental group was fed a diet mixed with a subunit vaccine for yellow catfish calicivirus. Yellow catfish fry received their first vaccination two days after stocking, and were fed once daily for seven consecutive days. Three weeks later, a second vaccination was performed, mixed with the diet and fed once daily for five consecutive days. Blank diet was used for the remainder of the period. Survival rates were calculated for each experimental group and pond at the end of the culture period. The statistical results are shown in Table 2.

[0063] Table 2 Clinical immunization trials of calicivirus subunit vaccines against yellow catfish in farms in different regions

[0064]

[0065] As shown in Table 2, the average survival rate of the unvaccinated control group at Farm A was 53.35%, while the average survival rate of the experimental group immunized with the calicivirus subunit vaccine was 85.65%. The average survival rate of the unvaccinated control group at Farm B was 36.50%, while the average survival rate of the experimental group immunized with the calicivirus subunit vaccine was 65.30%. The average survival rate of the unvaccinated control group at Farm C was 40.65%, while the average survival rate of the experimental group immunized with the calicivirus subunit vaccine was 70.53%. The average survival rate of the blank control group at the three different locations was 43.50%, while the average survival rate of the vaccine-immunized group was 73.83%. These results indicate that oral administration of this calicivirus subunit vaccine in the feed can increase the survival rate of cultured yellow catfish by approximately 30%.

[0066] Therefore, the yellow catfish calicivirus subunit protein and yellow catfish calicivirus subunit vaccine provided by the present invention can effectively prevent and control the occurrence of yellow catfish calicivirus disease, and are of great significance to promoting the healthy development of the yellow catfish aquaculture industry.

Claims

1. A subunit protein of yellow catfish calicivirus, characterized in that: The amino acid sequence of the yellow catfish calicivirus subunit protein is shown in SEQ ID NO.

1.

2. A gene encoding the yellow catfish calicivirus subunit protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The preparation method of the yellow catfish calicivirus subunit protein according to claim 1, characterized in that: Including: The coding gene was ligated to the vector pET-30a(+) using BamHI and HindⅢ as restriction sites to obtain a recombinant expression plasmid containing the target gene fragment; the recombinant expression plasmid containing the target gene fragment was transformed into Escherichia coli BL21(DE3), and the expression of the target protein was induced by IPTG, and the yellow catfish calicivirus subunit protein was obtained after purification.

4. A subunit vaccine against yellow catfish calicivirus, characterized in that: By mass, it includes 1-3 parts of the yellow catfish calicivirus subunit protein described in claim 1, 40-45 parts of an immune enhancer, 10-15 parts of mannitol, 10-15 parts of polyvinylpyrrolidone, and 340-350 parts of basic excipients.

5. The subunit vaccine of yellow catfish calicivirus according to claim 4, characterized in that: By mass, the immune enhancer includes the following raw materials: 10-20 parts of ammonium sulfate, 10-20 parts of astragalus polysaccharide, 1-5 parts of honeysuckle pollen; the particle fineness of the immune enhancer is above 200 mesh.

6. The yellow catfish calicivirus subunit vaccine according to claim 4, characterized in that: By mass, the basic excipients include the following raw materials: 2-5 parts of garlic powder, 2-10 parts of broken wall yeast powder, 5-20 parts of wheat bran, 2-5 parts of mixed oil, 0.5-1 part of table salt, 5-20 parts of fish meal, 5-15 parts of soluble dried distillers grains, 5-15 parts of peanut residue; the particle fineness of the basic excipients is above 200 mesh.

7. The yellow catfish calicivirus subunit vaccine according to claim 6, characterized in that: By mass, the basic excipients include the following raw materials: 3 parts of garlic powder, 5 parts of broken wall yeast powder, 10 parts of wheat bran, 2 parts of mixed oil, 1 part of table salt, 10 parts of fish meal, 10 parts of soluble dried distillers grains, 10 parts of peanut residue.

8. The preparation method of the yellow catfish calicivirus subunit vaccine according to claim 4, characterized in that: The spray-dried yellow catfish calicivirus subunit protein, immune enhancer, mannitol, and polyvinylpyrrolidone were sequentially added to a stirrer and stirred evenly, and then the basic excipients were added and stirred evenly to form an oral vaccine of yellow catfish calicivirus subunit.

9. Use of the yellow catfish calicivirus subunit protein described in claim 1 or the yellow catfish calicivirus subunit vaccine described in claim 4 in the preparation of a feed for preventing yellow catfish calicivirus.

10. The application according to claim 9, wherein: The yellow catfish calicivirus subunit vaccine is added to the feed for preventing yellow catfish calicivirus at a mass percentage of 0.5%-1%.

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