Pelteobagrus fulvidraco calicivirus subunit protein as well as preparation method and application thereof

Abstract

The invention belongs to the field of prevention and control of aquatic pathogenic microorganisms, and particularly relates to pelteobagrus fulvidraco calicivirus subunit protein as well as a preparation method and application thereof. The protein has strong immunogenicity, can effectively stimulate the immune response of pelteobagrus fulvidraco and effectively reduce the morbidity of pelteobagrus fulvidraco calicivirus diseases, and is expressed by escherichia coli, low in production cost and easy for large-scale production. Besides, the vaccine prepared by taking the protein as an active substance can be used for immunization in a fish feed mixing and feeding oral administration mode, so that the tedious operation of fish catching and injection immunization is avoided.

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] Yellow catfish belongs to the order Siluriformes, family Catfish, genus Pelteobagrus. As a famous freshwater aquaculture species in my country, the aquaculture industry has developed rapidly. In recent years, outbreaks of death have occurred in the main breeding areas of yellow catfish in Guangdong, Guangxi, Zhejiang, Hubei, Sichuan, Jiangsu and other places in my country, causing huge economic losses to the yellow catfish aquaculture industry. Researchers from the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences confirmed that the cause of the recent outbreak of death of yellow catfish is yellow catfish calicivirus disease, which mainly occurs in spring. Once infected, the mortality rate is high and the harm is serious.

[0003] The yellow catfish calicivirus belongs to the Caliciviridae family in the order Picornaviridae. The virus particles are spherical with a diameter of about 30-40 nm. The virus is highly contagious and can infect fish of all sizes. The mortality rate in ponds with severe disease can be as high as 90%. The acute onset period is 3-5 days, and the chronic disease lasts for a month or even longer. Under intensive breeding conditions, the disease resistance of yellow catfish is easily affected by various adverse factors and reduced, so vaccines are needed regularly to improve immunity and disease resistance. However, traditional vaccines have problems such as weak immunogenicity and high production costs. Therefore, the study of a vaccine for yellow catfish calicivirus with strong immunogenicity and low cost is of great significance for aquaculture. Summary of the invention

[0004] In view of the problems in the prior art, the present invention provides a yellow catfish calicivirus subunit protein, which has strong immunogenicity, can effectively stimulate the immune response of yellow catfish, and effectively reduce the incidence of yellow catfish calicivirus disease. The protein is expressed in Escherichia coli, has low production cost, and is easy to mass produce. In addition, the vaccine made with the protein as the active substance can be immunized by mixing it with fish feed and feeding it orally, avoiding the cumbersome operation of catching fish for injection immunization.

[0005] The purpose of the present invention can be achieved through the following technical solutions: 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 as SEQ ID NO.1.

[0006] The second aspect of the present invention provides a gene encoding the above-mentioned Pelteobagrus calicivirus subunit protein, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] 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.

[0008] The fourth aspect of the present invention provides a yellow catfish calicivirus subunit vaccine, which comprises, by weight, 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 auxiliary materials.

[0009] Furthermore, the immunoenhancer includes the following raw materials, measured by weight: 10-20 parts of ammonium sulfate, 10-20 parts of astragalus polysaccharide, and 1-5 parts of honeysuckle pollen; the particle size of the immunoenhancer is above 200 mesh.

[0010] Furthermore, the basic auxiliary materials include the following raw materials, measured 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 residues; the particle fineness of the basic auxiliary materials is above 200 mesh.

[0011] Furthermore, the basic auxiliary materials include the following raw materials, measured by mass: 3 parts of garlic powder, 5 parts of broken yeast powder, 10 parts of wheat bran, 2 parts of mixed oils, 1 part of salt, 10 parts of fish meal, 10 parts of soluble dry distiller's grains, and 10 parts of peanut residues.

[0012] Among them, garlic powder has antibacterial and immune-enhancing effects.

[0013] Cracked yeast powder, as a source of protein and B vitamins.

[0014] Wheat bran, as a source of fiber.

[0015] Mixed with fats and oils as a source of energy.

[0016] Salt, as an electrolyte and flavoring.

[0017] Fish meal, soluble dried distiller's grains, and peanut residue are used as protein sources.

[0018] 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, immunopotentiator, mannitol, and polyvinyl pyrrolidone are added to a blender in sequence, stirred thoroughly, and then basic auxiliary materials are added, and stirring is continued to form a yellow catfish calicivirus subunit oral vaccine.

[0019] A sixth aspect of the present invention provides a use of the above-mentioned yellow catfish calicivirus subunit protein or the above-mentioned yellow catfish calicivirus subunit vaccine in the preparation of feed for preventing yellow catfish calicivirus.

[0020] Furthermore, the yellow catfish calicivirus subunit vaccine is added to yellow catfish calicivirus prevention feed at a mass percentage of 0.5% to 1%.

[0021] The beneficial effects achieved by one or more technical solutions of the present invention are as follows: (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 Escherichia coli expression. The production cost is low and it is easy to produce on a large scale. (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; (3) The vaccine made with this protein as the active ingredient can be mixed with fish feed and fed orally for immunization, avoiding the tedious operation of catching fish for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the SDS-PAGE diagram for verifying the induced expression of the target protein of the yellow catfish calicivirus subunit protein of the present invention; Figure 2 This is the SDS-PAGE diagram for the purification verification of the yellow catfish calicivirus subunit protein of the present invention. DETAILED DESCRIPTION

[0023] 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 in conjunction with specific embodiments.

[0024] Unless otherwise specified, the materials, reagents and experimental equipment involved in the embodiments of the present invention are all commercially available products.

[0025] Example 1 A yellow catfish calicivirus subunit protein, its amino acid sequence (SEQ ID NO.1): SYSLVNRFKTVLMYFFPKMKCFRGKRTDLVEPITVVSMGTSFNGFLTPQNEFYCPDFYGESITLSDPPRPRLLELRGDTGGCSITCRACGVKIKKSNLLKHLQVCPKRTQPPRPPCPPKMTYKKTPAGQLKVKQAGPKQIAPIPMRDDPKGKGRWWWISSNLAAYSPAHADQHKGLACGDRGKSLKWTDVVEK SYTFNEVGDLLVLGTGLVESPYAVVATKDGLTSTGSTLPDIFHPVMCVGVNDRDEDDLDVTGSKDDSKLVITKMFDTPESHEKHRMVGSSITITVSGEAGRVNIAELTSGPEKDDLKTVHMTRLHQTDGHTAHNGKEGLYIISKPAHDHDPTNWMDNGDVIPALTSLVSMEDGGADKEHNITGGGGSRKDVY Gene sequence optimized by codons for E. coli expression (SEQ ID NO. 2): The preparation method of the yellow catfish calicivirus subunit protein is as follows: Based on the above gene sequence, restriction enzyme sites BamHI and HindⅢ were introduced, which were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and cloned into the corresponding restriction enzyme sites of vector pET-30a (+) through the restriction enzyme sites BamHI and HindⅢ at both ends of the gene sequence. The strain was transformed into Escherichia coli Top10 clone strain by heat shock method, and grown on LB solid medium containing 50 μg / ml Kan+. Transformants containing recombinant plasmids were screened by colony PCR method, inoculated in LB liquid medium containing 50 μg / ml Kan+, and cultured at 37°C with shaking at 200 rpm for 14 hours. The recombinant plasmid was extracted using the common plasmid extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0026] 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.

[0027] Use a sterile toothpick to pick up a single colony of BL21-DE3 bacteria containing the recombinant plasmid grown on the Kan+ resistant plate, inoculate it into 10 ml of LB liquid culture medium containing 50μg / ml Kan+, and culture it overnight in a constant temperature shaker at 37°C and 200 rpm. The next day, transfer the bacterial liquid to LB liquid culture medium containing 50μg / 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 to culture for 5h to induce the expression of the target protein. The SDS-PAGE image of the induced expression verification of the yellow catfish subunit target protein is shown in the figure Figure 1 As shown (where M: 180kDa color pre-stained protein molecular weight standard; lane 1: IPTG-induced bacterial solution; lane 2: uninduced bacterial solution).

[0028] Purification of Yellow Catfish Calicivirus Subunit Protein: The E. coli fermentation broth expressing the recombinant protein was centrifuged at 5000rpm for 8 minutes to collect the cells, and the cells were resuspended in PBS with 1 / 20 volume of the fermentation broth. The cells were homogenized and broken by a high-pressure homogenizer. Appropriate protease inhibitors were added during the breaking process to prevent protein degradation. The broken liquid mixture was centrifuged again, centrifuged at 13000rpm and 4℃ for 10 minutes, and the precipitate was collected. The target protein (precipitate) was resuspended in PBS containing 0.1% Triton-X100 and 0.5M urea, and the precipitate was collected by centrifugation at 10000rpm after oscillation at room temperature for 20 minutes. This was repeated twice to remove most of the soluble impurities. The washed precipitated protein was dissolved in a PBS solution containing 8M urea, oscillated 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 13000rpm and 4℃, and the supernatant was collected to obtain the purified protein. The purified yellow catfish subunit protein purification verification SDS-PAGE diagram is shown as follows Figure 2 As shown (wherein, M: 180 kDa color pre-stained protein molecular weight standard; lane 1: purified protein solution collected in tube 1; lane 2: purified protein solution collected in tube 2; lane 3: purified protein solution collected in tube 3), the purified protein obtained was spray-dried and stored at 5°C for later use.

[0029] Example 2 The present embodiment provides a yellow catfish calicivirus subunit vaccine, which includes, by weight, 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 auxiliary materials.

[0030] Among them, the immunoenhancer 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 immunoenhancer is 200 mesh.

[0031] In terms of 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 oils, 1 part of salt, 10 parts of fish meal, 10 parts of soluble dry distiller's grains, and 10 parts of peanut residues; the particle size of the basic auxiliary materials is 200 mesh.

[0032] 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 stirrer in sequence, stirring them evenly, then adding basic auxiliary materials, and continuing to stir them evenly to form the yellow catfish calicivirus subunit oral vaccine.

[0033] Example 3 Performance evaluation: Case 1: 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 naturally for more than 10 minutes to obtain feed evenly coated with the yellow catfish calicivirus subunit vaccine.

[0034] The preparation method of the feed mixed with yellow catfish calicivirus subunit protein is as follows: 0.25g of catfish calicivirus subunit protein is added to about 600ml of water, fully stirred, and then fully stirred and mixed with 20kg of feed, and naturally air-dried for more than 10 minutes to obtain the feed mixed with fish rhabdovirus binding protein.

[0035] 1. Test methods 720 healthy yellow catfish fry weighing 8-10g were selected to detect no yellow catfish calicivirus infection. The experiment was randomly divided into 4 groups, with 3 parallels in each group and 60 fish in each parallel. Group 1 was a blank control group, which was fed with blank feed throughout the whole process; Group 2 was fed with feed mixed with yellow catfish calicivirus subunit vaccine, which was fed continuously for 7 days for the first immunization, once a day, and mixed with feed for 5 days after an interval of 3 weeks, once a day, and blank feed was fed at other times; Group 3 was fed with feed mixed with yellow catfish calicivirus subunit protein, which was fed continuously for 7 days for the first immunization, once a day, and mixed with feed for 5 days after an interval of 3 weeks, once a day, and blank feed was fed at other times; Group 4 was an injection immunization control group, which was immunized by injection.

[0036] The vaccine antigen for injection is prepared by emulsifying the purified inactivated antigen of the yellow catfish calicivirus subunit protein and mineral oil adjuvant in a ratio of 1:2. The antigen content is 100μg / mL, the injection dose is 100μL / tail, and the second vaccination with the same dose of vaccine is given 28 days after the first vaccination.

[0037] The feeding amount of each group was calculated as 4% of body weight, 2 times / day, and the mortality of fish was observed and recorded. 14 days after the end of feeding and immunization (19 days after the second immunization of the 4th group of injection immunization control group), the calicivirus tissue poison was used to attack the virus. 0.1g of the visceral tissue diseased material of yellow catfish calicivirus-infected fish was fully ground, resuspended in an appropriate amount of PBS, centrifuged at 10000rpm, 4℃ for 5 minutes, and the supernatant was filtered and sterilized through a 0.22μm syringe filter. PBS was made up to 100ml as the virus liquid. 50 fish were taken from each parallel group, and the attack dose was 100μL / fish. After that, they were observed for 14 consecutive days and the statistical test results were recorded.

[0038] According to the formula RPS = ( )×100% to calculate the relative immune protection rate.

[0039] 3. Test results 14 days after the feeding immunization, 50 fish from each group were taken from each parallel group to challenge the virus. 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.

[0040] Table 1 Comparison of relative immune protection rates

[0041] From the results in Table 1, it can be seen that the relative immune protection rate of the group immunized with the calicivirus subunit vaccine of yellow catfish was 69.53%, the relative immune protection rate of the group immunized with the calicivirus subunit protein of 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 of yellow catfish can be close to 70%, which is slightly lower than that of injection immunization, but the operation is relatively simple and can meet the clinical immune effect requirements of yellow catfish farming.

[0042] Case 2: Three yellow catfish farms in different regions where yellow catfish calicivirus often occurs were selected, namely farms A, B, and C. Six ponds were selected from each farm, with three parallel blank control groups and three experimental groups. The blank control group was fed with blank feed throughout the whole process; the experimental group was fed with feed mixed with yellow catfish calicivirus subunit vaccine. The yellow catfish fry were first immunized 2 days after being released into the pond, and were fed once a day for 7 consecutive days. After an interval of 3 weeks, the second immunization was carried out, and the mixed feed was fed for 5 consecutive days, once a day. Blank feed was fed at other times. The survival rate of each experimental group pond in each farm after the entire breeding period was calculated, and the statistical results are shown in Table 2.

[0043] Table 2 Clinical immunization trials of yellow catfish calicivirus subunit vaccines in farms in different regions

[0044] As shown in Table 2, the average survival rate of the non-immunized control group in farm A was 53.35%, and the average survival rate of the experimental group immunized with yellow catfish calicivirus subunit vaccine was 85.65%; the average survival rate of the non-immunized control group in farm B was 36.50%, and the average survival rate of the experimental group immunized with yellow catfish calicivirus subunit vaccine was 65.30%; the average survival rate of the non-immunized control group in farm C was 40.65%, and the average survival rate of the experimental group immunized with yellow catfish calicivirus subunit vaccine was 70.53%. The average survival rate of the blank control group in three different regions was 43.50%, and the average survival rate of the vaccine immunization group was 73.83%. The results show that the survival rate of yellow catfish culture can be increased by about 30% by oral immunization with the yellow catfish calicivirus subunit vaccine in clinical practice.

[0045] Therefore, the yellow catfish calicivirus subunit protein and the 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 breeding industry.

Claims

1. A yellow catfish calicivirus subunit protein, 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. A method for preparing the yellow catfish calicivirus subunit protein according to claim 1, characterized in that: include: The coding gene was connected 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, the expression of the target protein was induced by IPTG, and the yellow catfish calicivirus subunit protein was obtained after purification.

4. A yellow catfish calicivirus subunit vaccine, characterized in that: Calculated by mass, it comprises 1 to 3 parts of the yellow catfish calicivirus subunit protein according to claim 1, 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 auxiliary materials.

5. The yellow catfish calicivirus subunit vaccine according to claim 4, characterized in that: The immunoenhancer comprises the following raw materials in parts by mass: 10-20 parts of ammonium sulfate, 10-20 parts of astragalus polysaccharide, and 1-5 parts of honeysuckle pollen; the particle size of the immunoenhancer is above 200 meshes.

6. The yellow catfish calicivirus subunit vaccine according to claim 4, characterized in that: Calculated by weight, the basic auxiliary materials include the following raw materials: 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 residues; the particle size of the basic auxiliary materials is above 200 mesh.

7. The yellow catfish calicivirus subunit vaccine according to claim 6, characterized in that: In terms of 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 residues.

8. The method for preparing the yellow catfish calicivirus subunit vaccine according to claim 4, characterized in that: The spray-dried yellow catfish calicivirus subunit protein, immunopotentiator, mannitol and polyvinyl pyrrolidone are sequentially added into the blender, stirred evenly, and then the basic auxiliary materials are added, and stirred evenly to form the yellow catfish calicivirus subunit oral vaccine.

9. Use of the yellow catfish calicivirus subunit protein as claimed in claim 1 or the yellow catfish calicivirus subunit vaccine as claimed in claim 4 in preparing feed for preventing yellow catfish calicivirus.

10. The use according to claim 9, characterized in that: The yellow catfish calicivirus subunit vaccine is added to yellow catfish calicivirus prevention feed at a mass percentage of 0.5% to 1%.

Images