Construction method and application of mandarin fish ISKNV MCP vaccine hypertonic immersion immunity
The method of vaccine absorption in mandarin fish driven by hyperosmotic treatment and osmotic pressure difference solves the problem of low vaccine absorption rate in mandarin fish immersion immunization, realizes efficient and low-cost ISKNV immunization control in mandarin fish, and improves the protection rate of mandarin fish against ISKNV.
Patent Information
- Application Number
- CN202511083820.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-04
- Publication Date
- 2025-11-07
AI Technical Summary
Existing methods of immersion immunization with mandarin fish vaccines have problems such as low vaccine absorption rate, poor protective effect, high risk of disease outbreak, and high workload and cost associated with vaccination.
Hypertonic treatment with 1-2.5% NaCl solution was used to create an osmotic pressure difference, which caused the epidermal cells of mandarin fish to contract. The cells were then transferred to ISKNV MCP nucleic acid vaccine solution. The osmotic pressure difference was used to drive the efficient absorption of the vaccine. Combined with water temperature control at 24-26℃, the optimal concentration and time were screened.
It significantly increased vaccine intake, improved the immune protection rate of mandarin fish against ISKNV, provided a low-cost and efficient immune control approach, and reduced stress response and vaccine waste.
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Figure CN120899894A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of disease prevention and treatment of aquaculture, and particularly relates to a construction method and application of a high-osmotic immersion immunization of a mandarin fish ISKNV MCP vaccine. BACKGROUND
[0002] Mandarin fish is an important characteristic freshwater economic fish in China due to its high-quality meat and high nutritional value, but the rapid development of mandarin fish aquaculture is seriously threatened by infectious spleen and kidney necrosis virus (ISKNV), which spreads quickly and has a high mortality rate, directly affecting the economic benefits and sustainable development of the aquaculture industry. Aquaculture vaccines can specifically improve the anti-infection level of aquatic animals to specific pathogens, thereby effectively preventing diseases, and have the advantages of being green, environmentally friendly, not polluting the environment, and having no drug residues, and are currently the best choice for preventing diseases in aquatic animals.
[0003] Currently, fish vaccines can be inoculated by immersion, injection or oral administration. The extensive use of injection immunization has greatly limited the application and promotion of aquaculture vaccines in the aquaculture industry. Therefore, it is particularly important to develop vaccines and immunization methods that are simple to use and low in cost for the aquaculture industry. Studies have shown that immersion immunization is a vaccine inoculation strategy based on the mucosal system, which can stimulate the fish to produce an immune response and improve the survival ability of test animals after pathogen infection.
[0004] However, the immune protection rate of direct immersion immunization of mandarin fish is usually low, and high-osmotic immersion is an important strategy to improve the protection efficiency of mandarin fish vaccine immersion immunization. High-osmotic immersion refers to pre-treating mandarin fish in a high-osmotic sodium chloride solution for a period of time, and then immersing them in a normal-osmotic vaccine solution for a period of time. During the treatment of mandarin fish in the high-osmotic sodium chloride solution, the increase in the external environment osmotic pressure causes the mucosal epithelium to lose water and shrink, and some mandarin fish epithelial cells fall off to leave some cavities. After immersion in the normal-osmotic vaccine solution, the epithelial cells recover to their original state, and a large amount of water absorption greatly increases the intake of the vaccine, thereby improving the immune response. However, since the increase in osmotic pressure can cause great harm to the fish, it is important to select the salinity of the high-osmotic solution to reduce the harm during immunization while improving the immune effect. At present, there is still a lack of research on high-osmotic immersion of mandarin fish vaccines, and it is urgent to improve the high-osmotic immersion strategy of mandarin fish to provide technical support for specific defense against ISKNV infection in mandarin fish.
[0005] The immune prevention and control of mandarin fish diseases mainly relies on injection vaccines, but this method has the problems of high work intensity, strong stress on fish, and high cost. Direct immersion immunization of vaccines has the defects of low vaccine absorption rate, resulting in serious waste of vaccines, long immunization cycle, high risk of disease outbreaks, and thus increasing the cost of aquaculture. SUMMARY
[0006] Therefore, the present application aims to provide a construction method and application of high-osmotic immersion immunization of ISKNV MCP vaccine of Siniperca chuatsi, and solve the problem of synergistic effect of osmotic pressure regulation on vaccine absorption.
[0007] To achieve the above-mentioned purpose, the present application provides the following technical scheme:
[0008] The present application provides a construction method of high-osmotic immersion immunization of ISKNV MCP vaccine of Siniperca chuatsi, comprising the following steps,
[0009] S1, immerse the Siniperca chuatsi in 1-2.5% NaCl solution, promote the skin cells of the fish body to shrink through the high-osmotic environment, and form an osmotic pressure difference;
[0010] S2, transfer the immersed Siniperca chuatsi to ISKNV MCP nucleic acid vaccine immersion, and drive ISKNV MCP to be absorbed efficiently by using the osmotic pressure difference;
[0011] S3, after 4 weeks of immunization, infect the Siniperca chuatsi with ISKNV, and select the optimal high-osmotic immersion immunization concentration.
[0012] Further, during the construction process, the water temperature is maintained at 24-26℃, so as to guarantee the metabolic activity of the fish body and the stability of the vaccine.
[0013] Further, the selection method of the step S3 is that inject each Siniperca chuatsi with ISKNV in the abdominal cavity, record the number of dead Siniperca chuatsi in the three groups after the infection for two weeks, and then calculate the relative protection rate.
[0014] Further, in the step S1, the immersion time of the Siniperca chuatsi in the NaCl solution is 5-10 minutes.
[0015] Further, in the step S2, the immersion time of the Siniperca chuatsi in the vaccine solution with normal osmotic pressure is 20-30 minutes.
[0016] Further, the application of the ISKNV MCP vaccine of Siniperca chuatsi in preventing the infection of the Siniperca chuatsi with infectious spleen and kidney necrosis virus.
[0017] The present application has the following beneficial effects:
[0018] 1. In the present application, the purpose of putting the Siniperca chuatsi into 1%-2.5% NaCl solution is high-osmotic treatment, promote the skin cells of the fish body to shrink through the high-osmotic environment, and form an osmotic pressure difference; transfer the high-osmotic treated Siniperca chuatsi to ISKNV MCP nucleic acid vaccine immersion with normal osmotic pressure, and drive ISKNV MCP to be absorbed efficiently by using the osmotic pressure difference. Compared with direct immersion, the vaccine intake amount is obviously increased, the specific immune response of the Siniperca chuatsi is better induced, and thus the protection rate against ISKNV infection is better.
[0019] 2、The present application solves the problems of low absorption rate and poor protection effect of traditional immersion immunization method.
[0020] 3、The present application significantly improves the ISKNV MCP vaccine intake through high osmotic immersion in 1%-2.5% NaCl solution, thereby improving the protection effect of immersion immunization, and providing a convenient, fast, low-cost, low-stress, high-throughput and efficient technical approach for the immunoprevention and control of ISKNV disease in Siniperca chuatsi.
[0021] Other advantages, objects and features of the present application will be set forth in the following specification and will be apparent to those skilled in the art from the teachings of the present application. The objects and other advantages of the present application will be realized and attained by the structure particularly pointed out in the specification. BRIEF DESCRIPTION OF DRAWINGS
[0022] In order to make the purpose, technical scheme and beneficial effects of the application more clear, the present application provides the following drawings for illustration:
[0023] Figure 1 Figure 1 shows the immune protection effect of high-osmotic immersion ISKNV vaccine in Siniperca chuatsi. DETAILED DESCRIPTION
[0024] As shown in Figure 1 , the present application provides a high-osmotic immersion method for ISKNV MCP vaccine in Siniperca chuatsi, which includes high-osmotic immersion group (1-2.5% NaCl), direct immersion group (DI) and non-immunized blank group (Blanck).
[0025] Example 1:
[0026] Preparation of ISKNV nucleic acid vaccine:
[0027] According to the sequence of ISKNV MCP gene (Gene ID: KC775382), primers P1 / P2 (Table 1) were designed and enzyme cutting sites (underlined) and protective bases (P1: CG; P2: GC) were added, and ISKNV DNA was used as a template to amplify the full-length sequence of MCP; the MCP sequence and the eukaryotic expression plasmid pcDNA3.1(+) were double-digested and then ligated to realize the cloning of MCP sequence into the eukaryotic expression plasmid pcDNA3.1(+), thereby constructing ISKNV MCP DNA vaccine pcMCP, and the total mass of eukaryotic expression plasmid is 400 mg, which is used for high-osmotic immersion of pcMCP in Siniperca chuatsi.
[0028] Table 1 MCP full-length amplification primers
[0029]
[0030] Mandarin fish immersion in hypertonic solution to immunize against PCMCP:
[0031] Hypertonic immersion immunization group I (1% NaCl), hypertonic immersion immunization group II (2% NaCl), hypertonic immersion immunization group III (2.5% NaCl), direct immersion immunization group (200mg pcMCP) and non-immunized blank group were set up respectively. Then, 10g mandarin fish were randomly divided into 5 groups, with 45 fish in each group.
[0032] The first group of mandarin fish was immersed in the direct immersion immunization group (200mg pcMCP) for 30 minutes;
[0033] The second, third, and fourth groups of mandarin fish were first soaked in hypertonic immersion immunization group I (1% NaCl), hypertonic immersion immunization group II (2% NaCl), and hypertonic immersion immunization group III (2.5% NaCl) for 10 minutes, respectively, and then transferred to 200mg pcMCP solution for immunization for 30 minutes.
[0034] The fifth group of mandarin fish was not immunized and served as a blank control.
[0035] The water temperature for immunizing each group of mandarin fish was controlled at 25℃ throughout the process.
[0036] Four weeks after immunization, each group of mandarin fish was challenged by intraperitoneal injection of the same dose of ISKNV. Figure 1 As shown, the results indicated that the hypertonic immersion immunization groups I, II, and III were significantly more effective than the direct immersion immunization group, with the relative protection rate of hypertonic immersion immunization group II reaching 75%. Figure 1 The relative protection rate of the direct immersion immunization group was 25%. (Relative percent survival (RPS)% = [1 - (mortality rate in the vaccine group / mortality rate in the control group)] * 100%)
[0037] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for constructing a high-osmotic immersion immunization of a mandarin fish ISKNV MCP vaccine, characterized by: Comprising the following steps, S1, immerse the mandarin fish in 1-2.5% NaCl solution, promote the shrinkage of the epidermal cells of the fish body through the hypertonic environment, and form the osmotic pressure difference; S2, transfer the immersed mandarin fish to the ISKNV MCP nucleic acid vaccine solution, and drive the efficient absorption of ISKNV MCP by using the osmotic pressure difference; S3, after 4 weeks of immunization, the mandarin fish is attacked by ISKNV to screen the best hypertonic immersion immunization concentration.
2. The method according to claim 1, wherein the method is characterized by the following steps: During the construction process, the water temperature is maintained at 24-26℃ throughout the process to ensure the metabolic activity of the fish body and the stability of the vaccine.
3. The method according to claim 2, wherein the method is characterized by the following steps: The screening method of step S3 is to inject ISKNV into the abdominal cavity of each mandarin fish, record the number of deaths of the mandarin fish in the three groups two weeks after the attack, and then calculate the relative protection rate.
4. The method according to claim 3, wherein the method is characterized by: In step S1, the immersion time of the mandarin fish in the NaCl solution is 5-10 minutes.
5. The method for constructing a hypertonic immersion immunization method for mandarin fish ISKNV MCP vaccine according to claim 4, characterized in that: In step S2, the immersion time of the mandarin fish in the normal osmotic pressure vaccine solution is 20-30 minutes.
6. The mandarin fish ISKNV MCP vaccine according to any one of claims 1-5 for preventing the infection of the mandarin fish with the infectious spleen and kidney necrosis virus.