Didyneciliate membrane protein vaccine as well as preparation method and application of didyneciliate membrane protein vaccine

Abstract

The invention relates to the technical field of biology, in particular to a pellicle membrane protein vaccine as well as a preparation method and application thereof. According to the specific technical scheme, the preparation method of the pellicle membrane protein vaccine is characterized in that pellicle membrane protein and an adjuvant are mixed to prepare the pellicle membrane protein vaccine, and the pellicle membrane protein is obtained by extracting the pellicle membrane protein through an ultracentrifugation method. According to the invention, the vaccine with immunogenicity is prepared by extracting and purifying the pellicle membrane protein, and the vaccine is used for preventing or treating infection caused by pellicle.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a scuticociliate membrane protein vaccine, a preparation method thereof and an application thereof. Background Art

[0002] Scuticociliates belong to facultative parasites and are currently recognized as pathogenic parasites of marine fish. They are widely present in the marine environment and can infect a variety of aquatic animals, especially fish, resulting in serious diseases and economic losses. At present, the prevention and control measures for scuticociliate infection are limited and mainly rely on chemical drugs. However, long-term use is likely to lead to drug resistance, environmental pollution and food safety risks. Immunization has become an important way for the prevention and control of fish diseases. Developing a safe and effective scuticociliate vaccine has important application prospects in the prevention and control of this disease.

[0003] Membrane proteins play an important role in the immune response of pathogens. Because they are located on the cell surface, they are easily recognized by the host immune system. By extracting the membrane proteins of scuticociliates and preparing vaccines, the present invention can induce the host to produce specific immune responses, thereby effectively preventing or treating scuticociliate infection. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a scuticociliate membrane protein vaccine, a preparation method thereof and an application thereof. By extracting and purifying scuticociliate membrane proteins, a vaccine with immunogenicity is prepared for preventing or treating infections caused by scuticociliates.

[0005] To achieve the above object, the present invention is realized through the following technical solutions:

[0006] The present invention discloses a preparation method of a scuticociliate membrane protein vaccine, which is prepared by mixing scuticociliate membrane proteins and an adjuvant. The scuticociliate membrane proteins are the membrane proteins of scuticociliates extracted by an ultracentrifugation method.

[0007] Preferably, the extraction process of the scuticociliate membrane proteins is as follows: Scuticociliates are cultured to the logarithmic growth phase, and scuticociliate cells are collected by centrifugation at 12,000 rpm for 2 min. The ciliate cells are suspended in a lysis buffer and centrifuged at 4°C and 12,000 rpm for 10 min to obtain a precipitate. Extraction Buffer is added to the precipitate and centrifuged at 4°C and 12,000 rpm for 10 min to obtain a supernatant, and the supernatant is the membrane protein.

[0008] Preferably, scuticociliates are inoculated into an L-15 medium containing 7% chicken serum and cultured at 20°C for 48 - 72 hours until the logarithmic growth phase is reached.

[0009] Preferably, 1 μL of protease inhibitor and 1 μL of 1 M DTT are added to each milliliter of the lysis buffer.

[0010] Preferably, the concentration of the scuticociliate membrane protein is 0.6 mg / mL, and the concentration of the adjuvant is 0.3 mg / mL.

[0011] Preferably, the adjuvant is Freund's adjuvant or aluminum adjuvant.

[0012] Preferably, the volume ratio of the scuticociliate membrane protein to the adjuvant is 1:1.

[0013] Correspondingly, a scuticociliate membrane protein vaccine prepared by the above preparation method.

[0014] Correspondingly, an application of a scuticociliate membrane protein vaccine prepared by the above preparation method in preventing or treating scuticociliate infection.

[0015] Preferably, the scuticociliate membrane protein and the adjuvant are mixed at a volume ratio of 1:1 to prepare a scuticociliate membrane protein vaccine. The concentration of the scuticociliate membrane protein is 0.6 mg / mL, and the adjuvant is Freund's adjuvant or aluminum adjuvant with a concentration of 0.3 mg / mL.

[0016] The present invention has the following beneficial effects:

[0017] By extracting the scuticociliate membrane protein, the present invention prepares a vaccine with high immunogenicity. This method is simple and efficient, can effectively prevent or treat infections caused by scuticociliates, and has broad application prospects. Description of the Drawings

[0018] Figure 1 It is a microscopic image of the mucus at the wound of the control group turbot 48 h after virus challenge;

[0019] Figure 2 It is a microscopic image of the mucus at the wound of the experimental group turbot 48 h after virus challenge;

[0020] Figure 3 It is a picture of the turbot 48 h after virus challenge after abrasion. Detailed Embodiments

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0023] The present invention discloses a preparation method of a Scuticociliates membrane protein vaccine, comprising the following steps:

[0024] (1) Culturing and collecting Scuticociliates;

[0025] Inoculate Scuticociliates into L-15 medium containing 7% chicken serum (inoculation ratio: 1 mL of ciliate solution + 100 mL of medium, calculate the required volume of ciliate solution according to the concentration of 300 ind / μL of ciliate solution at the logarithmic growth phase), culture at 20 °C for 48 - 72 hours until reaching the logarithmic growth phase. Then centrifuge at 12000 rpm for 2 min to collect Scuticociliates cells, and wash 3 times with phosphate buffer (PBS) to remove the residual medium.

[0026] (2) Extracting the Scuticociliates membrane protein;

[0027] Suspend the collected Scuticociliates cells in lysis buffer (Lysis Buffer, add 1 μL of protease inhibitor and 1 μL of 1 M DTT to each milliliter of Lysis Buffer before use), centrifuge at 4 °C and 12000 rpm for 10 min to take the precipitate, add extraction buffer to the precipitate and centrifuge at 4 °C and 12000 rpm for 10 min to take the supernatant, and the supernatant is the membrane protein.

[0028] (3) Measuring the concentration of the membrane protein;

[0029] The concentration of the membrane protein is measured by the Bradford method. The operation process is as follows:

[0030] 1) Prepare a set of bovine serum albumin (BSA) solutions with concentrations of 0 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, and 1.5 mg / mL as protein standards;

[0031] 2) Take 5 μL of protein standards with different concentrations and add them to a 96-well plate;

[0032] 3) Take 5 μL of the sample (membrane protein to be measured) to the 96-well plate;

[0033] 4) Add 250 μL of G250 staining solution to each well;

[0034] 5) Measure the absorbance at 595 nm with an enzyme-linked immunosorbent assay (ELISA) reader, make a standard curve (linear) equation from the values measured for the above protein standards, and then substitute the value measured for the sample into the equation to calculate the concentration of the sample.

[0035] (4) Preparation and evaluation of immunization effect of the vaccine.

[0036] The vaccine is prepared by mixing the membranous protein of Aspidisca with an adjuvant. The concentration of the membranous protein of Aspidisca is 0.6 mg / mL, and the adjuvant is Freund's adjuvant or aluminum adjuvant with a concentration of 0.3 mg / mL. They are mixed in a volume ratio of 1:1 to prepare a vaccine preparation. Finally, through emulsification or freeze-drying technology, the vaccine is prepared into a dosage form suitable for storage and use.

[0037] The present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1

[0039] Preparation of the membranous protein vaccine of Aspidisca includes the following steps:

[0040] (1) Cultivation and collection of Aspidisca

[0041] Aspidisca is inoculated into 500 mL of L-15 medium containing 7% chicken serum and cultured at 20 °C for 60 hours, and then the cells are collected. Then, the cells are washed 3 times with cold PBS (pH 7.4), and the residual medium is removed by centrifugation at 12000 rpm for 2 min each time.

[0042] (2) Extraction of membranous protein

[0043] (2.1) Collect no less than 1×10 7 ind of Aspidisca cells. Add 1 mL of Lysis Buffer (add 1 μL of protease inhibitor and 1 μL of 1 M DTT to each milliliter of Lysis Buffer before use) to the cell sample, vortex for 30 s, place on ice for 1 min, and repeat 5 times. After cell disruption, microscopic examination should be carried out, and the cell disruption rate should be no less than 90%.

[0044] (2.2) Centrifuge at 12000 rpm for 10 min at 4 °C, and transfer the supernatant to a new centrifuge tube, which is the cytoplasmic protein, and aliquot and store frozen.

[0045] (2.3) Take the precipitate, add 200 μL of cold extraction Buffer, vortex to mix evenly for 30 s, and then place on ice for 5 min, and repeat 5 times.

[0046] (2.4) Centrifuge at 12000 rpm for 10 min at 4 °C, and take the supernatant and transfer it to a new centrifuge tube, which is the cell membrane protein.

[0047] (3) Determination of membranous protein concentration

[0048] The concentration of the extracted membranous protein is determined by the Bradford method, and the result is 1.8 mg / mL.

[0049] (4) Preparation of Vaccine

[0050] Dilute the extracted membrane protein to 0.6 mg / mL with PBS (pH 7.4). Mix the diluted membrane protein with Freund's adjuvant (0.3 mg / mL) at a volume ratio of 1:1 to prepare a vaccine preparation.

[0051] Finally, prepare the vaccine into a suitable dosage form for storage through emulsification technology. Specifically: perform blowing emulsification with syringes. After removing the needles of two syringes, tightly connect them with a rubber tube. One syringe sucks up the adjuvant in advance, and the other sucks up the membrane protein in advance, with the time controlled within 45 min - 60 min.

[0052] Specifically: Connect the needle positions of two 5 - mL syringes together with a 1.5 - cm rubber tube. The first pushing direction is from the membrane protein to the adjuvant, and then push back and forth. It should be noted here that the capacity of the syringe needs to be greater than the volume of the adjuvant + membrane protein to enable repeated pushing.

[0053] (5) Evaluation of Vaccine Immunization Effect

[0054] Inject the prepared vaccine into experimental animals (such as fish) and detect its protective effect.

[0055] The specific process is as follows: (1) Select turbot, divide the turbot into two groups. One group is the control group, injected with PBS + adjuvant; the other group is the experimental group, injected with membrane protein + adjuvant. The preparation methods of the vaccines injected into the two groups are the same; perform immunization twice successively. The second immunization is carried out 14 days after the first immunization, and each fish is injected with 0.1 mL of vaccine.

[0056] (2) Conduct an infection experiment 7 days after the second immunization. The infection method adopts the abrasion challenge method.

[0057] (2.1) Culture the parasite solution before challenge. Inoculate the parasite solution into 2 L of L - 15 medium containing 7% chicken serum for culture for 4 days.

[0058] (2.2) Make 2 wounds by abrasion at the same position on the tail of each fish, and the abrasion area of each wound is 1.5 cm × 1.5 cm.

[0059] (2.3) The challenge concentration is 3000 ind / mL.

[0060] The number of Uronema marinum on the wound mucus of the two groups of turbot at 24 h and 48 h after challenge is shown in Table 1.

[0061] Table 1 The number of Uronema marinum on the wounds of the two groups of turbot at 24 h and 48 h after infection

[0062]

[0063] Note: The data in Table 1 are the average number of ciliates infected in each group ± standard deviation, unit: ind / cm 2 。

[0064] The microscopic image of the mucus at the wound of the control group turbot 48 h after virus challenge is shown as Figure 1 follows. The scuticociliates in the figure are pointed out by arrows. The microscopic image of the mucus at the wound of the experimental group turbot 48 h after virus challenge is shown as Figure 2 follows. The scuticociliates in the figure are pointed out by arrows. The picture of turbot 48 h after virus challenge following abrasion is shown as Figure 3 follows. The results show that the number of ciliates infecting the wound of the experimental group turbot is significantly less than that of the control group, indicating that the prepared vaccine helps to improve the immune effect of turbot and makes it less susceptible to ciliate infection.

[0065] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a scuticociliate membrane protein vaccine, characterized in that: It is prepared by mixing scuticociliate membrane protein and an adjuvant, and the scuticociliate membrane protein is the membrane protein of scuticociliates extracted by the ultracentrifugation method.

2. The preparation method of a scuticociliate membrane protein vaccine according to claim 1, characterized in that: The extraction process of the scuticociliate membrane protein is as follows: The scuticociliates are cultured to the logarithmic growth phase, and the scuticociliate cells are collected by centrifugation at 12,000 rpm for 2 min. The ciliate cells are suspended in the lysis buffer and centrifuged at 4°C and 12,000 rpm for 10 min to obtain the precipitate. Extraction Buffer is added to the precipitate and centrifuged at 4°C and 12,000 rpm for 10 min to obtain the supernatant, and the supernatant is the membrane protein.

3. The preparation method of a scuticociliate membrane protein vaccine according to claim 2, characterized in that: The scuticociliates are inoculated into the L-15 medium containing 7% chicken serum and cultured at 20°C for 48 - 72 hours until the logarithmic growth phase is reached.

4. The preparation method of a scuticociliate membrane protein vaccine according to claim 2, characterized in that: 1 μL of protease inhibitor and 1 μL of 1 M DTT are added to each milliliter of the lysis buffer.

5. The preparation method of a scuticociliate membrane protein vaccine according to any one of claims 1 to 4, characterized in that: The concentration of the scuticociliate membrane protein is 0.6 mg / mL, and the concentration of the adjuvant is 0.3 mg / mL.

6. The preparation method of a scuticociliate membrane protein vaccine according to claim 5, characterized in that: The adjuvant is Freund's adjuvant or aluminum adjuvant.

7. The preparation method of a membranous protein vaccine of Aspidisca according to claim 6, characterized in that: The volume ratio of the scuticociliate membrane protein to the adjuvant is 1:

1.

8. A scuticociliate membrane protein vaccine prepared by the preparation method according to any one of claims 1 to 7.

9. Use of a scuticociliate membrane protein vaccine prepared by the preparation method according to any one of claims 1 to 7 in the prevention or treatment of scuticociliate infection.

10. The application according to claim 9, wherein: The scuticociliate membrane protein vaccine is prepared by mixing the scuticociliate membrane protein and the adjuvant at a volume ratio of 1:

1. The concentration of the scuticociliate membrane protein is 0.6 mg / mL, and the adjuvant is Freund's adjuvant or aluminum adjuvant with a concentration of 0.3 mg / mL.

Images