A membrane protein vaccine from scutiformis, its preparation method and application

Abstract

This invention relates to the field of biotechnology, specifically to a scutocilius membrane protein vaccine, its preparation method, and its application. The specific technical solution is as follows: a method for preparing a scutocilius membrane protein vaccine involves mixing scutocilius membrane protein with an adjuvant. The scutocilius membrane protein is extracted from the scutocilius membrane using ultracentrifugation. This invention prepares an immunogenic vaccine by extracting and purifying scutocilius membrane protein for the prevention or treatment of infections caused by scutocilius.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a scuticociliate membrane protein vaccine and a preparation method and application thereof. BACKGROUND

[0002] Scuticociliates are facultative parasites, which are currently recognized as the pathogen of sea water fish parasitic disease, widely exist in sea water environment, can infect a variety of aquatic animals, especially fish, and cause serious diseases and economic losses. At present, the prevention and control means for scuticociliate infection are limited, mainly relying on chemical drugs, but long-term use is easy to cause drug resistance, environmental pollution and food safety risk. Immunization has become an important way for fish disease prevention and control, and the development of a safe and effective scuticociliate vaccine has important application prospects in the disease prevention and control.

[0003] Membrane proteins play an important role in the immune response of pathogens, because they are located on the cell surface and are easily recognized by the host immune system. The present application extracts the membrane protein of scuticociliates to prepare a vaccine, which can induce specific immune response of the host, thereby effectively preventing or treating scuticociliate infection. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a scuticociliate membrane protein vaccine and a preparation method and application thereof, which extracts and purifies scuticociliate membrane protein to prepare a vaccine with immunogenicity for preventing or treating infection caused by scuticociliates.

[0005] To achieve the above object, the present application is realized by the following technical scheme:

[0006] The present application discloses a preparation method of a scuticociliate membrane protein vaccine, which is prepared by mixing scuticociliate membrane protein and adjuvant, wherein the scuticociliate membrane protein is extracted by ultracentrifugation.

[0007] Preferably, the extraction process of the scuticociliate membrane protein is as follows: the scuticociliates are cultured to logarithmic growth phase, centrifuged at 12000 rpm for 2 min to collect the scuticociliate cells, the ciliates are suspended in lysis buffer, centrifuged at 12000 rpm for 10 min at 4℃ to obtain the precipitate, and the extraction buffer is added to the precipitate, centrifuged at 12000 rpm for 10 min at 4℃ to obtain the supernatant, and the supernatant is the membrane protein.

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

[0009] Preferably, 1 μL protease inhibitor and 1 μL 1M DTT are added per mL of the lysis buffer.

[0010] Preferably, the concentration of the membrane protein of the platyhelminth 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 membrane protein of the platyhelminth to the adjuvant is 1:1.

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

[0014] Correspondingly, the platyhelminth membrane protein vaccine prepared by the preparation method is used for preventing or treating platyhelminth infection.

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

[0016] The present application has the following advantages:

[0017] The present application extracts the membrane protein of the platyhelminth to prepare a vaccine with high immunogenicity. The method is simple and efficient, can effectively prevent or treat infection caused by the platyhelminth, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a picture of the mucus at the wound of the scophthalmus maximus in the control group 48 hours after the challenge;

[0019] Figure 2 It is a picture of the mucus at the wound of the scophthalmus maximus in the experimental group 48 hours after the challenge;

[0020] Figure 3 It is a picture of the scophthalmus maximus 48 hours after the challenge after the wound. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

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

[0023] This invention discloses a method for preparing a scutiformis membrane protein vaccine, comprising the following steps:

[0024] (1) Culture and collection of shield ciliates;

[0025] Shield-ciliates were inoculated into L-15 medium containing 7% chicken serum (inoculation ratio: 1 mL sciliate culture + 100 mL medium; the required culture volume was calculated based on the sciliate concentration reaching 300 ind / μL at the logarithmic growth phase). The cells were cultured at 20°C for 48-72 hours until the logarithmic growth phase was reached. The cells were then collected by centrifugation at 12000 rpm for 2 min and washed three times with phosphate-buffered saline (PBS) to remove any residual culture medium.

[0026] (2) Extraction of membrane proteins from Shield-ciliates;

[0027] The collected Shield-ciliated ciliate cells were suspended in lysis buffer (Lysis Buffer, with 1 μL of protease inhibitor and 1 μL of 1M DTT added per mL of Lysis Buffer before use), and centrifuged at 4°C and 12000 rpm for 10 min to collect the precipitate. Extraction buffer was added to the precipitate, and the mixture was centrifuged at 4°C and 12000 rpm for 10 min to collect the supernatant, which is the membrane protein.

[0028] (3) Determination of membrane protein concentration;

[0029] The concentration of the membrane protein was determined using the Bradford method. The procedure 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) Add 5 μL of protein standards of different concentrations to a 96-well plate;

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

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

[0034] 5) Measure the absorbance at 595 nm using an ELISA reader, and use the values ​​obtained from the protein standard to create a standard curve (linear) equation. Then, substitute the measured values ​​of the sample into the equation to calculate the concentration of the sample.

[0035] (4) Preparation of vaccines and evaluation of immunization effects.

[0036] The vaccine is prepared by mixing shield-ciliate membrane protein with an adjuvant. The shield-ciliate membrane protein has a concentration of 0.6 mg / mL, and the adjuvant is Freund's adjuvant or aluminum adjuvant with a concentration of 0.3 mg / mL. The mixture is prepared into a vaccine formulation at a 1:1 volume ratio. Finally, the vaccine is prepared into a dosage form suitable for storage and use using emulsification or freeze-drying techniques.

[0037] The present invention will be further described below with reference to specific embodiments.

[0038] Example 1

[0039] The preparation of the shield-ciliate membrane protein vaccine includes the following steps:

[0040] (1) Culture and collection of shield ciliates

[0041] The scutellaria were inoculated into 500 mL of L-15 medium containing 7% chicken serum and cultured at 20°C for 60 hours. The cells were then collected. The cells were then washed three times with cold PBS (pH 7.4), centrifuged at 12,000 rpm for 2 min each time to remove residual medium.

[0042] (2) Extraction of membrane proteins

[0043] (2.1) Collect no less than 1×10 7 Take only one (ind) of Shield-ciliated ciliate cells, add 1 mL of LysisBuffer (add 1 μL of protease inhibitor and 1 μL of 1M DTT to each mL of LysisBuffer before use), vortex for 30 seconds, place on ice for 1 minute, repeat 5 times, and examine under a microscope after cell disruption. The cell disruption rate should not be less than 90%.

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

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

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

[0047] (3) Determination of membrane protein concentration

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

[0049] (4) Vaccine preparation

[0050] The extracted membrane proteins were diluted to 0.6 mg / mL with PBS (pH 7.4). The diluted membrane proteins were then mixed with Freund's adjuvant (0.3 mg / mL) at a volume ratio of 1:1 to prepare the vaccine formulation.

[0051] Finally, the vaccine is prepared into a suitable dosage form for storage using emulsification technology. Specifically: two syringes are used for back-blowing emulsification. After removing the needles, the syringes are tightly connected with tubing. One syringe is used to draw up the adjuvant in advance, and the other syringe is used to draw up the membrane protein in advance. The time is controlled between 45 and 60 minutes.

[0052] Specifically: Connect the needles of two 5mL syringes together with a 1.5cm tubing. The first push should be made from the membrane protein toward the adjuvant, and subsequent pushes should be repeated. Note that the syringe volume must be greater than the volume of the adjuvant plus the membrane protein to achieve repeated pushes.

[0053] (5) Evaluation of the immunization effect of the vaccine

[0054] The prepared vaccine is injected into experimental animals (such as fish) and its protective effect is tested.

[0055] The specific process is as follows: (1) Select turbot and divide turbot into two groups. One group is the control group, which is injected with PBS + adjuvant; the other group is the experimental group, which is injected with membrane protein + adjuvant. The vaccine preparation method is the same for both groups. Two immunizations are carried out in succession. The second immunization is carried out 14 days after the first immunization. Each fish is injected with 0.1 mL of vaccine.

[0056] (2) An infection experiment was conducted 7 days after the second immunization, using the abrasion challenge method.

[0057] (2.1) Before challenge, the worm fluid was inoculated into 2L of L-15 medium containing 7% chicken serum and cultured for 4 days.

[0058] (2.2) Each fish has two abrasions at the same location on its tail, and the abrasion area of ​​each abrasion is 1.5cm × 1.5cm;

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

[0060] The number of scutiformes in the wound mucus of turbot in the two groups 24 and 48 hours after infection is shown in Table 1.

[0061] Table 1. Number of scutiformis at the wound site in two groups of turbot 24 and 48 hours after infection.

[0062]

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

[0064] The image below shows the mucus at the wound site of turbot in the control group 48 hours after the virus challenge. Figure 1 As shown in the image, the scutellum ciliates are indicated by arrows. The microscopic image of the mucus at the wound site of the turbot in the experimental group 48 hours after infection is shown below. Figure 2 As shown in the image, the scutellum is indicated by an arrow. The image shows the result of 48 hours after trepanation and viral infection of a turbot. Figure 3 As shown in the figure. The results showed that the number of ciliates infecting the wounds of turbot in the experimental group was significantly less than that in 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 merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method of preparing a galea membrane protein vaccine of galea, characterized by: The galea membrane protein vaccine is prepared by mixing the galea membrane protein extracted by ultracentrifugation and an adjuvant, wherein the adjuvant is Freund's adjuvant or aluminum adjuvant. The galea membrane protein is extracted by the following process: inoculating galea into L-15 medium containing 7% chicken serum and culturing at 20℃ for 48-72 hours until the logarithmic growth phase is reached; collecting galea cells by centrifugation at 12000 rpm for 2 min; suspending the galea cells in lysis buffer, adding 1 μL protease inhibitor and 1 μL 1M DTT per milliliter of the lysis buffer; centrifuging the mixture at 4℃ and 12000 rpm for 10 min to obtain the precipitate; adding extraction buffer to the precipitate and centrifuging at 4℃ and 12000 rpm for 10 min to obtain the supernatant, which is the membrane protein; the concentration of the galea membrane protein is 0.6 mg / mL, and the concentration of the adjuvant is 0.3 mg / mL.

2. The method for preparing a scutiformis membrane protein vaccine according to claim 1, characterized in that: The volume ratio of the galea membrane protein to the adjuvant is 1:

1.

3. Use of the galea membrane protein vaccine prepared by the preparation method of claim 1 or 2 in the preparation of a drug for preventing galea infection.

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