CN116731149B - Grass carp complement activation molecule carbon terminal peptide C5a-CP and application - Google Patents
Grass carp complement activation molecule carbon terminal peptide C5a-CP and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
Abstract
The invention belongs to the field of immunology, and particularly relates to a grass carp complement activation molecule C5a-CP and application thereof. The invention takes grass carp mixed immunized by C5a-CP and inactivated aeromonas hydrophila as an experimental group, detects the concentration and titer change of IgM of the grass carp after immunization, then adopts aeromonas hydrophila to infect the immunized grass carp, and detects the tissue bacterial load and survival rate of different groups of grass carp. The result shows that the concentration and titer of the IgM of the grass carp in the experimental group after immunization are obviously higher than those of the control group, the bacterial load of the grass carp tissue in the experimental group after the aeromonas hydrophila attacks the virus is obviously lower than that of the control group, and the immune protection rate is higher than that of the control group, so that the novel grass carp vaccine adjuvant and the vaccine related by the invention can obviously improve the antibody level in the grass carp body, can continuously and obviously improve the immune effect of the vaccine, and provide stronger immune protection for the fish body.
Description
Technical Field
The invention belongs to the field of immunology, and particularly relates to a grass carp complement activation molecule C5a-CP and application thereof.
Background
As the aquaculture environment deteriorates, the disease problem of aquaculture continues to increase, and it has become a significant challenge for aquaculture. Research shows that fish has a perfect immune system, so that development of a high-efficiency vaccine and improvement of disease resistance of fish body by means of immunoprophylaxis are key to solving disease problems in the future. The vaccine adjuvant is a substance capable of non-specifically changing or enhancing the specific immune response of an organism to an antigen and playing an auxiliary role, can induce the organism to generate a long-term and efficient specific immune response, improves the organism protection capability, can reduce the dosage of immune substances, reduces the production cost of the vaccine, and has almost the same importance as the vaccine to a certain extent. However, the types of adjuvants permitted to be used in commercial fish vaccines have been limited so far, and furthermore, among the vaccine adjuvants for fish permitted to be used, in particular, oil-based adjuvants such as Freund's adjuvant have been proved to have adverse side effects on fish bodies. Therefore, development of a novel, safe and efficient vaccine adjuvant for fishing is urgent.
C5a is a small molecule generated in the activation process of the complement system, and researches show that the teleosts C5a molecule has chemotactic function and immunoregulatory activity, for example, the C5a molecule of rainbow trout (Oncorhynchus mykiss) can chemotaxis white blood cells and enhance respiratory burst of the white blood cells. The fusion expression of the rainbow trout C5a and the antigen protein can be used as a molecular adjuvant to enhance the immunogenicity of the antigen. However, since teleosts have 3 pairs of disulfide bonds in the C5a molecule, it is extremely difficult to express and purify the active whole grass carp C5a molecule in a soluble form, and the chemical synthesis of the whole full-length molecule is time-consuming and laborious and costly. Based on the research, we creatively design and synthesize the carbon end alpha-helical peptide of grass carp C5a, namely a section of polypeptide with only 37 amino acids, to develop molecular adjuvant function research, and find that the section of polypeptide has strong molecular adjuvant function, has the obvious advantages of small dosage, obvious effect, low synthesis cost, easy acquisition and the like, and can be widely popularized and used.
Therefore, the invention focuses on developing a novel, safe, high-efficiency grass carp vaccine adjuvant which is easy to obtain at low cost in production, helping to prevent grass carp diseases, and promoting the development of immunity prevention and control technology of grass carp infectious diseases taking vaccination as a core.
Disclosure of Invention
Aiming at the problems of poor effect, low safety coefficient, large side effect and the like of grass carp vaccine adjuvants in the prior art, the invention provides a grass carp complement activation molecule carbon terminal peptide C5a-CP, wherein the terminal peptide is shown as SEQ ID NO. 1.
The invention also aims to provide application of the grass carp complement activation molecule carbon terminal peptide C5a-CP in preparing vaccine adjuvants, and the terminal peptide C5a-CP can obviously enhance the immune response of grass carp after combined immunization of grass carp vaccines, thereby providing stronger and more durable immune protection.
In order to achieve the above object, the present invention adopts the following technical measures:
a grass carp complement activating molecule C5a-CP carbon end peptide is shown in SEQ ID NO. 1.
Polynucleotides encoding the carbon-terminal peptides are also within the scope of the invention.
Substances which express the protein shown in SEQ ID No.1, including but not limited to expression cassettes, recombinant vectors or recombinant microorganisms comprising the polynucleotide encoding SEQ ID No.1, are also within the scope of the present invention.
The application of the carbon-terminal peptide, the polynucleotide or the substance expressing the protein shown in SEQ ID NO.1 in preparing grass carp vaccine adjuvant.
In the above application, preferably, the grass carp vaccine is a grass carp aeromonas hydrophila (Aeromonas hydrophila) inactivated vaccine.
Compared with the prior art, the invention has the following advantages:
1. the invention effectively intercepts the C5a carbon-terminal peptide, and a section of polypeptide with only 37 amino acids, overcomes the problem that the C5a is difficult to apply in actual production, and is easy to obtain by artificial synthesis or microbial fermentation.
2. The applicant develops a molecular adjuvant function research aiming at the truncated polypeptide for the first time, and discovers that the polypeptide has a powerful molecular adjuvant function, has the obvious advantages of small dosage, obvious effect, low synthesis cost, easy acquisition and the like, and can be widely popularized and used.
Drawings
FIG. 1 is a graph showing the change in IgM concentration in serum after immunization of grass carp groups as determined by ELISA in example 2 of the present invention;
wherein ∈r represents P <0.05, ++.r represents P <0.01, +.r represents P <0.001.
FIG. 2 is a graph showing the changes in the titers of antigen-specific IgM antibodies in serum after immunization of different groups of grass carp using ELISA in example 2 of the present invention;
wherein ∈r represents P <0.05, ++.r represents P <0.01, +.r represents P <0.001.
FIG. 3 shows the results of measuring the bacterial load in the head kidney and spleen tissues of grass carp of different immune groups 72 hours after challenge infection with Aeromonas hydrophila in example 3 of the present invention;
wherein ∈r represents P <0.05, ++.r represents P <0.01, +.r represents P <0.001.
FIG. 4 shows the results of ELISA for determining IgM antibody concentration and titer in various immunized groups of grass carp infected with Aeromonas hydrophila in example 3 of the present invention;
wherein ∈r represents P <0.05, ++.r represents P <0.01, +.r represents P <0.001.
FIG. 5 shows the calculation of the relative immunoprotection rate of each group by continuously monitoring the number of deaths in different immunized groups of grass carp after Aeromonas hydrophila infection in example 3 of the present invention.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention. The technical scheme of the invention is conventional in the field unless specifically stated otherwise, and the reagents or materials are commercially available unless specifically stated otherwise.
Example 1:
synthesis of grass carp C5a carbon-terminal polypeptide (C5 a-CP)
C5a-CP was designed and synthesized from the amino acid sequence of grass carp complement component C5a disclosed on NCBI (https:// www.ncbi.nlm.nih.gov /), synthesized by Jier Biochemical (Shanghai) Inc., and the polypeptide with a purity of greater than 95% was selected by mass spectrometry and liquid chromatography analysis, the amino acid sequence of the polypeptide being: EKTTDYWKKRCQRAFLECCEFAIKLRKDSVDKIILSR (SEQ ID NO. 1).
Example 2:
application of grass carp C5a carbon terminal polypeptide (C5 a-CP) in preparation of vaccine adjuvant
(1) Experimental animal
Grass carp used in the experiment has a weight of 20+/-5 g, is purchased from a Wuhan and Huang farm, is temporarily cultured at a culture base of the university of agricultural aquatic products in China, and is provided with a constant-temperature culture system. Feeding for 1 time every day in the temporary culture period, oxygenating for 24 hours, feeding the grass carp with daily feeding quantity being 2% of the mass of the grass carp, and selecting healthy grass carp for experiment after two weeks of continuous temporary culture.
(2) Immune grass carp and sampling
Inactivated aeromonas hydrophila is selected as an immune antigen, grass carp immunized by adding C5a-CP is taken as an experimental group, grass carp immunized by not adding C5a-CP is taken as a negative control group, grass carp immunized by only PBS is taken as a blank control group, and intraperitoneal injection immunization (200 mu L/tail) is carried out on the grass carp (200+/-20 g), wherein 60 grass carp are taken as each group. The Aeromonas hydrophila is inactivated by heat treatment (65deg.C, 1 h), and the concentration of the inactivated Aeromonas hydrophila by injection is 2×10 8 cfu/tail, C5a-CP was used in an amount of 0.12. Mu.g/tail. Immunization protocols are shown in table 1. Taking peripheral blood from 0 th, 7 th, 14 th, 21 st and 28d th after immunization, standing at 4 ℃ overnight after the blood is coagulated, centrifuging at 3000r/min and 4 ℃ for 10min, and sucking the supernatant into a new centrifuge tube to obtain grass carp serum.
TABLE 1 grass carp immunization protocol
(3) Immune effect detection
(1) IgM concentration detection
IgM antibody concentrations in the serum of grass carp of different immune groups were detected by ELISA. Diluting serum 40 times by using alkaline coating liquid, coating ELISA plates, incubating overnight at 4 ℃, washing 5 times, sealing for 2 hours (250 mu L/hole) by using 5% skimmed milk powder at 37 ℃, and washing the plates; mu.L of mouse anti-grass carp IgM monoclonal antibody (1:4000 dilution) is added to each hole, and the plates are washed after incubation for 1.5h at 37 ℃; 100 mu L of goat anti-mouse-HRP enzyme-labeled antibody (Proteintech) is added to each hole, and the plates are washed after incubation for 1h at 37 ℃; 100. Mu.L TMB color development solution (Biosharp) was added to each well, developed for 30min in the dark, and finally 50. Mu.L 2mol/L H was added to each well 2 SO 4 The stop solution stops the chromogenic reaction and is detected 450 using a microplate readerAbsorbance at nm. OD was measured using purified IgM at known concentrations as standard 450 The values are converted to IgM concentration.
The IgM concentration changes after immunization are shown in fig. 1, and serum IgM concentrations of grass carp in different groups on different days are shown in table 1, wherein IgM data of one non-immunized grass carp are shared on day 0. The results show that the serum IgM concentration of grass carp in the experimental group is significantly increased compared with the negative control group and the blank control group, which indicates that the addition of C5a-CP can stimulate the fish body to generate a strong immune response.
TABLE 1 serum IgM concentration of grass carp after immunization (μg/mL)
Note that: each data represents the average value of 4 fish (2) IgM titer assays
The titer of IgM antibodies in grass carp serum was detected by ELISA. Resuspension of inactivated Aeromonas hydrophila (final concentration 1×10) with alkaline coating solution 9 cfu/mL) was coated on 96-well plates, 100 μl per well, incubated overnight at 4deg.C, washed 5 times the next day, blocked with 5% nonfat dry milk at 37deg.C for 2h (250 μl per well), and the plates were washed; 100 mu L of serum diluted 40 times with PBS is added to each well, and the plates are washed after incubation for 3 hours at 37 ℃; mu.L of mouse anti-grass carp IgM monoclonal antibody (1:4000 dilution) is added to each hole, and the plates are washed after incubation for 1.5h at 37 ℃; adding 100 mu L of goat anti-mouse-HRP enzyme-labeled antibody into each hole, incubating at 37 ℃ for 1h, and washing the plate; adding 100 μL TMB color development liquid into each hole, developing for 30min in dark, and adding 50 μL 2mol/LH into each hole 2 SO 4 The stop solution stops the color reaction, and the absorbance at 450nm wavelength is detected using an enzyme-labeled instrument.
The IgM titer change after immunization is shown in fig. 2, where IgM data from one non-immunized grass carp was shared at day 0. Compared with the negative control group and the blank control group, the serum IgM titer of grass carp in the experimental group is obviously increased, and the rising trend is always shown in the detection time period, which shows that the addition of the C5a-CP adjuvant can continuously stimulate the fish body to generate strong specific antibody response.
TABLE 2 detection of serum IgM titres of grass carp after immunization (OD 450 value)
Note that: each data represents the average of 4 fish measurements example 4:
tissue bacterial load detection and relative immune protection rate calculation after aeromonas hydrophila infection
At 23d after immunization, 30 healthy grass carp were selected for aeromonas hydrophila challenge (8×10) 6 cfu/mL, 100. Mu.L/tail), 72h after challenge, head and kidney, liver and blood were taken from each group of surviving grass carp (4 tail/group) under aseptic conditions, and the determination of tissue-borne bacteria and the determination of antibody concentration and titer in serum after infection were performed, respectively.
Each tissue was weighed and homogenized in PBS using a tissue mill, and the tissue homogenate was serially diluted with PBS and smeared on aeromonas selective medium (hopebio). After incubation at 28℃for 12-16h, single colonies of Aeromonas hydrophila present on the plates were counted.
Antibody concentration and titer determination of IgM in serum was the same as in example 2.
After the toxicity is attacked, the survival number of fish bodies is continuously monitored, the relative immune protection rate is calculated, and the relative immune protection rate is calculated in the following way: rps= (1-immune group mortality/control group mortality) ×100%
The condition of the bacteria-carrying amount of the head kidney and the spleen tissue is shown in figure 3, and the result shows that the bacteria-carrying amount of the head kidney and the spleen tissue of the grass carp in the experimental group is obviously lower than that in the negative control group and the blank control group; the concentration and titer of IgM antibodies in serum after virus challenge are shown in figure 4, which shows that when immunized grass carp is infected with aeromonas hydrophila again, compared with a control group, the concentration and titer of IgM antibodies in grass carp serum of an experimental group are obviously higher, and stronger immune protection can be generated; the result of continuously monitoring the death number of the grass carp after the toxicity attack is shown in fig. 5, and the result shows that the relative immune protection rate of the grass carp in the experimental group reaches 96.3 percent and is obviously higher than that of the negative control group (80.07 percent) and the blank control group.
Claims (7)
1. A grass carp complement activating molecule C5a-CP, the carbon end peptide is shown in SEQ ID NO. 1.
2. A polynucleotide encoding the carbon-terminal peptide of claim 1.
3. A substance expressing the protein shown in SEQ ID NO.1, wherein the substance is an expression cassette, a recombinant vector or a recombinant microorganism containing the polynucleotide encoding SEQ ID NO. 1.
4. Use of the carbon-terminal peptide of claim 1 for the preparation of a grass carp vaccine adjuvant.
5. Use of the polynucleotide of claim 2 in the preparation of a grass carp vaccine adjuvant.
6. Use of the substance of claim 3 for the preparation of a grass carp vaccine adjuvant.
7. The use according to claim 4, 5 or 6, wherein the grass carp vaccine is aeromonas hydrophilaAeromonas hydrophila) And (5) inactivating the vaccine.
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