CN108659116B - Immunogen synthesis method for preparing aquatic pathogenic bacterium aeromonas cross-type antibody - Google Patents
Immunogen synthesis method for preparing aquatic pathogenic bacterium aeromonas cross-type antibody Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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Abstract
The invention relates to an immunogen synthesis method for preparing an aquatic pathogenic bacterium aeromonas cross-type antibody, belonging to the technical field of immunoassay. The conserved polypeptide PepF1 on the OmpF of the Aeromonas Outer membrane protein (Omp) is chemically and covalently coupled with Bovine Serum Albumin (BSA) to synthesize the Aeromonas conserved polypeptide epitope antigen. Mouse serum or egg yolk antibody immunized by the immunogen under the specific polypeptide sequence PepF1 and coupling reaction conditions has potency on 3 strains of aeromonas hydrophila and 4 strains of aeromonas bacteria, and simultaneously has potency on common E.coli and E.coli O157: h7, eel vibrio, parahemolytic vibrio, Vibrio harveyi, wound vibrio, cholera vibrio, alternate pseudomonas and Edwardsiella tarda have no cross reaction. And the mouse serum or yolk antibody immunized by the thallus has cross reaction to the common E.coli CICC21530 and DH5 alpha. The novel immunogen can be used for preparing an aquatic pathogenic bacterium aeromonas cross monoclonal antibody or an ovomyosin.
Description
Technical Field
The invention relates to an immunogen synthesis method for preparing an aquatic pathogenic bacterium aeromonas cross-type antibody, belonging to the technical field of immunoassay.
Background
Aeromonas hydrophila is one of the main pathogenic bacteria of fish culture septicemia, and brings huge economic loss to freshwater fish and aquaculture industry. Aeromonas hydrophila is widely distributed in various water bodies in the nature, is a primary pathogenic bacterium of various aquatic animals, is a conditional pathogenic bacterium, and is a typical pathogenic bacterium of human-animal-fish co-morbidity. Aeromonas hydrophila can produce highly toxic exotoxins such as: hemolysin, histotoxin, necrotoxin, enterotoxin and protease cause diseases of aquatic animals. In actual production, there are many fulminant hemorrhagic diseases caused by infection with Aeromonas hydrophila, and the symptoms vary depending on the serotype of Aeromonas hydrophila and the subject of infection. Such as silver carp fulminant hemorrhagic disease, turtle septicemia, eel hemorrhagic disease, eel red fin disease, etc. The bacterium is a conditional pathogen, and when the environment suddenly changes and the water quality deteriorates, the bacterium is often mixed with other bacteria (such as aeromonas sobria, vibrio and the like) to infect so as to aggravate the disease. Diseases infected by Aeromonas hydrophila are generally violent in disease symptoms, are mostly malignant infectious diseases, and have high mortality rate.
Currently, the main means of controlling aeromonas hydrophila disease include antibiotics and vaccines. At present, the disease prevalence and antibiotic abuse of poultry and aquaculture industry in China are common, so that the problem of antibiotic residue in meat, fish and environment is prominent, and great threats are brought to food safety, export trade, environmental protection and public health in China. The total amount of Chinese antibiotics used in 2013 is reported to be 16.2 ten thousand tons, 48% of which are human antibiotics and the rest are veterinary antibiotics. While antibiotics for human use are mostly used in the treatment of diseases and are on the decline, antibiotics for veterinary use are mostly used in feed. Because the veterinary antibiotics are low in price, a plurality of farmers do not use the antibiotics in order to prevent diseases. After the cattle, sheep, chickens, ducks, fishes and the like eat the veterinary antibiotics, the veterinary antibiotics enter water, soil and crops through excretion, and finally react with human beings to cause the problems of drug-resistant bacteria and 'super bacteria', so that a malignant ecological chain circulation system is formed.
Meanwhile, because the pathogenic bacteria have various serotypes, the common bacterial vaccines have great difference in efficacy, and often have good effect on immune strains, but have no effect or general effect on other serotypes. The recombinant protein vaccine based on the conservative outer membrane protein has a good effect, but the problems that the vaccination approach is mainly fish-by-fish injection immunization, the time and labor are wasted, the cost is high, the fish death is easy to cause and the like are faced; moreover, the veterinary vaccine approval process is very complex, usually requires 5-10 years, and can not meet the market demand by matching with a pharmaceutical-grade GMP (good Manufacturing practice) production workshop.
The yolk globulin (IgY) as food component and active immune recognizing matter has green, broad spectrum and continuous aquatic disease controlling effect. The poultry source antibody is immunoglobulin which is obtained by immunizing laying poultry with a specific antigen and has antigen specificity and rake tropism from yolk of the immunized poultry egg in a physical mode, and simultaneously has better edible safety as a food raw material; the FDA in the united states has considered aviary antibodies as the first choice "drug" to replace antibiotics and has recognized that aviary antibodies are more advantageous as food than as drugs and is classified in the category of "Generally Accepted as Safe (GRAS)". Compared with the traditional control method which adopts antibiotics and vaccines to prevent and treat related diseases, the poultry-derived antibody has the following characteristics: (1) high efficiency and strong pertinence, and only inhibits corresponding pathogeny by utilizing antigen-antibody specific immunoreaction. (2) The egg yolk antibody is green and safe, is an egg yolk component, has no side effect on animals and human bodies, is degradable in the environment, and has no residue problem. (3) Compared with vaccines, the vaccine has the advantages of simplified research and development and application programs, simple use method in prevention and treatment of specific pathogens, low use cost due to the fact that the vaccine is usually used in oral administration, spraying and other modes. (4) The application range is wide, and the yolk antibody provides a new choice for developing functional foods on a large scale, diagnosing diseases, and preventing and treating bacterial infection and virus infection. (5) Easy to obtain and has good persistence, can immunize live chickens after obtaining pathogenic strains or conservative antigens, and has long egg laying period and high yield. The uniform identification and specificity of the high-immunity chicken yolk globulin (IgY) to aquatic pathogenic bacteria lay a foundation for biological identification, proliferation inhibition and green prevention and control of aquatic diseases by using the IgY as a feed additive.
Disclosure of Invention
The invention aims to provide an immunogen synthesis method for preparing an aquatic pathogenic bacterium aeromonas cross antibody, and provides a method and an idea for preparing a broad-spectrum monoclonal antibody and a yolk antibody in aquatic pathogenic bacterium aeromonas hydrophila.
In order to achieve the purpose, the invention adopts a conservative polypeptide epitope PepF1 of an Aeromonas outer membrane protein OmpF with Cys modified at the N end as an antigen, and synthesizes the Aeromonas polypeptide antigen and an artificial antigen of a carrier protein BSA by an SMCC method.
The types of the aeromonas surface antigens are various, the specific structures of the aeromonas surface antigens are also variable due to different bacterial strain serotypes, and the conservation and the surface exposable polypeptide epitope are the basis for preparing the aeromonas specific antibody. However, the molecular weight of polypeptide antigens is usually between 1000-2000, which belongs to molecular haptens, and cannot be directly immunized. After the bifunctional coupling agent SMCC is coupled with carrier protein BSA, the antibody has immunogenicity, and can be prepared.
The Aeromonas cross type mouse antibody or yolk antibody can be obtained by immunizing BALB/c mice and laying hens (Roche chickens) with the Aeromonas conserved polypeptide epitope PepF1-SMCC-BSA immunogen.
A method for synthesizing immunogen for preparing aquatic pathogenic bacterium Aeromonas cross-type antibody comprises the following steps:
(1) modification of the polypeptide PepF1: modifying cysteine Cys at the N end of the OmpF conserved polypeptide PepF1 of the aeromonas outer membrane protein obtained by screening;
(2) derivatization of BSA: reacting a bifunctional coupling agent 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid Sulfo succinimide ester sodium salt Sulfo-SMCC with bovine serum albumin BSA according to a reaction molar ratio of 40-80:1 in 0.05-0.15M/L phosphate buffer solution at room temperature for 1-2h to obtain SMCC-BSA, and removing redundant Sulfo-SMCC by ultrafiltration;
(3) preparation of immunogen: reacting the polypeptide PepF1 obtained in the step (1) with the derivative BSA obtained in the step (2) for 2-6h at room temperature in 0.05-0.15M/L phosphate buffer solution according to the reaction molar ratio of 30-60:1, and dialyzing to obtain the immunogen PepF1-SMCC-BSA of the aeromonas cross-type antibody.
The Aeromonas outer membrane protein OmpF conserved polypeptide PepF1 is specifically Tyr Lys Gly Glu Gly Arg Gly Tyr Glu Leu Ala Ala.
The immunogen synthesis method comprises the following specific steps: dissolving 10mg of carrier protein BSA in 0.1M PB buffer solution, adding a bifunctional coupling agent Sulfo-SMCC dissolved in N, N-Dimethylformamide (DMF) into a BSA solution according to a reaction molar ratio of Sulfo-SMCC: BSA of 40-80:1, and reacting at room temperature for 1-2h under magnetic stirring; after the reaction is finished, carrying out ultrafiltration for 2-4 times by using a Millipore ultrafiltration tube to obtain SMCC-BSA, and removing unreacted bifunctional coupling agent; adding 0.05-0.15M/L PB buffer solution dissolved Aeromonas conserved polypeptide epitope PepF1 with cysteine Cys modified at the N-terminal into the reaction solution according to the molar ratio of the polypeptide PepF1: BSA of 30-60:1, and reacting at room temperature for 2-6h under magnetic stirring; and then dialyzing the reaction solution by using 0.01-0.05M/L PBS, replacing the dialyzate once for 4-6h, and dialyzing for 2-3 days to obtain the immunogen PepF1-SMCC-BSA of the Aeromonas cross-type antibody.
The invention has the beneficial effects that: the invention provides a preparation method of immunogen for preparing specific mouse antibody or yolk antibody with wide internal spectrum and external specificity of aquatic disease bacterium aeromonas; the invention screens the conserved amino acid sequence PepF1 of Aeromonas on the outer membrane protein OMPF. And the polypeptide PepF1-SMCC-BSA artificial immunogen is synthesized for the first time, the artificial polypeptide antigen PepF1-SMCC-BSA is used for immunizing laying hens and BALB/c mice, and indirect ELISA tests show that the immunized egg yolk antibody IgY and mouse serum have titer on 3 strains of aeromonas hydrophila (CICC 10500, MCCC 1A00190, MCCC 1A 0007) and 4 strains of aeromonas bacteria (aeromonas bourbonis, aeromonas spot, aeromonas jodongpensis and aeromonas bivalve) which are tested, and meanwhile, the artificial immunogen has titer on common E.coli and E.coli O157: h7, eel vibrio, parahemolytic vibrio, Vibrio harveyi, wound vibrio, cholera vibrio, alternate pseudomonas and Edwardsiella tarda have no cross reaction and good specificity. And the mouse serum and the yolk antibody which directly immunize the aeromonas hydrophila thallus have relatively serious cross reaction on the common E.coli CICC21530 and DH5 alpha, which shows that the polypeptide immunogen adopted by the invention has the advantages of conservation and specificity. The uniform identification and specificity of the high-immunity chicken ovovitellin (IgY) in aeromonas and the safety of the IgY serving as a food component lay a foundation for green prevention and control of aquatic disease septicemia.
Drawings
FIG. 1 is a polyacrylamide gel electrophoresis (SDS-PAGE) characterization of the synthetic PepF1-SMCC-BSA artificial antigen. 1, BSA; 2. BSA-SMCC; 3. PepF 1-SMCC-BSA;
FIG. 2 is a cross reaction of synthetic PepF1-SMCC-BSA artificial antigen immunized mouse serum with 3 strains of Aeromonas hydrophila, 4 strains of Aeromonas bacteria.
3 of them (1, ASP CICC 10500, 4, ASP MCCC 1A00190, 2, ASP MCCC 1A 0007) and 4 of bacteria of the genus Aeromonas (5, Aeromonas boldii, 3, Aeromonas sporum, 6, Aeromonas Zygdonovani, 7, Aeromonas bivalvensis).
Detailed Description
The aeromonas strains described in the examples were purchased from the China center for Industrial microbiological Collection center (CICC), the China Center for General Microbiological Collection Center (CGMCC), and the China center for Marine microbiological Collection (MCCC). The polypeptide sequence was synthesized by Biotechnology engineering (Shanghai) GmbH.
The invention is further illustrated by the following examples.
The instrument comprises the following steps: table high speed refrigerated centrifuge, hokken instruments & equipments ltd; ELGA ultra pure water machine, alder, uk; WD-9405B type horizontal shaking table, Beijing, six instruments; a 96-hole 8X 12 high-adsorption detachable enzyme label plate, Wuxi Guosheng bioengineering Co., Ltd; multiskan FC microplate reader, Thermo Fisher Scientific; 200 μ L, 1mL pipette, 300 μ L of an 8-channel pipette, Thermo Labsystems Inc.; XW-80A vortex mixer, Shanghai Qingpu Huxi apparatus, Millipore Ultrafiltration tube (Cut off: 10000) available from Millipore-Merck.
Reagent: tetramethylbenzidine (TMB), Sigma reagent ltd; all other reagents were analytically pure reagents.
The method comprises the following steps:
1. screening and design of polypeptide sequences: the outer membrane protein is an important structure of gram-negative bacteria cell membrane, wherein the outer membrane protein OmpF is an important adhesion factor of aeromonas hydrophila and is a key component of a two-component regulation system which is crucial to the survival of the bacteria under adverse conditions. Meanwhile, the homology of the Aeromonas hydrophila outer membrane protein OmpF in the Aeromonas hydrophila is more than 83%. OmpF is a promising immune target. Through the comparison of an amino acid sequence of an OmpF protein of Aeromonas, the antigenicity, alpha-helix, beta-folding region, hydrophilicity, hydrophobicity and surface exposure of the OmpF amino acid sequence are analyzed by combining a bioinformatics tool, a polypeptide sequence with better conservation and surface exposure is selected as a candidate antigen, and the length of the polypeptide sequence is generally 10-20 AA. And selecting the N-terminal or C-terminal modified cysteine Cys for subsequent coupling.
2. Preparation of immunogen: 10mg of the carrier protein BSA was dissolved in 0.1M PB buffer, and Sulfo-SMCC dissolved in N, N-Dimethylformamide (DMF) was added to the BSA solution at a reaction molar ratio of 70:1, and reacted at room temperature for 1 hour under magnetic stirring. After the reaction, the reaction mixture was ultrafiltered 3 times using a Millipore ultrafilter tube (Cut off:10000 MW) to remove the unreacted coupling agent. 0.1M PB buffer solution dissolved Aeromonas conserved polypeptide epitope PepF1 is added into the reaction solution according to the reaction molar ratio of 60:1, and the mixture is magnetically stirred and reacted for 1 hour at room temperature. And then dialyzing the reaction solution by using 0.01M PBS, replacing the dialyzate once after 6 hours, and dialyzing for 2 days to obtain the immunogen of the aeromonas cross-type antibody.
3. Preparation of immune serum or yolk antibody
(1) Experimental animals: 6, 8-week-old BALB/c mice and 6, 20-week-old Roche layers were selected for immunization.
(2) Antigen preparation: diluting the immunogen with physiological saline to prepare a solution with a required concentration;
(3) emulsification: emulsifying the solution with equal amount of complete or incomplete Freund's adjuvant by mixing and stirring, and injecting into mouse or Roche egg chicken subcutaneously at multiple points after complete emulsification;
(4) the immunization method comprises the following steps: mice were immunized according to the following immunization protocol: 80 μ g of the first immune, 80 μ g of the second immune and 40 μ g of the third immune. Immunizing the laying hens according to the following immunization process: 500 μ g of primary immune, 500 μ g of secondary immune and 300 μ g of tertiary immune. 3, after the immunization, measuring the titer of the bacteria in the aeromonas and other bacteria outside the aeromonas by using an indirect enzyme-linked immunosorbent assay (ELISA) method;
(5) collecting blood of a white mouse: performing tail-breaking blood collection 1 week after the second and third immunizations; egg laying chicken IgY: collecting eggs 1 week after the second and third immunizations, and separating yolk to prepare IgY crude extract. Antiserum titers and cross-reactions were determined by indirect ELISA.
4. The ELISA reaction process comprises the following steps:
(1) antibody titer determination step: the test bacterial solution was boiled for inactivation and coated with 96-well elisa plates using coating buffer as serial dilution, 100 μ L/well, in a refrigerator at 4 ℃ overnight. The next day, the microplate was taken out and returned to room temperature, 200. mu.L of PBST solution was injected into each well, the shaking table was shaken for 3min, the washing solution was forcibly thrown off, patted dry on absorbent paper, and washing was continued for 2 times. The following washing methods were the same;
(2) after full washing, sealing the enzyme label plate by using a sealing buffer solution, incubating for 2h in an incubator at 37 ℃, taking out and drying for later use;
(3) serially diluting the positive serum and correspondingly adding the diluted positive serum into the first 7 ranks of the ELISA plate, adding the negative serum into the 8 th rank of the ELISA plate, incubating at the temperature of 37 ℃ for 1h, washing and patting dry;
(4) adding 100 mu L of HRP-labeled goat-anti-mouse secondary antibody diluted at 1:3000 or HRP-labeled goat-anti-chicken secondary antibody diluted at 1:3000 into each hole, incubating at 37 ℃ for 1h, washing, and patting dry;
(5) adding 100 μ L of color developing solution (TMB/substrate solution ratio is 1:5) into each well, reacting at 37 deg.C in dark for 15 min, taking out, adding 50 μ L of stop solution (2 mol/L sulfuric acid) into each well, and measuring light absorption value A with enzyme-labeling instrument450. The specific results are shown in FIG. 2.
The strain comprises 3 strains of aeromonas hydrophila (CICC 10500, MCCC 1A00190 and MCCC 1A 0007) and 4 strains of aeromonas bacteria (aeromonas bordetella, aeromonas spot, aeromonas Zhaodong and aeromonas bivalve).
Claims (2)
1. A method for synthesizing immunogen for preparing aquatic pathogenic bacterium Aeromonas cross-type antibody is characterized by comprising the following steps:
(1) modification of the polypeptide PepF1: modifying cysteine Cys at the N end of the OmpF conserved polypeptide PepF1 of the aeromonas outer membrane protein obtained by screening;
the Aeromonas outer membrane protein OmpF conserved polypeptide PepF1 is specifically Tyr Lys Gly Glu Gly Arg Gly Tyr Glu Leu Ala Ala;
(2) derivatization of BSA: reacting a bifunctional coupling agent 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid Sulfo succinimide ester sodium salt Sulfo-SMCC with bovine serum albumin BSA according to a reaction molar ratio of 40-80:1 in 0.05-0.15mol/L phosphate buffer solution at room temperature for 1-2h to obtain SMCC-BSA, and removing redundant Sulfo-SMCC by ultrafiltration;
(3) preparation of immunogen: reacting the polypeptide PepF1 obtained in the step (1) with the derivative BSA obtained in the step (2) for 2-6h at room temperature in 0.05-0.15mol/L phosphate buffer solution according to the reaction molar ratio of 30-60:1, and dialyzing to obtain the immunogen PepF1-SMCC-BSA of the aeromonas cross-type antibody.
2. The method for synthesizing the immunogen for preparing the cross-type antibody of the aquatic pathogenic bacterium aeromonas according to the claim 1 is characterized by comprising the following steps:
dissolving 10mg of carrier protein BSA in 0.1M PB buffer solution, adding a bifunctional coupling agent Sulfo-SMCC dissolved in N, N-dimethylformamide DMF into a BSA solution according to a reaction molar ratio of Sulfo-SMCC to BSA of 40-80:1, and reacting at room temperature for 1-2h under magnetic stirring; after the reaction is finished, ultra-filtering for 2-4 times by using an ultra-filtration tube to obtain SMCC-BSA, and removing unreacted bifunctional coupling agent; adding 0.05-0.15mol/L of PB buffer solution dissolved Aeromonas conserved polypeptide PepF1 of cysteine Cys modified by N-terminal into the reaction solution according to the molar ratio of the polypeptide PepF1: BSA of 30-60:1, and reacting at room temperature for 2-6h under magnetic stirring; and then dialyzing the reaction solution by using 0.01-0.05 mol/L PBS, replacing the dialyzate once for 4-6h, and dialyzing for 2-3 days to obtain the immunogen PepF1-SMCC-BSA of the Aeromonas cross-type antibody.
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