CN108659116A - A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody - Google Patents

Abstract

The invention relates to an immunogen synthesis method for preparing the cross-type antibody of the aquatic pathogen Aeromonas, belonging to the technical field of immune analysis. It uses the conserved peptide PepF1 on OmpF of the outer membrane protein (Omp) of Aeromonas to chemically covalently couple with bovine serum albumin (BSA) to synthesize the conserved peptide epitope of Aeromonas antigen. Under the conditions of specific polypeptide sequence PepF1 and coupling reaction, the serum of mice immunized with the immunogen or egg yolk antibody of laying hens were effective against 3 strains of Aeromonas hydrophila and 4 strains of Aeromonas bacteria, while for common E .coli, E.coli O157: H7, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio harveii, Vibrio vulnificus, Vibrio cholerae, Pseudomonas alternata, Edwardsiella tarda no cross-reaction. However, the serum or egg yolk antibody of the immunized mice had cross-reaction to common E.coli CICC21530 and DH5α. The novel immunogen can be used to prepare cross-type monoclonal antibody or yolk globulin of aquatic pathogen Aeromonas.

Description

An immunogen synthesis for preparing cross-type antibody against aquatic pathogen Aeromonas method

technical field

The invention relates to an immunogen synthesis method for preparing cross-type antibodies of the aquatic pathogen Aeromonas, belonging to the technical field of immune analysis.

Background technique

Aeromonas hydrophila is one of the main pathogens of fish farming sepsis, which brings huge economic losses to freshwater fish and aquaculture industry. Aeromonas hydrophila is widely distributed in various water bodies in nature. It is the primary pathogenic bacterium of many aquatic animals, and it is an opportunistic pathogenic bacterium. It is a typical pathogen of human-animal-fish comorbidity. Aeromonas hydrophila can produce highly toxic exotoxins, such as: hemolysin, histotoxin, necrotic toxin, enterotoxin and protease, which can make aquatic animals pathogenic. In actual production, there are many fulminant hemorrhagic diseases infected by Aeromonas hydrophila. Because there are many serotypes of Aeromonas hydrophila, and the infected objects are different, the symptoms are also different. Such as silver carp fulminant hemorrhagic disease, soft-shelled turtle septicemia, eel hemorrhagic disease, eel red fin disease, etc. The bacterium is an conditional pathogenic bacterium. When the environment changes suddenly and the water quality deteriorates, it will often be mixed with other bacteria (such as Aeromonas temperatus, Vibrio, etc.) to aggravate the condition. Diseases infected by Aeromonas hydrophila are generally severe, mostly malignant infectious diseases, and have a high mortality rate.

Currently, the main means of controlling Aeromonas hydrophila include antibiotics and vaccines. At present, the prevalence of poultry and aquaculture diseases and the abuse of antibiotics are relatively common in China, leading to prominent problems of antibiotic residues in meat, fish, and the environment, posing a huge threat to my country's food safety, export trade, environmental protection, and public health. According to reports, in 2013, the total amount of antibiotics used in China was 162,000 tons, of which 48% were for human use and the rest were for veterinary use. The vast majority of antibiotics for humans are used for the treatment of diseases and are showing a downward trend, while most antibiotics for animals are used in feed. Due to the low price of veterinary antibiotics, many farmers use them without restraint in order to prevent diseases. But cattle, sheep, chickens, ducks, fish, etc. eat veterinary antibiotics and excrete them into water, soil, and crops, and finally react on humans, leading to the problem of drug-resistant bacteria and "super bacteria", forming a vicious ecological chain cycle system.

At the same time, due to the variety of serotypes of the pathogenic bacteria, the efficacy of common bacterial vaccines varies greatly, and the effect on immune strains is often better, but has no or general effect on other serotypes. Recombinant protein vaccines based on conserved outer membrane proteins are more effective, but they face the problems of immunization by individual fish injection, time-consuming, laborious, high cost, and easy to cause fish death; not only that, the approval process of veterinary vaccines is very difficult. It is complicated and often takes 5-10 years, and it needs to be equipped with pharmaceutical-grade GMP (Good Manufacturing Practice) production workshops, which cannot meet market demand.

As a food ingredient and immune recognition active substance, vitellin globulin (IgY) has green, broad-spectrum and sustainable characteristics in aquatic disease control. Poultry-derived antibodies refer to immunoglobulins with antigen specificity and targeting properties that are obtained physically from the yolk of immunized poultry eggs after immunizing laying poultry with a specific antigen. Good; the US FDA has regarded avian-derived antibodies as the preferred "drug" to replace antibiotics, and believes that avian-derived antibodies are more beneficial as food than as drugs, and have included them in "Generally Accepted as Safe (GRAS) "category. Compared with traditional control methods that use antibiotics and vaccines to prevent and control related diseases, avian-derived antibodies to prevent and control diseases have the following characteristics: (1) High efficiency and strong pertinence, using antigen-antibody-specific immune responses to inhibit only corresponding pathogens. (2) Green and safe, yolk antibody is a component of egg yolk, has no side effects on animals and humans, can be degraded in the environment, and has no residual problems. (3) Compared with vaccines, the research and development and application procedures are simplified, and the methods of use in the prevention and treatment of specific pathogens are simple, mostly through oral administration, spraying, etc., and the use cost is low. (4) Wide range of applications, yolk antibody provides a new option for mass development of functional food and disease diagnosis, prevention and treatment of anti-bacterial infection and anti-viral infection. (5) It is easy to obtain and has good sustainability. Live chickens can be immunized after obtaining pathogenic strains or conserved antigens, with long laying cycle and high yield. The uniform recognition and specificity of high-immune chicken yolk globulin (IgY) to aquatic pathogens laid a foundation for it to be used as a feed additive for biological identification, proliferation inhibition, and green prevention and control of aquatic diseases.

Contents of the invention

The purpose of the present invention is to provide an immunogen synthesis method for preparing the cross-type antibody of the aquatic pathogen Aeromonas genus, and provide a method for preparing the broad-spectrum monoclonal antibody and the yolk antibody of the aquatic pathogen Aeromonas hydrophila. methods and ideas.

The technical scheme of the present invention, in order to achieve the above object, the present invention selects the conserved polypeptide epitope PepF1 of the Aeromonas outer membrane protein OmpF of the N-terminal modification Cys as antigen, and synthesizes the Aeromonas polypeptide antigen and Artificial antigen of the carrier protein BSA.

There are many kinds of Aeromonas surface antigens, and the specific structure varies with different strain serotypes. Conservative and surface-exposed polypeptide epitopes are the basis for preparing Aeromonas-specific antibodies. 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 the carrier protein BSA, it has immunogenicity, and antibodies can be prepared.

The Aeromonas conserved polypeptide epitope PepF1-SMCC-BSA immunogen is immunized to BALB/c mice and laying hens (Roche chickens) to obtain Aeromonas cross-type mouse antibodies or egg yolk antibodies.

A method for synthesizing an immunogen for preparing the cross-type antibody of the aquatic pathogen Aeromonas, the steps of which are as follows:

(1) Modification of the polypeptide PepF1: the N-terminus of the conserved polypeptide PepF1 of the Aeromonas outer membrane protein OmpF was modified with cysteine Cys;

(2) Derivation of BSA: the bifunctional coupling agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt Sulfo-SMCC and bovine serum Albumin-BSA was reacted in 0.05-0.15 M/L phosphate buffer at room temperature for 1-2 hours according to the reaction molar ratio of 40-80:1 to obtain SMCC-BSA, and ultrafiltration was used to remove excess Sulfo-SMCC;

(3) Preparation of immunogen: The polypeptide PepF1 obtained in step (1) and the derivatized BSA obtained in step (2) were reacted at room temperature in 0.05-0.15 M/L phosphate buffer according to the molar ratio of 30-60:1. -6h, the immunogen PepF1-SMCC-BSA of Aeromonas cross-type antibody was obtained after dialysis.

The Aeromonas outer membrane protein OmpF conservative polypeptide PepF1 is specifically Tyr Lys Gly Glu Gly ArgGly Tyr Glu Leu Ala Ala.

The specific steps of this immunogen synthesis method are as follows: take 10 mg of carrier protein BSA and dissolve it with 0.1M PB buffer, and dissolve the bifunctional coupling agent Sulfo-SMCC dissolved with N,N-dimethylformamide (DMF) according to the reaction mole The ratio of Sulfo-SMCC:BSA is 40-80:1, add it into the BSA solution, and react at room temperature under magnetic stirring for 1-2h; after the reaction, use Millipore ultrafiltration tube to ultrafilter 2-4 times to obtain SMCC-BSA, and remove unreacted The bifunctional coupling agent; the Aeromonas conservative polypeptide epitope PepF1 modified by the N-terminal cysteine Cys dissolved in 0.05-0.15M/L PB buffer according to the reaction molar ratio of polypeptide PepF1:BSA is 30 -60:1 was added to the reaction solution, and reacted at room temperature for 2-6 hours under magnetic stirring; then, the reaction solution was dialyzed with 0.01-0.05 M/L PBS, and the dialysate was changed once every 4-6 hours, and the dialysate was dialyzed for 2-3 days to obtain gas The immunogen PepF1-SMCC-BSA of the Monas spp. cross-type antibody.

Beneficial effects of the present invention: the present invention provides an immunogen preparation method for preparing the broad-spectrum within the genus Aeromonas and the specificity outside the genus of the genus Aeromonas or the immunogen preparation method of the egg yolk antibody; the present invention screens the outer membrane protein OMPF The conserved amino acid sequence PepF1 of Aeromonas. And synthesized the polypeptide PepF1-SMCC-BSA artificial immunogen for the first time, immunized laying hens and BALB/c mice with the artificial polypeptide antigen PepF1-SMCC-BSA, and the indirect ELISA test showed that the immunized chicken egg yolk antibody IgY and the mouse serum were against the tested 3 strains of Aeromonas hydrophila (CICC 10500, MCCC 1A00190, MCCC 1A0007), 4 strains of Aeromonas bacteria (Aeromonas bordetii, Aeromonas spotted, Aeromonas Zhaodong, Bivalve Aeromonas) are all effective, and for common E.coli, E.coli O157:H7, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio harveylius, Vibrio vulnificus, Vibrio cholerae, Alternative pseudomonas Bacillus and Edwardsiella tarda have no cross-reaction, and the specificity is good. However, the serum and egg yolk antibodies of mice directly immunized with Aeromonas hydrophila had serious cross-reactions to common E.coli CICC 21530 and DH5α, indicating that the polypeptide immunogen used in the present invention has conservation and specificity. advantage. The homogeneous recognition and specificity of high-immune chicken yolk globulin (IgY) to Aeromonas and its safety as a food ingredient laid the foundation for the green prevention and control of aquatic disease sepsis.

Description of drawings

Figure 1 is polyacrylamide gel electrophoresis (SDS-PAGE) characterization of artificially synthesized PepF1-SMCC-BSA artificial antigen. 1. BSA; 2. BSA-SMCC; 3. PepF1-SMCC-BSA;

Fig. 2 is the cross-reaction of synthetic PepF1-SMCC-BSA artificial antigen immunized mouse serum with 3 strains of Aeromonas hydrophila and 4 strains of Aeromonas bacteria.

Among them, 3 strains of Aeromonas hydrophila (1, ASP CICC 10500, 4, ASP MCCC 1A00190, 2, ASP MCCC1A0007), 4 strains of Aeromonas bacteria (5, Aeromonas bordetii, 3, spotted gas 6, Aeromonas Zhaodong, 7, Aeromonas bivalve).

Detailed ways

The Aeromonas strains described in the examples were purchased from China Industrial Microorganism Collection Center (CICC), China General Microorganism Collection Center (CGMCC), and China Marine Microorganism Collection Center (MCCC). The peptide sequence was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

The present invention is further illustrated by the following examples.

Instruments: Desktop high-speed refrigerated centrifuge, Hunan Hexi Instrument Equipment Co., Ltd.; ELGA ultra-pure water machine, Elgar, UK; WD-9405B horizontal shaker, Beijing Liuyi Instrument Factory; 96-well 8×12 high-adsorption detachable enzyme Standard plate, Wuxi Guosheng Bioengineering Co., Ltd.; Multiskan FC microplate reader, Thermo Fisher Scientific; 200μL, 1mL pipette gun, 300μL 8-channel pipette gun, Thermo Labsystems; XW-80A vortex mixer, Shanghai Qingpu Huxi Instrument factory; Millipore ultrafiltration tube (Cut off: 10000), purchased from Millipore-Merck Company.

Reagents: Tetramethylbenzidine (TMB), Sigma Reagent Co., Ltd.; other reagents are of analytical grade.

Proceed as follows:

1. Screening and design of polypeptide sequences: the outer membrane protein is an important structure of the cell membrane of Gram-negative bacteria, and the outer membrane protein OmpF is an important adhesion factor of Aeromonas hydrophila, which is the key factor for the bacteria to survive under unfavorable conditions. A key component of an important two-component regulatory system. At the same time, the homology of Aeromonas hydrophila outer membrane protein OmpF in Aeromonas hydrophila is more than 83%. OmpF is a promising immune target. After comparing the amino acid sequence of the OmpF protein of Aeromonas, combined with bioinformatics tools, the antigenicity, α-helix, β-sheet region, hydrophilicity, hydrophobicity, and surface exposure of the OmpF amino acid sequence were analyzed. A polypeptide sequence with conservative and better surface exposure is selected as a candidate antigen, and the length of the polypeptide sequence is generally 10-20 AA. Choose N-terminal or C-terminal modified cysteine Cys for subsequent coupling.

2. Preparation of immunogen: Dissolve 10 mg of carrier protein BSA in 0.1M PB buffer, add Sulfo-SMCC dissolved in N,N-dimethylformamide (DMF) to BSA at a molar ratio of 70:1 The solution was reacted at room temperature for 1 h under magnetic stirring. After the reaction, ultrafiltration was performed three times with Millipore ultrafiltration tubes (Cut off: 10000 MW) to remove unreacted coupling agent. The conserved peptide epitope PepF1 of Aeromonas dissolved in 0.1M PB buffer was added to the reaction solution at a reaction molar ratio of 60:1, and reacted at room temperature for 1 h under magnetic stirring. Subsequently, the reaction solution was dialyzed with 0.01M PBS, the dialysate was changed once every 6 hours, and dialyzed for 2 days, the immunogen of Aeromonas cross-type antibody was obtained.

3. Preparation of immune serum or egg yolk antibody

(1) Experimental animals: 6 8-week-old BALB/c mice and 6 20-week-old Roche laying hens were selected for immunization.

(2) Antigen configuration: Dilute the immunogen with normal saline to prepare a solution of the required concentration;

(3) Emulsification: Emulsify the above solution with an equal amount of complete or incomplete Freund's adjuvant by mixing and stirring, and inject mice or Roche laying hens subcutaneously at multiple points after emulsification is complete;

(4) Immunization method: Immunize mice according to the following immunization procedure: first immunization 80 μg, second immunization 80 μg, third immunization 40 μg. Immune laying hens according to the following immunization procedure: 500 μg for the first immunization, 500 μg for the second immunization, and 300 μg for the third immunization. 3 After immunization, use the indirect enzyme-linked immunoassay (ELISA) method to measure the titer against bacteria within the genus Aeromonas and other bacteria outside the genus;

(5) Blood collection from mice: blood collection by tail docking 1 week after the second and third immunizations; IgY from laying hens: 1 week after the second and third immunizations, eggs were collected and yolks were separated to prepare crude IgY extracts. Antiserum titers and cross-reactivity were determined by indirect ELISA.

4. ELISA reaction process:

(1) Antibody titer determination steps: Boil the test bacteria solution to inactivate it, and then use coating buffer for serial dilution to coat a 96-well microtiter plate, 100 μL/well, and store in a refrigerator at 4°C overnight. The next day, take out the ELISA plate and return to room temperature, inject 200 μL of PBST solution into each well, shake on the shaker for 3 minutes, shake off the washing solution vigorously, pat dry on absorbent paper, and continue to wash 2 times. The following washing methods are the same;

(2) After fully washing, block the microtiter plate with blocking buffer, 200 μL/well, incubate in a 37°C incubator for 2 hours, take it out and dry it for use;

(3) Add the serial dilutions of positive sera to the first 7 rows of the microtiter plate, add negative sera to the 8th row, 100 μL/well, incubate at 37°C for 1 hour, wash and pat dry;

(4) Add 100 μL of 1:3000 diluted HRP-labeled goat anti-mouse secondary antibody or 1:3000 diluted HRP-labeled goat anti-chicken secondary antibody to each well, incubate at 37 °C for 1 hour, wash and pat dry;

(5) Add 100 μL of chromogenic solution (the ratio of TMB to substrate solution is 1:5) to each well, react at 37 °C in the dark for 15 min, and add 50 μL of stop solution (2 mol/L sulfuric acid) to each well after taking it out. The absorbance value A ₄₅₀ was measured with a microplate reader. The specific results are shown in Figure 2.

These included 3 strains of Aeromonas hydrophila (CICC 10500, MCCC 1A00190, MCCC 1A0007), 4 strains of Aeromonas bacteria (Aeromonas bordetii, Aeromonas spotted, Aeromonas Zhaodong, Aeromonas bivalve).

Claims (3)

1. a kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody, it is characterised in that step Suddenly it is：

（1）The modification of polypeptide PepF1：The Aeromonas outer membrane protein OmpF conservative polypeptide PepF1 that screening obtains are repaiied in N-terminal Adorn cysteine Cys；

（2）The derivative of BSA：By bifunctional coupling agent 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinyls Imines ester sodium salt Sulfo-SMCC and bovine serum albumin(BSA) BSA are according to reacting molar ratio 40-80:1 in 0.05-0.15 M/L phosphorus 1-2h is reacted at room temperature in phthalate buffer, obtains SMCC-BSA, and ultrafiltration removes extra Sulfo-SMCC；

（3）The preparation of immunogene：By step（1）Gained polypeptide PepF1 and step（2）BSA after gained derives rubs according to reaction You compare 30-60:1 reacts at room temperature 2-6h in 0.05-0.15 M/L phosphate buffers, and Aeromonas friendship is obtained after dialysis The immunogene PepF1-SMCC-BSA of forked type antibody.

2. being used to prepare the immunogene synthesis side of aquatic pathogenic bacterium Aeromonas chiasma type antibody according to claim 1 Method, it is characterised in that：The Aeromonas outer membrane protein OmpF conservative polypeptides PepF1 is specially Tyr Lys Gly Glu Gly Arg Gly Tyr Glu Leu Ala Ala。

3. being used to prepare the immunogene synthesis side of aquatic pathogenic bacterium Aeromonas chiasma type antibody according to claim 1 Method, it is characterised in that be as follows：

The PB buffer solutions for taking carrier protein BSA 10mg 0.1M, it is double by being dissolved with n,N-Dimethylformamide DMF Function coupling agent Sulfo-SMCC is according to reaction molar ratio Sulfo-SMCC:BSA is 40-80:1 is added in BSA solution, magnetic Power stirring is lower to react at room temperature 1-2h；Millipore super filter tubes ultrafiltration 2-4 times is used after reaction, obtains SMCC-BSA, is removed not The bifunctional coupling agent of reaction；By the gas list through N-terminal modification cysteine Cys of the PB buffer solutions of 0.05-0.15M/L Born of the same parents bacterium conservative polypeptide epitope PepF1 is according to reaction molar ratio polypeptide PepF1:BSA is 30-60:1 is added in reaction solution, 2-6h is reacted at room temperature under magnetic agitation；The PBS hemodialysis reaction solution of 0.01-0.05 M/L, 4-6h is then used to replace dialyzate one Secondary, dialysis 2-3 days is to get to the immunogene PepF1-SMCC-BSA of Aeromonas chiasma type antibody.