CN102206257B - Edwardsiella tarda immunogenic protective antigen, and related expression vector, vaccine and application - Google Patents

Abstract

The present invention provides an immunoprotective antigen of Edwardsiella tarda, which is a flagellar-associated protein of Edwardsiella tarda, and the amino acid sequence of the flagellar-associated protein of Edwardsiella tarda is shown in SEQ ID NO:2. Preferably, the nucleotide sequence of the flagellar-associated protein of Edwardsiella tarda is shown in SEQ ID NO: 1, and related recombinant expression vectors are also provided, the subunit vaccine of Edwardsiella tarda produced, and preparation of the tarda Use of Edwardsiella subunit vaccines. The Edwardsiella tarda immunoprotective antigen of the present invention has good immunogenicity, provides a safe, effective and economical vaccine for Edwardsiella disease in cultured fish, has practical commercial development and application value, and is suitable for large-scale promotion application.

Description

Edwardsiella tarda immune protective antigen, related expression vector, vaccine and application

technical field

The present invention relates to the technical field of antigens, more specifically to the technical field of immune protective antigens, in particular to an Edwardsiella tarda immune protective antigen, related expression vectors, vaccines and applications.

Background technique

With the continuous and steady development of fish farming, various disease problems have become increasingly prominent, which have seriously affected the production and growth of aquaculture. In my country, sudden and explosive diseases of seawater cage culture and factory culture fish developed in recent years occur frequently. At present, the average mortality loss rate of mariculture in my country is over 30%, and the annual economic loss is as high as 16 billion yuan. The disease problem has become an important factor restricting the healthy development of mariculture industry.

For the occurrence of various diseases, chemotherapy represented by antibiotics has played an active role in the control and prevention of diseases. However, the environmental pollution caused by such disease control measures, the emergence of a large number of drug-resistant pathogens, and the negative effects of drug residues in aquatic products are becoming more and more serious. Antibiotic-based chemicals have gradually been banned in aquaculture in the European Union, the United States, and Canada.

In order to curb the increasingly serious development trend of farmed fish diseases caused by various environmental factors and the rapid increase in breeding density, etc., and promote the sustainable development of the mariculture industry, the World Food and Agriculture Organization combined the successful experience of fishery development in developed countries (Ormonde P. Fisheries resources: trends in production, utilization and trade. In: Nomura I (ed.). The State of World Fishery and Agriculture 2002. Rome: FAO Information Division, 2002, p3-p45.), advocating "system management approaches" (System management approaches, SMA) breeding mode to prevent the occurrence of various diseases. One of the main contents of this measure is to promote the application of various immune prevention and control technologies represented by vaccination. The adoption of these measures will greatly reduce the use of chemical drugs, which not only avoids environmental pollution, but also increases the consumption safety of aquatic products. As a cost-effective disease control strategy and means in line with the environment-friendly and sustainable development strategy, vaccination is becoming the main frontier and application field of research and development in the modern aquaculture practice.

Vaccines have the characteristics of strong pertinence, long disease resistance cycle, lifelong immunity, remarkable effect and active prevention and control. The inactivated vaccine (Kill vaccine) based on the inactivated body of pathogenic bacteria provides an effective means for the prevention and treatment of aquaculture diseases, but the inactivated vaccine generally has the disadvantage of inconvenient administration (injection administration has better immune protection). The technical application defect is extremely inconvenient for the fish farming industry that needs to immunize tens of thousands of numbers, and the cost of drug administration is often not affordable for the aquaculture industry. And then can't implement injection administration for the fish fry that disease takes place serious and juvenile fish, simultaneously, to many disease inactivated vaccines often not good effect or invalid. All these have brought hindrance to the widespread application of immune control technology for aquaculture diseases.

The industrial characteristics of the aquaculture industry require that the disease control technology must be economical and easy to apply and implement. Therefore, in addition to the technical requirements of high titer in the development of vaccine products, the cost of immunization must be low and cannot exceed the bearing capacity of the aquaculture industry. Live attenuated vaccines have the advantages of convenient administration (can be soaked for administration), high immune titer (can reduce dosage), low cost, and can develop broad-spectrum vaccines (live bacterial vaccines often have cross-protection). , has become a hotspot and frontier field in the research and development of vaccines for aquaculture in the world.

Edwardsiella genus is a common pathogen that causes bacterial diseases in freshwater and marine fish, and is specifically divided into Edwardsiella tarda, Edwardsiella ictaluri and Edwardsiella hoshinae ). The fish hemorrhagic septicemia caused by it is collectively known as Edwardsiellosis. The disease has a wide spread area, no obvious seasonality, high infection rate and mortality rate, and many types of damage, including carp, tilapia, eel, mullet, salmon, trout, flounder, etc. Most of them have high economic value species of fish. In addition, Edwardsiella tarda infects shellfish, reptiles, amphibians, birds, and mammals. Notably, Edwardsiella tarda is also an important zoonotic pathogen, being the only member of the Edwardsiella genus that infects humans. At present, Edwardsiella tarda is the most serious pathogen of Edwardsiella disease in farmed fish in my country, and there is a huge threat of pathogen transfer to human body. The U.S. patent has the report (Evans J J, Klesius, P H and Shoemaker C A2006 Modified live Edwardsiella tarda vaccine for aquatic animals; United States) that utilizes rifampicin to screen and obtain the natural attenuated mutant of the wild strain as an attenuated live vaccine Patent 7067122). At present, there are no effective control measures for this disease in our country.

Edwardsiella tarda wild strain EIB202 of the present invention is separated from the diseased fish of the Edwardsiella disease that broke out in the aquaculture fishery in the East China Sea, and is a highly toxic Edwardsiella tarda EIB202 (Edwardsiella tardaEIB202, Xiao Jingfan, etc. , "Isolation and identification of fish pathogen Edwardsiella tarda from marine culture in my country", "Aquaculture Research" Vol.40, 2009), has been preserved in the China Type Culture Collection Center, address: Wuhan University, Wuhan, China, and the preservation number is: CCTCC NO : M 208068, the preservation date is May 1st, 2008, specifically refer to the Chinese invention patent application CN200910052707.6.

Because there is still a lack of systematic understanding of the pathogenic mechanism of Edwardsiella tarda, its multi-host adaptability also makes the control of this bacteria particularly difficult. At present, the control of Edwardsiella mainly relies on the use of antibiotics and chemotherapy. Because antibiotics are widely recognized, their use in aquaculture is increasingly limited. In addition, pathogenic Edwardsiella tarda is often found to be naturally resistant to multiple antimicrobial complexes, increasing the difficulty of antibiotic-based therapy. In contrast, vaccines are a safer and more effective method of disease control from a long-term perspective. Due to the weak antigenicity of inactivated vaccines and the unstable safety of attenuated vaccines, it is particularly necessary for the aquaculture industry to develop subunit vaccines that are easy to use, efficient, easy to commercialize and produce on a large scale, and effective.

Contents of the invention

The main purpose of the present invention is to provide a kind of Edwardsiella tarda immunoprotective antigen, related expression vector, vaccine and application for the above existing problems and deficiencies. The immunogenicity of the Edwardsiella tarda immunoprotective antigen is better, for Edwardsiosis in cultured fish provides a safe, effective and economical vaccine, which has practical commercial development and application value and is suitable for large-scale promotion and application.

In order to achieve the above object, in the first aspect of the present invention, a kind of Edwardsi tarda immunoprotective antigen is provided, it is characterized in that, described Edwardsi tarda immunoprotective antigen is Edwardsi tarda flagellar-associated protein, so The amino acid sequence of the Edwardsiella tarda flagellar-associated protein is shown in SEQ ID NO: 2.

Preferably, the nucleotide sequence of the Edwardsiella tarda flagellar-associated protein is shown in SEQ ID NO: 1.

In the second aspect of the present invention, a recombinant expression vector is provided, which is characterized in that it includes the expression vector and the exogenous nucleotide sequence encoding the above-mentioned Edwardsiella tarda immunoprotective antigen integrated in the expression vector .

Preferably, the exogenous nucleotide sequence is shown in SEQ ID NO:1.

In the third aspect of the present invention, a subunit vaccine of Edwardsiella tarda is provided, which is characterized in that it is prepared from the above-mentioned immunoprotective antigen of Edwardsiella tarda.

In the fourth aspect of the present invention, there is provided the application of the above-mentioned Edwardsiella tarda immunoprotective antigen in the preparation of a subunit vaccine of Edwardsiella tarda.

Preferably, the Edwardsiella tarda is Edwardsiella tarda EIB202, and the preservation number is: CCTCC NO: M 208068.

The beneficial effect of the present invention is specifically that: the Edwardsiella tarda immunoprotective antigen of the present invention is the Edwardsiella tarda flagellar-associated protein, and the amino acid sequence of the Edwardsiella tarda flagellar-associated protein is shown in SEQ ID NO: 2, the experiment It shows that the immune protective antigen of Edwardsiella tarda has a better immune protective effect on Edwardsiella tarda, and at the same time, for the development of subunit vaccines, the immune protective antigen of Edwardsiella tarda will also be the first choice for subsequent vaccine design The target is to provide a safe, effective and economical vaccine for Edwardsiosis in farmed fish, which has practical commercial development and application value and is suitable for large-scale promotion and application.

Description of drawings

Fig. 1 is the SDS-PAGE figure after purification of the immunoprotective antigen of Edwardsiella tarda expressed by a specific embodiment of the recombinant expression vector of the present invention, swimming lane 1: protein Marker; swimming lane 2 and 3: FD protein expression result; swimming lane 4 and 5: FD protein purification results.

Fig. 2 is an analysis of the relative immune protection after immunizing zebrafish with the immune protective antigen of Edwardsiella tarda of the present invention.

Fig. 3 is an analysis of the relative immune protection of turbot immunized with the Edwardsiella tarda immune protective antigen of the present invention.

Fig. 4 is an analysis of the bactericidal activity of the Edwardsiella tarda immunoprotective antigen immune turbot serum of the present invention.

Fig. 5 is the titer analysis of specific antibodies in the serum of turbot immunized with Edwardsiella tarda immune protective antigen of the present invention.

Detailed ways

In order to better understand the contents of the present invention, the following examples are given for further description.

The preparation of embodiment 1 recombinant protein vaccine

1. Experimental materials

1. Strains and plasmids

Escherichia coli TOP10, Escherichia coli BL21 (DE3) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Edwardsiella tarda E. tarda EIB202, the preservation number is: CCTCC M 208068; pET 28a (+) vector Purchased from Bao Biological Engineering (Dalian) Co., Ltd.

2. Medium and culture conditions

Medium:

LB: 1% Tryptone, 0.5% Yeast extract, 1% NaCl. Add 2% ager to the solid medium. Autoclave at 121°C for 20 minutes. For the cultivation of Escherichia coli.

TSB: 3% TSB. Add 2% ager to the solid medium. Autoclave at 121°C for 20 minutes. For EIB202 culture.

DHL medium: 60g of gallic acid milk agar medium was dissolved in 1L of deionized water, boiled repeatedly to dissolve until clear, and made a plate after cooling.

Training conditions:

Escherichia coli was cultured statically on plate in LB solid medium or shaker in LB liquid medium at 37°C

Edwardsiella tarda was inoculated in TSB liquid medium and shaken overnight at 28°C and 200r/min. The Edwardsiella tarda solution was spread or streaked on the DHL solid medium, and placed upside down in a constant temperature incubator at 37°C for overnight culture, and black-core colonies with translucent edges formed on the DHL solid medium.

3. Experimental reagents

Routine molecular biology reagents and kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; restriction endonucleases Nde I and Xho I were purchased from Bao Bioengineering (Dalian) Co., Ltd. Routine reagents for protein expression and purification were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. The affinity chromatography medium Ni-Sepharose FF was purchased from GE Healthcare. Monoclonal antibody against turbot IgM was purchased from Aquatic Diagnostics, and goat anti-mouse monoclonal antibody was purchased from Tiangen.

4. Antigen protein gene sequence

According to the complete genome sequence of E. tarda EIB202, the DNA sequence (SEQ ID NO: 1) of the corresponding Edwardsiella tarda flagellar-associated protein FD gene was obtained. Its amino acid sequence is shown in SEQ ID NO: 2.

5. Antigen protein primers

Primer Premier 5 software was used to design specific primers for the FD gene. The primers were synthesized by Shanghai Bioengineering Company and were purified by PAGE.

FDf: GGGAGATCATATGGCCATTTCGGTATCG (Nde I)

FDr: GCCGCTCGAGTAAGATCTGGCGTACGT (Xho I)

6. Experimental animals and immune reagents

Zebrafish, purchased from a Shanghai breeding farm, have a body length of 4 cm and a weight of about 0.4 g. Before the experiment, put them in the aquarium to adapt to training for 2 weeks, and then move them to the fish feeding system for 2 weeks to adapt. The fish breeding system flows and replaces the water body every day, and the breeding temperature is about 22°C. Feed 2 times a day and remove feces in time.

Turbot, purchased from a farm in Weifang, Shandong Province, weighed 30 g and had a body length of 10 cm, was placed in a water tank for domestication for 2 weeks and was set aside. In the experiment, natural seawater was used to replace 2/3 volume of aquaculture water and aerated every day, and the water temperature was 14-16°C. Before and after immunization, they were managed according to the conventional breeding procedures.

Fish anesthetic tricaine methanesulphonate (MS-222) was purchased from Sigma. Fish adjuvant MONTANIDE ^TM ISA 763A was purchased from SEPPIC, France.

2. Experimental method

1. Construction of FD recombinant antigen protein gene

The vector plasmid pET28a was extracted according to the operating instructions of TIANprep Mini Plasmid Kit. The genome of Edwardsiella tarda EIB202 was extracted according to the operating instructions of the TIANamp Bacteria DNA Kit. The genome is used for PCR reaction, and the primers used are the designed specific primers FDf/FDr to obtain the target product containing Nde I and Xho I restriction endonuclease sites at both ends. Restriction endonucleases Nde I and Xho I were used for double digestion, and the digested target gene and vector were subjected to agarose gel electrophoresis. After the target band was cut, the DNA was recovered using an agarose gel DNA recovery kit.

After the target gene and plasmid with cohesive ends are obtained, the two are connected under the action of DNA ligase to form a recombinant plasmid containing the target gene. The recombinant plasmid can be used for transcloning Escherichia coli BL21 (DE3) host bacteria. The clone on the plate was determined to be positive by sequencing and enzyme digestion verification, that is, the recombinant Escherichia coli BL21(DE3)/pET-28a-FD of Edwardsiella tarda FD protein.

2. Expression and purification of FD recombinant antigen protein

Inoculate the expression strain with successful sequencing, grow to a certain extent and add the inducer IPTG to induce expression. After the collected bacterial solution was centrifuged, the wall was broken by ultrasonic method, and the supernatant was collected after centrifugation. Protein electrophoresis was used to detect the induced expression of FD recombinant protein, as shown by the arrow in Figure 1. The results of protein electrophoresis showed that FD recombinant protein was expressed in large quantities.

Both ends of the FD recombinant protein have a His tag, which can specifically bind to Ni. Therefore, the Ni column can be used to specifically purify the target protein. The eluted protein was put into a dialysis bag with a molecular weight cutoff of 14kD, dialyzed in PBS solution, and stored at -70°C.

Embodiment 2: Taking zebrafish as the experimental animal's immune protection test by injection

Protein Concentration Determination

The OD ₂₈₀ of the nucleic acid quantification instrument NanoDrop ND-1000 spectrophotometer was used to determine the concentration of the recombinant protein, and the protein was diluted to a predetermined concentration with PBS buffer.

Immunization of zebrafish

The purified FD recombinant protein was mixed with the adjuvant ISA 763A at a ratio of 7:3, and the final protein concentration was 0.3 μg/μl. Zebrafish were cultured in random groups, 30 fish/group, and each zebrafish was immunized by tail intramuscular injection. Then feed them normally, observe the activity and death of the fish. Immunization time is one month. The negative control was the mixed group of PBS and adjuvant.

Before the injection operation, the zebrafish were soaked in 100ng/ml MS-222 for anesthesia. After the experimental period, the remaining zebrafish were euthanized by immersing in 300ng/ml MS-222 for more than 10 minutes.

Zebrafish challenge injection

Collect the cells of Edwardsiella tarda EIB202, wash them three times with sterilized PBS, measure the cell density after resuspension with a spectrophotometer, quantify the OD ₆₀₀ as 1, and dilute the cell solution with PBS to the desired concentration gradient; The experimental zebrafish were immunized by tail intramuscular injection at a dose of 5 μl/fish; the morbidity and death of zebrafish in each group were observed and recorded.

Determination of immune protection of recombinant protein

After the zebrafish were immunized and bred for 4 weeks, an appropriate dose was selected according to the LD50 value of the wild strain to challenge the immunized zebrafish; the morbidity and mortality of the zebrafish in each group were observed and recorded.

Calculate the relative percent survival (RPS) according to the formula (1):

RPS=(1-mortality rate of immunization group/mortality rate of control group)×100% (1)

The mortality of zebrafish within 8 days after challenge is shown in Figure 2. A very small number of fish died on the first day after the zebrafish was challenged, and then the fish body stabilized. On the third day, there was a large number of deaths, and it was found that the zebrafish moved slowly, and the tail swung from side to side before death, and the body color of the infected part turned black. On the 8th day, the death situation has stabilized. Among them, the mortality rate of the blank control group was 81%, and that of the immunized group was 23%. It was calculated that the relative protection rate of FD protein was 71%.

After dissecting the diseased fish, it was found that the spleen of the fish was enlarged, the blood loss was pink, and the liver had diffuse hemorrhage. The diseased fish tissue was properly treated and diluted, then coated on a DHL plate and cultured overnight at 28°C. There were a large number of black colonies on the plate, indicating that the cause of the disease in the zebrafish was indeed infected with Edwardsiella tarda.

Embodiment 3: Taking turbot as the experimental animal's immune protection test by injection

Immunization of turbot

Turbot were cultured in random groups, 30 fish/group, and each fish was intramuscularly injected into the tail at a dose of 100 μl/fish. After the purified protein was dialyzed overnight, according to its concentration, it was mixed with the adjuvant ISA 763A at a ratio of 7:3 until the protein concentration reached 0.3 μg/μl. PBS and adjuvant ISA 763A immunization group were negative controls. Then feed them normally, observe the fish's activities and whether they die. The immunization time is 10 weeks.

Before the injection operation, soak the turbot in 100ng/ml MS-222 for anesthesia. After the experimental period, the remaining turbot were euthanized by immersion in 300ng/ml MS-222 for more than 10 minutes.

Turbot attack

The challenge method of turbot is the same as that of zebrafish. After the turbot is immunized, at the 10th week, according to the LD50 value of the wild strain, select the appropriate dose to carry out group challenge experiments on the immunized turbot.

Analysis of the immune protection of antigenic protein in turbot

The mortality of turbot within 8 days after challenge is shown in Figure 3. The experimental results showed that turbot began to die in large numbers on the third day after being challenged, and had obvious symptoms: the subcutaneous hyperemia and redness of the operculum margin, the base of the membranous fin and the ventral surface of the diseased fish, followed by pus in the bleeding lesions Ulcers, forming subcutaneous abscesses. The death situation on the 7th day has stabilized. The mortality rate of the blank control group was 80%, and that of the immunized group was 30%. It was calculated that the relative protection rate of FD protein was 62.5%.

Comparing the experimental results obtained by immunizing FD protein between zebrafish and turbot, it shows that the candidate antigen protein FD obtained by the primary screening on the first-level animal model zebrafish also has a better immune protection effect on the second-level animal model turbot. This aspect lays a good foundation for further re-screening experiments in the future, and also confirms that zebrafish can become a new animal model for screening protective antigens. At the same time, for the development of subunit vaccines, FD will also be the preferred target for subsequent vaccine design.

Embodiment 4: FD antigen protein immune level analysis

Blood collection and preparation of antiserum from turbot

After the turbot was immunized with FD recombinant protein, blood was collected from the tail vein of the experimental fish at the 8th and 10th week respectively, and about 0.3ml of blood was taken from each tail. The whole blood was allowed to stand at room temperature and then centrifuged to collect the supernatant serum, which was stored at -80°C for later use.

antiserum bactericidal test

Inoculate Edwardsiella tarda EIB202 and culture overnight. Wash with PBS three times, take a certain amount of bacterial suspension mixed with immune serum and incubate, and take samples at 1, 2, 3, 4, and 5 hours to coat the plate; the blank control sample is mixed with bacterial suspension and non-immune serum. Three groups of parallel controls were set up for each group. All the plates were cultured for 20 hours, colonies were counted, and the bactericidal ability of the antiserum at different time points was calculated. The antiserum obtained from turbot immunized with FD recombinant protein was incubated with EIB202, and the bactericidal ability of the antiserum was analyzed at different incubation time points. The results are shown in Figure 4.

It can be seen from the results in the above figure that the number of viable bacteria was the least after the antiserum was incubated with Edwardsiella tarda for 4 hours, which means that the serum has the best bactericidal ability at this time. After incubation with live bacteria for 4 hours, the survival rate of Edwardsiella tarda in the 8th week serum was 41%, and the survival rate of bacteria in the 10th week serum was 53%. Obviously, the antiserum in the 8th week has more bactericidal properties.

Serum Antibody Level Determination and Analysis

Determination of specific antibody levels produced by experimental fish turbot after immunization Conventional indirect ELISA method detection:

Incubate the purified recombinant protein FD-coated plate overnight. Wash the plate 3 times and pat dry; add blocking solution and block at 22°C for 2h. Wash the plate 3 times, pat dry; add fish immune serum, 100μl/well, add to each well at 2-fold serial dilution, the initial dilution is 1:4, the maximum dilution is 1:512, and the blank serum is the same treatment as a control. Incubate at 22°C for 3h. Wash the plate 5 times and pat dry; add monoclonal antibody against turbot IgM and incubate at 22°C for 1h. Wash the plate 5 times and pat dry; add goat anti-mouse monoclonal antibody (HRP-labeled) and incubate at 22°C for 1h. Wash the plate 5 times and pat it dry; add the soluble TMB substrate washing solution, and place it in the dark at room temperature for 10 minutes; add the reaction termination solution, and place it in a microplate reader for detection at OD ₄₅₀ wavelength.

Antibody titers were determined based on P/N values. Test serum absorbance value (P) = test serum OD value - blank serum OD value, negative control serum absorbance value (N) = negative serum OD value - blank OD value, when the ratio of the two is greater than 2.1, the highest dilution of antiserum The multiple is its final antibody titer.

The specific antibody level produced by turbot immunized with FD recombinant protein was determined by conventional indirect ELISA method, and the results are shown in Fig. 5 . The results showed that the absorbance value of the antiserum was significantly greater than that of the blank serum. Certain specific antibodies were produced in turbot immunized with FD recombinant protein.

In summary, the Edwardsiella tarda immunoprotective antigen of the present invention has better immunogenicity, provides a safe, effective and economical vaccine for Edwardsiella in cultured fish, and has practical commercial development and application value. It is suitable for large-scale popularization and application.

In this specification, the invention has been described with reference to specific embodiments thereof. However, it is obvious that various modifications and changes can be made without departing from the spirit and scope of the invention. Accordingly, the specification and drawings are to be regarded as illustrative rather than restrictive.

Claims (3)

1. a subunit vaccine for tardy Edwardsiella disease in fish, characterized in that, it is prepared from tardy Edwardsiella immunoprotective antigen, and said tardy Edwardsiella immunoprotective antigen is tardy Edwardsiella Bacteria flagella-related protein, the amino acid sequence of the Edwardsiella tarda flagella-related protein is shown in SEQ ID NO:2. 2. the application of a tardy Edwardsiella immunoprotective antigen in the preparation of subunit vaccines for tardy Edwardsiella disease, characterized in that, said tardy Edwardsiella immunoprotective antigen is tardy Edwardsiella Bacteria flagella-related protein, the amino acid sequence of the Edwardsiella tarda flagella-related protein is shown in SEQ ID NO:2. 3. application according to claim 2, is characterized in that, described Edwardsiella tarda is Edwardsiella tarda EIB202, and preservation number is: CCTCC NO: M208068.

Images