CN104857507B - Preparation method and application of oral yeast carrying DNA fragment as novel aquatic product oral DNA vaccine - Google Patents

Abstract

The invention relates to a preparation method for inducing aquatic animals to generate immune response by targeting living bodies of DNA fragments carried by oral yeast to intestinal immune cells of the aquatic animals. The vaccine has low production cost and simple use method, is suitable for large-scale production, and has no toxicity threat to animals compared with the traditional attenuated vaccine. The DNA vaccine can be used for developing and applying various bacterial, viral and parasitic oral DNA vaccines of aquatic animals such as fishes and shellfishes, so that the aquatic animals grow healthily, the loss of aquaculture in the aspect of diseases is reduced, and the defects of the existing DNA vaccine are avoided.

Description

Preparation method and application of oral yeast carrying DNA fragment as novel aquatic product oral DNA vaccine

Technical Field

The application relates to an oral DNA vaccine of yeast carrying DNA fragments, belonging to the field of genetic engineering.

Background

The DNA vaccine is characterized in that pathogen antigen genes are inserted into plasmids capable of being expressed in eukaryotic organisms to obtain recombinant plasmids, after the plasmid DNA is inoculated to fish bodies, the plasmids can be replicated, transcribed and translated into corresponding antigen proteins in the fish bodies, and the immune system of the fish bodies is stimulated by different paths and modes so as to generate immune response to the antigens and play a role in continuous immune protection.

Compared with the traditional fish vaccine, the DNA vaccine has the advantages of simple and convenient preparation process, higher efficiency, attenuated vaccine and no risk of reversion, thereby being more and more valued by scientific researchers. At present, the fish DNA vaccine is injected intramuscularly, but intramuscular injection is the most effective immunization mode of the DNA vaccine, is time-consuming and labor-consuming, has certain limitation in practical application, and is not suitable for popularization and application in large-scale fish culture. In contrast, oral immunization is safe and convenient, and is not limited by the size of the fish body, so that the oral DNA vaccine has more practical advantages. So far, only DNA vaccines aiming at fish infectious hematopoietic necrosis virus are licensed and put into commercial use in Canada all over the world, and domestic licensed commercial oral DNA vaccines applied to aquatic products are not reported yet.

For oral vaccines, the effective carrier can prevent the protective antigen from being digested in the digestive tract of the fish body, thereby ensuring the immune effect of the vaccine. Yeast, as an edible microorganism, has been used for a long time in the preparation of foods such as bread, steamed bread, and beer, and has a high safety; the cell wall of yeast contains a large amount of glucan, and is a high-quality immune polysaccharide; the yeast has stronger immune adjuvant effect; importantly, studies have demonstrated that yeast have a strong resistance to gastrointestinal secretions. Thus, yeast is an ideal carrier for oral vaccines.

Disclosure of Invention

The invention relates to a preparation method for inducing aquatic animals to generate immune response by targeting living bodies of DNA fragments carried by oral yeast to intestinal immune cells of the aquatic animals. The vaccine has low production cost and simple use method, is suitable for large-scale production, and has no toxicity threat to animals compared with the traditional attenuated vaccine. The DNA vaccine can be used for developing and applying various bacterial, viral and parasitic oral DNA vaccines of aquatic animals such as fishes and shellfishes, so that the aquatic animals grow healthily, the loss of aquaculture in the aspect of diseases is reduced, and the defects of the existing DNA vaccine are avoided.

Egg albumin Ovalbumin (OVA) is a good model antigen and is often used in a variety of immunological studies. In this study, the OVA gene was used as an example, and the recombinant yeast contains eukaryotic gene expression vector pRS426-CMV-OVA, which is not expressed in yeast but is expressed in mammalian cells. Eukaryotic gene expression vectors can replicate as yeast plasmids with yeast cells. The recombinant yeast is mixed into fish feed to feed red crucian carp of about 25g, the generation of specific antibody can be detected in the serum of the red crucian carp after immunization for 14 days, and the antibody reaches the peak value in 21 days. The research proves that the oral yeast can carry DNA fragments to target fish immune cells to cause immune response. In this process, the yeast not only plays the role of presenting antigen, but also plays the role of adjuvant. The invention has the function of using the gene engineering yeast as a DNA delivery carrier to target and regulate the intestinal immune cells of aquatic animals, can replace OVA segments of the carrier used in the experiment with antigen genes of various aquatic animal viruses or bacteria and the like, can be applied to the development and production of novel oral DNA vaccines, and realizes the regulation of the immunity of the aquatic animals and the prevention and treatment of diseases.

Drawings

FIG. 1: the PCR products were visualized by 1% agarose gel electrophoresis, in which lane M is Trans2K Plus DNAmarker; lanes 1 and 2 are PCR amplification products of CMV and OVA gene fragments.

FIG. 2: plasmid map of pRS426-CMV-OVA vector.

FIG. 3: pRS426-CMV-OVA, and cleaved with SacII/BamHI/XhoI enzyme, wherein lane M is DNA Marker; lane 1 is a SacII/BamHI/XhoI-digested pRS426-CMV-OVA product

FIG. 4: OVA SDS-PAGE of expression of cells transformed with JMB84-CMV-OVA plasmid, wherein lane 1 is western blot with Easy See protein marker (available from Olympic Ltd.), lane 2 is a protein cleaved from empty cells, and lane 3 is a cellular protein transfected with pRS 426-CMV-OVA.

FIG. 5: OVA protein SDS-PAGE run, where lane 1 is Blue Plus II protein pre-stained marker (from whole gold), lane 2 is 20. mu.L OVA loading, and lane 3 is 30. mu.L OVA loading.

FIG. 6: western Blot to detect the production of OVA antibody in fish serum, wherein lane 1 is Easy See protein marker, lane 2 is PBS control, lane 3 is JMY1 empty yeast control, and lane 4 is an experimental group immunized with pRS426-CMV-OVA yeast.

FIG. 7: ELISA detection result chart for detecting antibody titer at different time after immunization.

FIG. 8 titers of antibodies produced in OVA ① group for 21 days.

Detailed Description

In order to facilitate the understanding of the technical scheme of the invention, the following specific test case combining OVA gene is further used for explaining the method for expressing exogenous gene by oral recombinant yeast (i.e. the method for carrying functional DNA fragment to aquatic animals in a targeted way by utilizing oral genetically engineered fermentation yeast). The present invention will be described in further detail with reference to specific examples.

Example 1 construction of Yeast eukaryotic expression vector pRS426-CMV-OVA

Firstly, PCR (polymerase chain reaction) is carried out to amplify a CMV fragment by taking a carrier plasmid pEGFP-C1(clontech) as a template; the cDNA sequence of OVA (GenBank: J00895.1) was amplified using chicken genomic DNA as a template, followed by an HA tag sequence (YPYDVPDYA).

1. Primer design and Synthesis

Using a vector plasmid pEGFP-C1 as a template, utilizing Clone manager V7 software to design primers for amplifying CMV as CMV-F and CMV-R, wherein a SacII enzyme cutting site is added into the CMV-F, a BamHI enzyme cutting site is added into the CMV-R, and the sequences of the primers are as follows:

CMV-F(SacII):5'TCCccgcggTAGTTATTAATAGTAATCAATTACG 3'

CMV-R(BamHI):5'CGCggatccAGCTCTGCTTATATAGACCTC 3'

designing and amplifying primers OVA-F and OVA-R of OVA by taking chicken genome DNA as a template, introducing a BamHI enzyme cutting site and an HA tag sequence into an upstream primer OVA-F, and introducing an XhoI enzyme cutting site into a downstream primer OVA-R. The length of the amplified fragment was designed to be 1194 bp. The primer sequences were designed as follows:

OVA-F(BamHI)：

CGCggatccATGTACCCATACGACGTTCCAGACTACGCTGGCTCCATCGGCGCAGCAAG

OVA-R(XhoI)：CCGctcgagTTAAGGGGAAACACATCTGCC

wherein: the red font of the 5' end of the primer is a protective base, the lower case letters are corresponding enzyme cutting sites, and the underlined part in the OVA-F primer sequence is an HA tag sequence.

The designed primers were synthesized by Nanjing Kingsler Biotech Co.

PCR amplification of CMV promoter and Chicken Gene OVA fragments

Respectively taking pEGFP-C1 vector plasmid and chicken genome DNA as templates, and CMV-F/CMV-R and OVA-F/OVA-R as primers, amplifying CMV and OVA gene fragments by PCR, wherein the PCR reaction system is shown in Table 1, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min for 20s, and 35 cycles; finally, extension is carried out for 10min at 72 ℃.

TABLE 1 PCR reaction System for amplification of CMV and OVA

The correct size of the PCR product was checked by 1% agarose gel electrophoresis as shown in FIG. 1. Wherein lane M is Trans2K Plus DNA Marker; lanes 1 and 2 are PCR amplification products of CMV and OVA gene fragments.

3. Construction of Yeast expression vector pRS426-CMV-OVA

The target fragment (the enzyme cutting system is shown in the table 2) obtained by recovering the double-enzyme cutting glue of SacII/BamHI and BamHI/XhoI is connected with the yeast eukaryotic expression vector pRS426(Addgene) subjected to double enzyme cutting of BamHI/XhoI, the enzyme cutting system is shown in the table 3, the connecting system is shown in the table 4 and is connected overnight at the temperature of 16 ℃, a plasmid pRS426-CMV-OVA is constructed, then escherichia coli DH5 α competent cells are transformed, LB/Amp plates are coated, and a single clone is picked and cultured for 8 hours at the temperature of 37 ℃ in an LB/Amp liquid culture medium.

TABLE 2 amplified BamHI/XhoI double digestion reaction System for CMV/OVA fragment

TABLE 3 BamHI/XhoI double digestion reaction System of pRS426

TABLE 4 ligation reaction System for CMV, OVA and pRS426

The plasmid map of the constructed pRS426-CMV-OVA yeast expression vector is shown in FIG. 2, and it can be seen that the yeast expression vector is a CMV promoter which can promote the expression of the following gene (OVA) in aquatic animals but cannot express it in yeast. The eukaryotic gene expression cassette replicates as a yeast plasmid with yeast cells.

Extracting plasmid pRS426-CMV-OVA, and carrying out SacII/BamHI/XhoI enzyme digestion and identification, wherein a lane M is a DNA Marker as shown in figure 3; lane 1 is the product of digestion of pRS426-CMV-OVA with SacII/BamHI/XhoI. And (4) sending the positive plasmids to Nanjing Jinslei Biotechnology limited for sequencing analysis, and storing the plasmids with correct sequencing for later use.

Example 2 HA tag antibody detection of pRS426-CMV-OVA expression in HEK293T cells

After the pRS426-CMV-OVA plasmid was sequenced correctly, the plasmid was transfected into HEK293T cells to test whether the OVA protein could be expressed correctly. The HA tag in the expression vector pRS426-CMV-OVA is utilized, the inserted foreign gene fragment can be fused and expressed, and then the HA tag antibody is used for detecting the expression condition of the target protein.

pRS 426-CMV-OVA-transfected HEK293T cells

The HEK293T cell line was cultured in DMEM, 10% fetal bovine serum, 100. mu.g/mL streptomycin in medium at 37 ℃ in a 5% CO2 incubator. Transfection of HEK293T cell line: for example, HEK293T cells were seeded into 12-well plates and replaced with fresh medium when the cell density approached 70%. The medium was aspirated from 12 wells, 500. mu.L of fresh medium preheated at 37 ℃ was added to each well, and transfection was started after 2-4 hours. Two 1.5mL sterile EP tubes were taken, one added with about 2. mu.g of plasmid, followed by the addition of Opti-MEM to a total volume of 50. mu.L; another EP tube was filled with 2. mu.L of So-Fast transfection reagent and Opti-MEM to 50. mu.L. The mixture in both EP tubes was gently mixed, and then the transfection reagent-containing Opti-MEM was slowly added to the plasmid-containing EP tubes with gentle shaking to mix them well. After the addition, the mixture was left at room temperature for 20min, then a transfection mixture was added dropwise to one of the 12 wells, gently shaken several times, the plates were returned to the incubator, and the fresh medium was changed after 12 hours.

Extraction of cellular proteins with RIPA lysis buffer

(1) The cultured cells were collected by centrifugation at 800g at 4 ℃ for 5 minutes, and the volume of the cells after centrifugation was estimated (10)⁶cells＝～20μLPCV,10⁷cells＝～100μL PCV)；

(2) Adding 5 times of volume of RIPA lysis buffer (250-500 mu L) into 50-100 mu L PCV, placing in an ice bath for 10 minutes, and oscillating on a vortex mixer for 30 seconds every 5 minutes;

(3) centrifuging at 12000g and 4 deg.C for 10min, and transferring the supernatant into a new centrifuge tube to obtain total cell protein product;

(4) heating at 95 deg.C for 5min, rapidly ice-cooling for 5min, centrifuging at 12000g and 4 deg.C for 10min, and transferring the supernatant into a new centrifuge tube to obtain total cell protein product.

Western Blot to detect OVA expression in cells

SDS-PAGE separates the cell protein extracts (empty cells and cells transfected with pRS 426-CMV-OVA), the concentration of the concentrated gel of the upper layer of the SDS-PAGE is 5%, the concentration of the separated gel of the lower layer is 12%, the protein loading amount is 30uL, the concentrated gel is firstly used for 30min by 80V and then used for 60min by 100V, and then the concentrated gel is transferred to a PVDF membrane (firstly soaked for 20min by methanol). Sealing the film for 1h at room temperature by using 5 percent of skimmed milk powder after film transfer; adding primary antibody (HA-tag antibody 1:2000), incubating at room temperature for 1h, washing with TBST for 3 times, each time for 10 min; adding secondary antibody (goat anti-mouse IgG 1:5000) and incubating at room temperature for 1h, decolorizing with TBST at room temperature, washing with shaking bed for 3 times, each time for 10 min; then adding a chemiluminescent substrate for developing.

Cells transformed with pRS426-CMV-OVA plasmid expressed OVA at approximately 36kD, as shown in FIG. 4. Wherein, lane 1 is western blot using Easy See protein marker (available from gold, Inc.), lane 2 is a protein obtained by cell lysis, and lane 3 is a cell protein transfected with pRS 426-CMV-OVA.

Example 3 transformation of yeast with pRS426-CMV-OVA plasmid

In a yeast strain JMY1(MAT α, his 3-. DELTA.1 trp1-289rad 1-. DELTA.ura 3-52) which was transformed with the correctly sequenced yeast expression vector pRS426-CMV-OVA by the method of LiAc, respectively:

inoculating single clone JMY1 into 3mLYPD culture medium, and shaking the strain overnight at 30 ℃ and 250 rpm/min; inoculating at 1:50 in the next day, shaking at 30 ℃ and 250rpm/min until OD is about 0.5; centrifuging at 3000rpm/min to collect thallus, washing twice with sterilized water, adding 900 μ L water and 100 μ L1M LiAC (purchased from Sigma), mixing, standing at 30 deg.C for 5min, centrifuging, and collecting thallus; gently resuspending the cells with a 50. mu.L volume of plasmid solution to be transfected (about 1. mu.g), adding 36. mu.L of 1M LiAC and 25. mu.L of ssDNA (Salmon Sperm DNA sigma; cat # 31149-10G-F) denatured at 94 ℃ for 10min on ice, mixing, adding 240. mu.L of 50% PEG600, and vortexing appropriately; centrifuging at 42 deg.C for 45min, collecting bacteria, and incubating at 200rpm/min with 1mLYPD30 deg.C for 1-2 h; the cells were collected by centrifugation, spread evenly on a basic SD solid Medium (Sigma, cat # Y2001-20G; glucose, West chemical Co., Ltd., cat # 14527; YNB (Yeast Nitrogen Base W/O Amino acids), Solambio, cat # Y8040-100; agar, Solambio, cat # A8190), and screened. The screened positive clones were inoculated into liquid SD medium. And extracting yeast plasmids, and carrying out enzyme digestion verification on the screened clones.

Example 4 addition of recombinant Yeast to aquatic feed for immunizing Fish

Inoculating the empty yeast JMY1 strain in YPD culture medium, shaking to OD value of about 1.7, inoculating the yeast with correct pRS426-CMV-OVA, shaking in URA-lacking SD culture medium, shaking to OD value of about 1.7 (1OD 1-2X 10)⁷cells) at 6000rpm/min for 5min, collecting yeast cells, re-suspending thallus with PBS, adding yeast which is re-suspended with PBS after the bought finished feed is crushed, stirring uniformly, granulating, air-drying, storing in a freezer at-20 ℃, manually preparing into granules with the diameter of about 2mm and the length of about 3mm before feeding, wherein the feed of a test group is divided into three groups of low (①), medium (②) and high (③) according to the proportion of the yeast cell number in the feed, and the yeast number in the low test group is about 1.0 multiplied by 10⁸cells/g feed, the number of yeasts in the pilot group was about 1.5X 10 in the feed⁸cells/g feed, high test group yeast count in feed was about 2.0X 10⁸cells/g feed. Each fish (about 25 g) was fed about 3% of body weight per day, so that the yeast feed ratio of the three test groups to fish was about 0.7X 10⁸、1.1×10⁸、1.5×10⁸cells/25g fish weight.

The test red crucian carp is purchased in the Xian aquarium market, healthy red crucian carps with the weight of about 25g are randomly distributed in 5 fish tanks, 5 fish in each fish tank are divided into groups, each group is named as a PBS group, a JMY1 group, an OVA ① group, an OVA ② group and an OVA ③ group, training feeding is carried out in a culture system for one week after the fish are divided for the first time, the fish are made to adapt to the test culture environment completely, conditioned reflex is formed on feeding, the test fish are fasted and disinfected 24 hours before the test feed is fed formally, the test fish are fed formally the next day, 09: 00 and 16: 30 days are fed fully for 2 times respectively, the feeding time is about 40 minutes, the illumination time is 12 hours (08: 00-20: 00) every day, water is changed every other day, and the water temperature is 26-29 ℃.

Example 5 blood sampling

Collecting blood every other week after feeding for 2 weeks, collecting blood from caudal vertebra vein with disposable vacuum blood collection tube (common tube) for about 1ml, immediately bathing in water for 30min, centrifuging at 3000rpm/min at 4 deg.C for 20min, collecting serum, and storing at-20 deg.C. The collected serum samples were sera immunized for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, and 10 weeks, respectively.

Example 6 Western Blot detection of OVA antibody production in Fish serum

OVA protein was purchased from Sigma (cat # A5503), dissolved in PBS to 1. mu.g/. mu.L, added to a sample buffer, boiled in a water bath for 5min, and immediately placed on ice for 5 min. SDS-PAGE gel run, the results of which are shown in FIG. 5. In this case, lane 1 is Blue Plus II protein pre-stained marker (available from Oncorhynchus gold), lane 2 is 20. mu.L OVA loading, and lane 3 is 30. mu.L OVA loading.

The concentration of the SDS-PAGE running OVA protein is 30 mu L, the concentration of the SDS-PAGE upper concentrated gel is 5 percent, and the concentration of the SDS-PAGE lower separated gel is 12 percent. Running at 80V for 30min, running at 100V for 60min, transferring to PVDF membrane (soaked in methanol for 20min) with semi-dry membrane transferring apparatus under 0.8mA/cm². Sealing the film for 1h at room temperature by using 5 percent of skimmed milk powder after film transfer; adding crucian serum (1:50) 35 days after immunization, and incubating overnight at 4 deg.C. Washing with TBST for 10min for 3 times the next day; adding a secondary antibody crucian monoclonal antibody (Aquatic Diagnostics Ltd. (ADL) company, F14), and incubating for 2h at room temperature; TBST washing for 3 times, each time for 10 min; three antibodies (goat anti-mouse IgG 1:5000) were added and incubated at room temperature for 1h, washed 3 times with TBST, 10min each time. Adding chemiluminescence substrate for developing. The results are shown in FIG. 6, in which lane 1 is Easy See protein marker, lane 2 is PBS control, lane 3 is JMY1 empty yeast control, and lane 4 is an immunized yeast containing pRS 426-CMV-OVA.

Example 7 measurement of antibody titer in serum by ELISA

1. Detection of antibody titres at different times after immunization

The purchased OVA protein is diluted to 20 mu g/ml by using prepared coating buffer, added into small holes of an ELISA plate, 100 mu L of each hole is added, the well is placed at 4 ℃ overnight, the coating solution in the reaction holes is poured out the next day and washed three times by using PBS washing buffer, then 100 mu L of red crucian serum to be detected (the dilution ratio is 1:10) is added into the reaction holes coated above, the red crucian serum is placed at room temperature for incubation for 2h, then washed four times by using PBS, 100 mu L of crucian monoclonal antibody is added into all the reaction holes, the red crucian monoclonal antibody is washed four times by using PBS after incubation for 2h, 100 mu L of TMB substrate solution (TIANGEN, catalog number is PA107) is added into each reaction hole after fresh dilution (1:5000) is added into all the reaction holes, 2M sulfuric acid is added into each reaction hole after incubation for 1h, the reaction holes are immediately added into each reaction hole after incubation for 15min, the reaction holes are added with 2M sulfuric acid sulfate solution, the reaction stop value is used as a control solution, the peak value of each reaction hole is immediately detected, and the result is compared with a blank ELISA plate after reaching 357 nm, and the result of the ELISA plate is compared with a blank ELISA plate after the result of the ELISA plate.

2. The titer of the antibody produced by OVA ① group in 21 days is detected

The ELISA assay was as above, where the primary antibody was OVA ① group serum at 21 days in double dilution (initial ratio 1:10, double dilution to 1: 1280). the OD450 values in each well are shown in FIG. 8, and it can be seen that the titer of antibody produced at 21 days in OVA ① group was greater than 1: 1280.

Claims (3)

1. The application of an oral yeast DNA vaccine in preparing a reagent for immunizing aquatic animals is characterized in that: the yeast is a fermentation yeast strain JMY1, the eukaryotic expression vector carried by the yeast is pRS426-CMV-OVA, and the aquatic animal is crucian.

2. Use of an oral yeast DNA vaccine according to claim 1 for the preparation of a reagent for immunizing aquatic animals, characterized in that: after the fermentation yeast JMY1 carries a eukaryotic expression vector pRS426-CMV-OVA to present an antigen OVA encoding fragment, the antigen protein OVA can be expressed in aquatic animal cells and cause the animals to generate specific immune response.

3. Use of an oral yeast DNA vaccine according to claim 2 for the preparation of a reagent for immunizing aquatic animals, characterized in that: the recombinant yeast is cultured according to the yeast number of 1.0 × 10⁸cells/g feed is added into aquatic feed, and the daily feeding amount of yeast feed is 3% of body weight.

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