CN112843225B - Riemerella anatipestifer DNA vaccine based on RA OmpA gene, and preparation method and identification method thereof - Google Patents
Riemerella anatipestifer DNA vaccine based on RA OmpA gene, and preparation method and identification method thereof Download PDFInfo
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Abstract
The invention discloses a RaompA gene riemerella anatipestifer DNA vaccine, which comprises eukaryotic expression plasmids pVAX1-ompA of the RA ompA gene, and when the vaccine is inoculated, 100 mug/recombinant eukaryotic expression plasmids PVAX1-ompA are used for carrying out subcutaneous immunization on the neck of a duckling, and also discloses a preparation method and an identification method. The eukaryotic expression plasmids pVAX1-OmpA and PVAX1-OmpA recombinant plasmids containing the RAOmpA gene are developed, specific immune antibodies can be induced and generated after the duckling is immunized, a stronger immune protection effect can be generated, the eukaryotic expression plasmids pVAX1-OmpA and PVAX1-OmpA gene can be used as candidate strains of novel DNA vaccines, and a foundation is laid for further developing and developing novel RA genetic engineering vaccines. Compared with the prior art, the Riemerella anatipestifer DNA vaccine has better immune protection effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Riemerella anatipestifer DNA vaccine based on an RA OmpA gene, a preparation method and an identification method thereof.
Background
Riemerella anatipestifer disease is a contact infectious disease caused by Riemerella Anatipestifer (RA), also called duck infectious serositis (Infectiosis), new duck disease, duck septicemia, duck epidemic syndrome, duck epidemic pasteurellosis and the like, is an acute or chronic contact infectious disease of various birds such as infected ducks, geese and the like, and has been called goose influenza or goose exudative septicemia after the infection of geese, and has higher morbidity and mortality. Riemerella anatipestifer disease is popular in China, almost all duck raising areas are covered, great loss is caused to the development of duck raising industry, and serious threat is brought to the raising duck industry in the western area.
At present, students at home and abroad aim at researching a novel effective vaccine with protection effect on each serotype RA, wherein DNA vaccine is an ideal development direction. The DNA vaccine can induce humoral and cellular immune responses simultaneously to generate persistent immune responses, can cross-protect different serotypes, and has the advantages of easy production, strong stability, convenient storage and transportation and the like. The key to DNA vaccine development is the selection of target gene sequences and expression vectors, and the selection of one or more antigen components with cross-protection can provide more comprehensive protection. OmpA is used as main outer membrane protein of RA, has high conservation of gene nucleic acid sequence, has good immunogenicity, can excite specific humoral immunity and cytotoxicity reaction, assist other antigen cross presentation, improve the immune response level of organisms, and play a role in immune cross protection on RA of different serotypes, and is a first choice target gene for RA nucleic acid vaccine development. The document "construction of coexpression RAOmpA and duck IL-2 recombinant plasmid and immune research" successfully constructs RAOmpA gene eukaryotic expression plasmid pcDNA3.1 (+) -OmpA, and the result shows that eukaryotic expression plasmid pcDNA3.1 (+) -OmpA can stimulate duck to generate RA specific antibody in vivo by detecting duck immune indexes after immunizing healthy duckling, and has certain cross immune protection effect on serum type 1 and serum type 2 RA.
The immune effect of eukaryotic expression plasmid induced organism is positively correlated with the ability of plasmid vector to express antigen protein, so that high-efficiency plasmid expression vector should be selected for constructing DNA vaccine. pVAX1 is used as an expression vector for efficiently expressing a target gene in a mammalian cell expression system, the size of the vector is 3.0kb, the vector is a novel eukaryotic expression vector reconstructed on the basis of pcDNA3.1, kanamycin is used for replacing Ampicillin resistance screening genes, the possibility of human genome recombination can be effectively reduced, the pVAX1 vector is a non-fusion vector, and the produced protein is almost the same as the naturally occurring protein in structure and function. Because of the advantages of BGHpolyA signal and strong human cytomegalovirus promoter (CMV), large expression capacity, small molecular weight and the like, pVAX1 has been used as eukaryotic expression vector for constructing recombinant nucleic acid vaccines for various infectious diseases or parasitic diseases. The recombinant plasmids pVAX1-Cal-SAG1 and pVAX1-SAG1 are constructed in the literature of the influence of a molecular adjuvant Cal on toxoplasma SAG1 subunit vaccine, and after mice are immunized, ELISA, CCK-8 method and mouse attack experiments prove that the DNA vaccine can induce the organism of the mice to generate humoral and cellular immunity. The recombinant plasmids PVAX1-Hsp65-Ag85B and PVAX1-Hsp65-ESAT6 are constructed in the literature of construction of a mycobacterium tuberculosis Hsp65-Ag85B, hsp-ESAT 6 fusion gene DNA vaccine strain and immunogenicity research, and after the expression of the recombinant plasmids is verified by in vitro transfection, the results of an immune C57BL/6 mouse experiment show that the Hsp65-Ag85B, hsp-ESAT 6DNA vaccine strain can excite stronger immune response.
Patent application CN201410098187.3 discloses a DNA vaccine based on OmpA gene Riemerella anatipestifer, which is characterized in that: eukaryotic expression plasmid pcDNA3.1 (+) -OmpA containing Riemerella anatipestifer OmpA gene with plasmid concentration of 2-5 mg/mL contains total gene coding sequence of Riemerella anatipestifer OmpA, the length of the gene is 1164 nucleotides, the type is nucleotides, and the total length is coding region. The Riemerella anatipestifer vaccine is safe and effective, can induce duck organisms to generate specific humoral immune response, and can effectively prevent the generation and the epidemic of the Riemerella anatipestifer.
However, the existing vaccine has an insufficient immune protection effect, so that in order to obtain a more eukaryotic expression recombinant plasmid of the riemerella anatipestifer outer membrane protein A gene, a scheme of the application is researched and obtained.
Disclosure of Invention
The invention provides a Riemerella anatipestifer DNA vaccine based on an RA OmpA gene, a preparation method and an identification method thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a DNA vaccine based on RA OmpA gene Riemerella anatipestifer comprises eukaryotic expression plasmid pVAX1-OmpA of RA OmpA gene.
Further, the eukaryotic expression plasmid pVAX1-OmpA was extracted using E.coli and prepared using pVAX1 as a vector, and 10 ten thousand. Mu.g of the recombinant plasmid PVAX1-OmpA was contained in each bottle of vaccine.
Furthermore, the DNA vaccine based on the RA OmpA gene Riemerella anatipestifer is used for subcutaneously immunizing the neck of the duckling by using 100 mug/recombinant eukaryotic expression plasmid PVAX1-OmpA during the inoculation.
Further, the preparation method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) Preparation of polyclonal antisera: constructing an RA OmpA gene into a pET32a+ prokaryotic expression vector, carrying out induction expression on recombinant expression plasmid pET32a+ -RA-OmpA transformed competent cells BL21, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, loading a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni+ column affinity purification on the recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, carrying out subcutaneous immunization for 0.5 mg/time, using Freund's incomplete adjuvant to emulsify the recombinant protein for boosting once every interval 14d, detecting the titer of white rabbit antiserum on the PA OPMA recombinant protein through an indirect ELISA method, collecting blood with the titer of 1:12800 white rabbit after 3 times immunization, and separating serum to obtain rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal serum;
(2) Primer design and synthesis: according to the RAOmpA gene sequence in GenBank, combining pVAX1 eukaryotic expression vector multiple cloning sites, designing a specific primer of the RA OmpA gene by utilizing Primerpremier5.0, and designing a synthetic primer by taking XhoI and BamHI as enzyme cutting sites;
(3) Construction of RA OmpA gene eukaryotic expression plasmid: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double digestion by restriction enzymes XhoI and BamHI, are connected and then are transformed into DH5 alpha competent cells, are coated on a kanamycin-resistant LB plate, are cultured overnight at 37 ℃, are selected from single white colonies on the transformed kanamycin LB plate, are inoculated into a kanamycin-containing LB liquid medium and are cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, double digestion identification is carried out, and the extracted recombinant plasmids are used for PCR identification, so that the Riemerella anatipestifer eukaryotic vaccine is obtained.
Further, in step (2), the specific primer used is synthesized by Shanghai Biotechnology, inc., the upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', underlined is the XhoI enzyme recognition site; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', underlined is the BamHI enzyme recognition site.
Further, the identification method based on the RA OmpA gene Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) transfection: extracting the concentrations and purities of eukaryotic expression plasmids PVAX1-OmpA and empty plasmid pVAX1 identified to be correct by using an endotoxin-free plasmid extraction kit, selecting plasmids with the OD260 to OD280 ratio of 1.8-2.0 to transfect DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, when the cultured cells grow to 80-100% of a monolayer by using a DMEM nutrient solution containing 10% fetal bovine serum, discarding the supernatant, washing the supernatant for 2-3 times by using a PBS buffer, respectively transfecting the recombinant plasmids PVAX1-OmpA and empty plasmids pVAX1 into DF-1 cells according to a liposome transfection reagent specification, and performing no treatment on a blank group at 37 ℃ and 5% CO 2 Culturing in an incubator for 5 hours, and then replacing a new DMEM culture solution containing 5% fetal bovine serum for further culturing for 48 hours;
(2) PCR amplification and electrophoresis detection: extracting total RNA of cells by using an RNA extraction kit, performing cDNA synthesis by referring to a HiFiScript cDNA first-strand synthesis kit instruction, performing PCR amplification by using cDNA as a template, and performing electrophoresis detection on a 1.2% agarose gel containing a nucleic acid dye by using a 6 mu LPCR amplification product;
(3) detecting expression of RA OmpA gene in DF-1 cells: collecting DF-1 cells 48h after transfection, and washing 3 times by precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with pre-cooled PBS for 3 times; adding 0.3% Triton X-100, permeabilizing at room temperature for 20min, discarding Triton X-100, and washing with precooled PBS for 3 times; adding the riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing with precooled PBS for 3 times; FITC-labeled goat anti-rabbit IgG is added, incubated for 1h at 37 ℃, FITC-labeled goat anti-rabbit IgG is removed, and the mixture is washed with precooled PBS for 3 times; adding nuclear dye DAPI, allowing the mixture to act at room temperature for 4min, and washing the mixture with precooled PBS for 3 times; observing the result under a fluorescence microscope;
(4) detecting expression of the recombinant protein: DF-1 cells 48h after transfection were collected for lysis, 12% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, and developed with ECL using rabbit anti-RA OmpA prokaryotic expression recombinant protein serum as primary antibody and goat anti-rabbit HRP-IgG as secondary antibody.
Further, in step (2), the PCR reaction system is: 25. Mu.L, 2 XPCRMIX 12.5. Mu.L, template 2. Mu.L, 10mol/L of each of the upstream and downstream primers 1. Mu.L, ddH were taken 2 O was made up to 25. Mu.L; the PCR reaction conditions were: pre-denatured at 94℃for 3min, denatured at 94℃for 45s, annealed at 52℃for 1min, extended at 72℃for 1min, and after 30 cycles, extended at 72℃for 10min.
Further, the nucleic acid dye is goldbiew.
Further, the solid-to-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1:500.
Further, the solid-to-liquid ratio of the goat anti-rabbit HRP-IgG is 1:2000.
The positive plasmid PUC57-OmpA used in the present application was constructed by Shanghai Biotechnology; the pVAX1 eukaryotic expression vector is presented by a fresh Simei teacher at the university of Guizhou animal science college; DF-1 cells were purchased from Torulous Biocompany; DNA gel recovery kit and plasmid miniprep kit are purchased from OMEGA; DNALigationKit, DNAMarkerIII, DNA restriction enzymes XhoI and BamHI were purchased from Takara; e.coli DH5 alpha, DL2000, hiFiScript cDNA first strand synthesis kit and endotoxin-free plasmid extraction kit are all purchased from Tiangen biochemical technology; goat anti-rabbit IgG-FITC was purchased from beijing boaosen. Trivalent inactivated vaccine for duck infectious serositis (ZJ 01 strain +2 HN01 strain +7 YC03 strain) is purchased from Holuanimal health products Co. HRP-labeled goat anti-duck IgG was purchased from KPL company in the united states. RA antibody negative duckling was purchased from the institute of ecological foods, inc.
The eukaryotic expression plasmid pVAX1-OmpA of the RA OmpA gene is constructed by taking pVAX1 as a eukaryotic expression vector, identification is carried out through plasmid PCR, double digestion and sequencing, the identified positive plasmid pVAX1-OmpA is transfected into DF-1 cells, and the RT-PCR, IFA, westernblot test proves that the eukaryotic expression plasmid pVAX1-OmpA can be expressed in DF-1 cells transiently. Meanwhile, in the IFA test, pVAX1-OmpA eukaryotic expression protein can be identified by polyclonal antibodies obtained by immunizing rabbits with OmpA gene prokaryotic expression protein, and combined with goat anti-rabbit IgG-FITC, green fluorescence is shown in cytoplasma, and a Westernblot result also shows that a band appears at about 41KD after a recombinant plasmid is transfected into cells, so that the eukaryotic expression protein has immunogenicity.
According to the application, PVAX1-OmpADNA is immunized against duckling, eukaryotic expression plasmids pVAX1-OmpA are successfully constructed, specific expression of OmpA protein is detected by both methods, the recombinant plasmids PVAX1-OmpA test group is extremely remarkable (P < 0.01) or is remarkably (P < 0.05) lower than an inactivated vaccine control group in 7d and 21d, the differences of 14d, 28d, 35d, 49d and 63d are not remarkable, the immune protection rate of the duckling reaches 100%, the result shows that the induced humoral immunity level of the recombinant plasmids PVAX1-OmpA group is equivalent to that of an inactivated vaccine group after 28d, and the toxicity attacking experiment result shows that the immune protection effect of the recombinant plasmids PVAX1-OmpA group on duckling can be higher than that of the inactivated vaccine group after the recombinant plasmids PVAX1-OmpA group are immunized for 21 d. The result shows that PVAX1-OmpA recombinant plasmid can induce specific immune antibody after immunization of duckling, can generate stronger immune protection effect, and can be used as a candidate strain of a novel DNA vaccine.
Because the invention adopts the technical proposal, the invention has the following beneficial effects:
based on the fact that Omp A has high conservation among isolated RA strains, cross immunogenicity exists among Omp A proteins of RA strains with different serotypes, an RA vaccine aiming at multiple serotypes can be prepared, eukaryotic expression plasmids pVAX1-ompA containing RA ompA genes are developed, specific immune antibodies can be induced and generated after duckling is immunized by PVAX1-ompA recombinant plasmids, a relatively strong immune protection effect can be generated, and the strain can be used as a candidate strain of a novel DNA vaccine, and lays a foundation for further developing and developing novel RA genetic engineering vaccines. Compared with the prior art, the Riemerella anatipestifer DNA vaccine has better immune protection effect.
Drawings
For a clearer description of an example of the invention or of a technical solution in the prior art, the drawings required in the description of the embodiment or of the prior art will be briefly described, it being obvious that the drawings in the description below are only some examples of the invention, from which, without the inventive development, other drawings can be obtained for a person skilled in the art:
FIG. 1 is a diagram showing the PCR identification result of eukaryotic expression plasmid pVAX1-OmpA of the present application;
FIG. 2 is a diagram showing the results of double cleavage identification of eukaryotic expression plasmid pVAX1-OmpA of the present application;
FIG. 3 is a diagram showing the results of RT-PCR detection of eukaryotic expression plasmid pVAX1-OmpA of the present application;
FIG. 4 is a graph showing the result of IFA detection after the recombinant eukaryotic expression plasmid PVAX1-OmpA is transfected into DF-1 cells for 48 hours;
FIG. 5 is a graph showing the detection results of Merge treatment IFA after the recombinant eukaryotic expression plasmid PVAX1-OmpA is transfected into DF-1 cells for 48 hours;
FIG. 6 is a diagram of the result of the Westernblot detection after transfection of eukaryotic expression plasmid PVAX1-OmpA of the present application;
FIG. 7 is a graph showing dynamic changes of antibodies of different immune groups according to the present application;
FIG. 8 is a graph showing the results of toxicity protection tests of PVAX1-OmpADNA vaccine of the present application against RA infection of duckling 21 d.
In the accompanying drawings: 1-PVAX1-OmpA; 2-negative control; 3-PVAX1-OmpA double enzyme digestion; 4-PVAX1; 5-normal cell control; transfecting DF-1 cells with 6-PVAX 1-OmpA; transfecting DF-1 cells with PVAX1 empty vector; M-DL2000; K-DL5000; P-D2000; R-ProteinMark.
Detailed Description
The following detailed description of the invention is provided in further detail, but the invention is not limited to these embodiments, any modifications or substitutions in the basic spirit of the present examples, which still fall within the scope of the invention as claimed.
Example 1
A DNA vaccine based on RA OmpA gene Riemerella anatipestifer comprises eukaryotic expression plasmid pVAX1-OmpA of RA OmpA gene; the eukaryotic expression plasmid pVAX1-OmpA is extracted by adopting escherichia coli, and is prepared by taking pVAX1 as a carrier, and each bottle of vaccine contains 10 ten thousand mug of recombinant plasmid PVAX1-OmpA; at the time of inoculation, the neck of the duckling was subjected to subcutaneous immunization with 100. Mu.g/recombinant eukaryotic expression plasmid PVAX1-OmpA.
The preparation method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) Preparation of polyclonal antisera: constructing an RA OmpA gene into a pET32a+ prokaryotic expression vector, carrying out induction expression on recombinant expression plasmid pET32a+ -RA-OmpA transformed competent cells BL21, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, loading a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni+ column affinity purification on the recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, carrying out subcutaneous immunization for 0.5 mg/time, using Freund's incomplete adjuvant to emulsify the recombinant protein for boosting once every interval 14d, detecting the titer of white rabbit antiserum on the PA OPMA recombinant protein through an indirect ELISA method, collecting blood with the titer of 1:12800 white rabbit after 3 times immunization, and separating serum to obtain rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal serum;
(2) Primer design and synthesis: according to the RA OmpA gene sequence in GenBank, combining pVAX1 eukaryotic expression vector multiple cloning sites, designing a specific primer of the RA OmpA gene by utilizing Primerpremier5.0, and designing a synthetic primer by taking XhoI and BamHI as enzyme cutting sites;
the specific primers used were synthesized by Shanghai Biotechnology, an upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', underlined is the XhoI enzyme recognition site; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', underlined are BamHI enzyme recognition sites;
(3) Construction of RA OmpA gene eukaryotic expression plasmid: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double digestion by restriction enzymes XhoI and BamHI, are connected and then are transformed into DH5 alpha competent cells, are coated on a kanamycin-resistant LB plate, are cultured overnight at 37 ℃, are selected from single white colonies on the transformed kanamycin LB plate, are inoculated into a kanamycin-containing LB liquid medium and are cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, double digestion identification is carried out, and the extracted recombinant plasmids are used for PCR identification, so that the Riemerella anatipestifer eukaryotic vaccine is obtained.
The identification method based on RA OmpA gene Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) transfection: extracting the concentrations and purities of eukaryotic expression plasmids PVAX1-OmpA and empty plasmid pVAX1 identified to be correct by using an endotoxin-free plasmid extraction kit, selecting plasmids with the OD260 to OD280 ratio of 1.8-2.0 to transfect DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, when the cultured cells grow to 80-100% of a monolayer by using a DMEM nutrient solution containing 10% fetal bovine serum, discarding the supernatant, washing the supernatant for 2-3 times by using a PBS buffer, respectively transfecting the recombinant plasmids PVAX1-OmpA and empty plasmids pVAX1 into DF-1 cells according to a liposome transfection reagent specification, and performing no treatment on a blank group at 37 ℃ and 5% CO 2 Culturing in an incubator for 5 hours, and then replacing a new DMEM culture solution containing 5% fetal bovine serum for further culturing for 48 hours;
(2) PCR amplification and electrophoresis detection: extracting total RNA of cells by using an RNA extraction kit, performing cDNA synthesis by referring to a HiFiScript cDNA first-strand synthesis kit instruction, performing PCR amplification by using cDNA as a template, and performing electrophoresis detection on a 1.2% agarose gel containing a nucleic acid dye by using a 6 mu LPCR amplification product;
(3) detecting expression of RA OmpA gene in DF-1 cells: collecting DF-1 cells 48h after transfection, and washing 3 times by precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with pre-cooled PBS for 3 times; adding 0.3% Triton X-100, permeabilizing at room temperature for 20min, discarding Triton X-100, and washing with precooled PBS for 3 times; adding the riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing with precooled PBS for 3 times; FITC-labeled goat anti-rabbit IgG is added, incubated for 1h at 37 ℃, FITC-labeled goat anti-rabbit IgG is removed, and the mixture is washed with precooled PBS for 3 times; adding nuclear dye DAPI, allowing the mixture to act at room temperature for 4min, and washing the mixture with precooled PBS for 3 times; observing the result under a fluorescence microscope;
(4) detecting expression of the recombinant protein: DF-1 cells 48h after transfection were collected for lysis, 12% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, and developed with ECL using rabbit anti-RA OmpA prokaryotic expression recombinant protein serum as primary antibody and goat anti-rabbit HRP-IgG as secondary antibody.
Further, the PCR reaction system is as follows: 25. Mu.L, 2 XPCRMIX 12.5. Mu.L, template 2. Mu.L, 10mol/L of each of the upstream and downstream primers 1. Mu.L, ddH were taken 2 O was made up to 25. Mu.L; the PCR reaction conditions were: pre-denaturation at 94℃for 3min, denaturation at 94℃for 45s, annealing at 52℃for 1min, extension at 72℃for 1min,30 cycles, extension at 72℃for 10min; the nucleic acid dye is GoldView; the solid-to-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1:500; the solid-to-liquid ratio of the goat anti-rabbit HRP-IgG is 1:2000.
Example 2
A DNA vaccine based on RA OmpA gene Riemerella anatipestifer comprises eukaryotic expression plasmid pVAX1-OmpA of RA OmpA gene; the eukaryotic expression plasmid pVAX1-OmpA is extracted by adopting escherichia coli, and is prepared by taking pVAX1 as a carrier, and each bottle of vaccine contains 10 ten thousand mug of recombinant plasmid PVAX1-OmpA; at the time of inoculation, the neck of the duckling was subjected to subcutaneous immunization with 100. Mu.g/recombinant eukaryotic expression plasmid PVAX1-OmpA.
The eukaryotic expression plasmid pVAX1-OmpA of the RA OmpA gene is identified by PCR, double digestion and sequencing, the result shows that the OmpA gene is connected with the PVAX1 vector correctly, the size of the OmpA gene fragment is about 1149bp, the recombinant eukaryotic expression plasmid which is identified as positive by the double digestion and sequencing result is named PVAX1-OmpA, and the identification result is shown in figures 1 and 2.
The preparation method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) Preparation of polyclonal antisera: constructing an RA OmpA gene into a pET32a+ prokaryotic expression vector, carrying out induction expression on recombinant expression plasmid pET32a+ -RA-OmpA transformed competent cells BL21, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, loading a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni+ column affinity purification on the recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, carrying out subcutaneous immunization for 0.5 mg/time, using Freund's incomplete adjuvant to emulsify the recombinant protein for boosting once every interval 14d, detecting the titer of white rabbit antiserum on the PA OPMA recombinant protein through an indirect ELISA method, collecting blood with the titer of 1:12800 white rabbit after 3 times immunization, and separating serum to obtain rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal serum;
(2) Primer design and synthesis: according to the RA OmpA gene sequence in GenBank, combining pVAX1 eukaryotic expression vector multiple cloning sites, designing a specific primer of the RA OmpA gene by utilizing Primerpremier5.0, and designing a synthetic primer by taking XhoI and BamHI as enzyme cutting sites;
the specific primers used were synthesized by Shanghai Biotechnology, an upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', underlined is the XhoI enzyme recognition site; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', underlined are BamHI enzyme recognition sites;
(3) Construction of RA OmpA gene eukaryotic expression plasmid: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double digestion by restriction enzymes XhoI and BamHI, are connected and then are transformed into DH5 alpha competent cells, are coated on a kanamycin-resistant LB plate, are cultured overnight at 37 ℃, are selected from single white colonies on the transformed kanamycin LB plate, are inoculated into a kanamycin-containing LB liquid medium and are cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, double digestion identification is carried out, and the extracted recombinant plasmids are used for PCR identification, so that the Riemerella anatipestifer eukaryotic vaccine is obtained.
The identification method based on RA OmpA gene Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) transfection: extracting the concentrations and purities of eukaryotic expression plasmids PVAX1-OmpA and empty plasmid pVAX1 which are identified to be correct by using an endotoxin-free plasmid extraction kit, selecting plasmids with the OD260 to OD280 ratio of 1.8-2.0 to be corresponding to the plasmids and transfecting DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, when the cultured cells grow to 80-100 percent of monolayers by using a DMEM nutrient solution containing 10 percent fetal bovine serum, discarding the supernatant, washing the cells for 2-3 times by using a PBS buffer solution, respectively transfecting the recombinant plasmids PVAX1-OmpA and the empty plasmid pVAX1 into the DF-1 cells according to a liposome transfection reagent instruction book,blank group without any treatment, 37 ℃, 5% CO 2 Culturing in an incubator for 5 hours, and then replacing a new DMEM culture solution containing 5% fetal bovine serum for further culturing for 48 hours;
(2) PCR amplification and electrophoresis detection: extracting total RNA of cells by using an RNA extraction kit, performing cDNA synthesis by referring to a HiFiScript cDNA first-strand synthesis kit instruction, performing PCR amplification by using cDNA as a template, and performing electrophoresis detection on a 1.2% agarose gel containing a nucleic acid dye by using a 6 mu LPCR amplification product;
(3) detecting expression of the RAOmpA gene in DF-1 cells: collecting DF-1 cells 48h after transfection, and washing 3 times by precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with pre-cooled PBS for 3 times; adding 0.3% Triton X-100, permeabilizing at room temperature for 20min, discarding Triton X-100, and washing with precooled PBS for 3 times; adding the riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing with precooled PBS for 3 times; FITC-labeled goat anti-rabbit IgG is added, incubated for 1h at 37 ℃, FITC-labeled goat anti-rabbit IgG is removed, and the mixture is washed with precooled PBS for 3 times; adding nuclear dye DAPI, allowing the mixture to act at room temperature for 4min, and washing the mixture with precooled PBS for 3 times; observing the result under a fluorescence microscope;
(4) detecting expression of the recombinant protein: DF-1 cells 48h after transfection were collected for lysis, 12% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, and developed with ECL using rabbit anti-RAOmpA prokaryotic expression recombinant protein serum as primary antibody and goat anti-rabbit HRP-IgG as secondary antibody.
Further, the PCR reaction system is as follows: 25. Mu.L, 2 XPCRMIX 12.5. Mu.L, template 2. Mu.L, 10mol/L of each of the upstream and downstream primers 1. Mu.L, ddH were taken 2 O was made up to 25. Mu.L; the PCR reaction conditions were: pre-denaturation at 94℃for 3min, denaturation at 94℃for 45s, annealing at 52℃for 1min, extension at 72℃for 1min,30 cycles, extension at 72℃for 10min; the nucleic acid dye is GoldView; the solid-to-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1:500; the solid-to-liquid ratio of the goat anti-rabbit HRP-IgG is 1:2000.
1. RT-PCR detection of eukaryotic expression plasmid transient expression results
After the eukaryotic expression plasmids PVAX1-OmpA and PVAX1 are transfected into DF-1 cells for 48 hours, total RNA of the cells is extracted, and the target gene expression is detected by an RT-PCR method, and the result is shown in figure 3.
2. IFA detection of eukaryotic expression plasmid transient expression results
The IFA detection result shows that after the eukaryotic expression plasmid PVAX1-OmpA is transfected into DF-1 cells for 48 hours, the cytoplasm of the cells can be seen to have green fluorescence, as shown in figure 4; performing Merge processing on the obtained protein fluorescence localization picture and the corresponding cell nucleus fluorescence picture by using Photoshop CS3 software, and judging subcellular localization of the recombinant protein through fluorescence co-localization, wherein the subcellular localization of the recombinant protein is shown in FIG. 5; the PVAX1 empty vector group and the normal cell group did not see green fluorescence, showing that the view field was black.
3. Westernblot detection of recombinant protein expression
48h after the recombinant eukaryotic expression plasmid pVAX1-OmpA is transfected into DF-1 cells, western blot is carried out after cell lysis is collected, and the result shows that the cell transfected recombinant plasmid has a band around 41KD, the size of which is consistent with that of a predicted result, and no band exists in an empty vector, as shown in FIG. 6.
4. Protection test of PVAX1-OmpADNA vaccine against duckling infection RA
Recombinant eukaryotic expression plasmids pVAX1-OmpA and PVAX1 empty plasmids were extracted using an endotoxin-free plasmid large extraction kit, and the nucleic acid concentration was determined. 1 day old RA antibody negative healthy duckling 120 were raised to 10 days old to adapt to the environment, and randomly divided into 4 groups, 30 groups, 100 μg/PVAX 1-OmpA plasmid immunized group, 100 μg/PVAX 1 plasmid blank control group, 0.2mL inactivated vaccine control group, and 0.2mL physiological saline control group. The duckling was vaccinated subcutaneously at the neck and immunized for 7d and then immunized for a second time. After the first immunization, 10 ducklings of each group were randomly collected at 7d, 14d, 21d, 28d, 49d and 63d, and serum was collected and separated.
Detecting the RA antibody level of the immunized duckling by adopting an indirect ELISA method, coating 5 mu g of RAOmpA prokaryotic expression recombinant protein on each hole of an ELISA plate, diluting the duckling serum by 1:100, incubating for 1h at 37 ℃, washing 3 times by PBS, adding HRP-labeled goat anti-duck IgG (1:2000), incubating for 45min at 37 ℃, washing 3 times by PBS, developing for 15min at room temperature by TMB, terminating reaction by 2M sulfuric acid, and measuring the OD450nm value. As a result, it was found that the recombinant eukaryotic expression plasmid pVAX1-OmpA test group was extremely remarkable (P < 0.01) or remarkably (P < 0.05) lower than the inactivated vaccine control group at 7d, 21d, while the differences of 14d, 28d, 35d, 49d, 63d were not remarkable, as shown in FIG. 7.
In the toxicity attack protection test, 10 test ducks which are subjected to the 14d after the two-stage treatment are randomly extracted from each group, 1-type RA bacterial suspension with the bacterial liquid concentration of 4 multiplied by 107CFU/mL (2 times of LD50 dosage) is adopted, the toxicity attack protection rate is measured through leg intramuscular injection inoculation, and the morbidity or mortality of ducklings in 7d after the toxicity attack is observed. The result shows that: the protection rate of the recombinant eukaryotic expression plasmid pVAX1-OmpA test group 21d reaches 100%, the protection rate of the inactivated vaccine control group 21d reaches 90%, and the results of the PVAX1 plasmid empty vector control group and the physiological saline control group show that: the 21d protection rates were 10% as shown in fig. 8.
Comparative example 1
The preparation of the riemerella anatipestifer DNA vaccine was carried out according to the method given in patent application CN 201410098187.3.
In order to further illustrate that the technical effect of the invention can be achieved, the following experiment is carried out:
the method comprises the steps of selecting 90 young ducks which are quite large, have quite reasonable month-old and physical signs and are all infected with the riemerella anatipestifer, randomly and averagely dividing the young ducks into three groups, wherein 30 young ducks in each group have quite equal proportion, respectively carrying out vaccine on the riemerella anatipestifer DNA vaccine prepared in the embodiment 1 and the comparative example 1 of the application on the young ducks, observing the morbidity or mortality of each group of young ducks, recording whether the vaccine is effective or not, and the experimental results are shown in the following table 1.
TABLE 1
| Group of | Morbidity (only) | Death (only) | Survival (only)) | Vaccine effective rate |
| Example 1 | 4 | 2 | 28 | 93.33％ |
| Example 2 | 3 | 1 | 29 | 96.67％ |
| Comparative example 1 | 12 | 8 | 22 | 73.33％ |
As shown in the experimental results of Table 1, compared with the prior art, the Riemerella anatipestifer DNA vaccine prepared by the application has better effect of resisting Riemerella anatipestifer and can play a good role in vaccine.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Sequence listing
<120> Riemerella anatipestifer DNA vaccine based on RA OmpA gene and preparation method thereof
<160> 2
<210> 1
<211> 27
<212> DNA
<213> upstream primer
<400> 1
ggatccatgt tgatgactgg acttggt 27
<210> 2
<211> 31
<212> DNA
<213> downstream primer
<400> 2
ctcgagttat tttcttttct tttttactac t 31
Claims (8)
1. A Riemerella anatipestifer DNA vaccine based on RA OmpA gene is characterized in that: eukaryotic expression plasmid pVAX1-OmpA including RA OmpA gene;
the eukaryotic expression plasmid pVAX1-OmpA is extracted by adopting escherichia coli, and is prepared by taking pVAX1 as a carrier, and each bottle of vaccine contains 10 ten thousand mug of recombinant plasmid PVAX1-OmpA;
at the time of inoculation, the neck of the duckling was subjected to subcutaneous immunization with 100. Mu.g/recombinant eukaryotic expression plasmid PVAX1-OmpA.
2. A method for preparing a RA OmpA gene-based riemerella anatipestifer DNA vaccine according to claim 1, comprising the steps of:
(1) Preparation of polyclonal antisera: constructing an RA OmpA gene into a pET32a+ prokaryotic expression vector, carrying out induction expression on a recombinant expression plasmid pET32a+ RA-OmpA transformed competent cell BL21, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, loading a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni+ column affinity purification on the recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, carrying out subcutaneous immunization for 0.5 mg/time, using Freund's incomplete adjuvant to emulsify the recombinant protein for boosting once every interval 14d, detecting the titer of white rabbit antiserum on the RA OPMA by an indirect ELISA method, collecting blood with the titer of 1:12800 white rabbits after 3 times of immunization, and separating serum to obtain rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal serum;
(2) Primer design and synthesis: according to the RA OmpA gene sequence in GenBank, combining pVAX1 eukaryotic expression vector multiple cloning sites, designing a specific primer of the RA OmpA gene by utilizing Primerpremier5.0, and designing a synthetic primer by taking XhoI and BamHI as enzyme cutting sites;
(3) Construction of RA OmpA gene eukaryotic expression plasmid: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double digestion by restriction enzymes XhoI and BamHI, are connected and then are transformed into DH5 alpha competent cells, are coated on a kanamycin-resistant LB plate, are cultured overnight at 37 ℃, are selected from single white colonies on the transformed kanamycin LB plate, are inoculated into a kanamycin-containing LB liquid medium and are cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, double digestion identification is carried out, and the extracted recombinant plasmids are used for PCR identification, so that the Riemerella anatipestifer DNA vaccine is obtained.
3. The method for preparing the Riemerella anatipestifer DNA vaccine based on the RA OmpA gene according to claim 2, which is characterized in that: in step (2), the specific primers used were synthesized by Shanghai Biotechnology, inc., the upstream primers: 5 '-GGGatCC_ A T G T T G A T G A C T G G A C T T G G T-3', GGatCC is the XhoI enzyme recognition site; a downstream primer: 5'-CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', CTCGAG is a BamHI enzyme recognition site.
4. A method for identifying a DNA vaccine of riemerella anatipestifer based on the RA OmpA gene as claimed in claim 1, comprising the steps of:
(1) transfection: the concentrations and the purities of the eukaryotic expression plasmids PVAX1-OmpA and empty plasmid pVAX1 which are identified to be correct are extracted by using an endotoxin-free plasmid extraction kit, and the OD260 to OD280 ratio is selectedPlasmid corresponding to 1.8-2.0 is transfected into DF-1 cells, DF-1 cells are inoculated into a 6-hole cell culture plate, when the cells are cultured to 80-100 percent of single layer by using DMEM nutrient solution containing 10 percent of fetal bovine serum, the supernatant is discarded, PBS buffer is washed for 2-3 times, recombinant plasmids PVAX1-OmpA and empty plasmid pVAX1 are respectively transfected into DF-1 cells according to liposome transfection reagent specifications, the blank group is not subjected to any treatment, the temperature is 37 ℃ and the concentration of CO is 5 percent 2 Culturing in an incubator for 5 hours, and then replacing a new DMEM culture solution containing 5% fetal bovine serum for further culturing for 48 hours;
(2) PCR amplification and electrophoresis detection: extracting total RNA of cells by using an RNA extraction kit, performing cDNA synthesis by referring to a HiFiScript cDNA first-strand synthesis kit instruction, performing PCR amplification by using cDNA as a template, and performing electrophoresis detection on a 1.2% agarose gel containing a nucleic acid dye by using a 6 mu LPCR amplification product;
(3) detecting expression of RA OmpA gene in DF-1 cells: collecting DF-1 cells 48h after transfection, and washing 3 times by precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with pre-cooled PBS for 3 times; adding 0.3% Triton X-100, permeabilizing at room temperature for 20min, discarding Triton X-100, and washing with precooled PBS for 3 times; adding the riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing with precooled PBS for 3 times; FITC-labeled goat anti-rabbit IgG is added, incubated for 1h at 37 ℃, FITC-labeled goat anti-rabbit IgG is removed, and the mixture is washed with precooled PBS for 3 times; adding nuclear dye DAPI, allowing the mixture to act at room temperature for 4min, and washing the mixture with precooled PBS for 3 times; observing the result under a fluorescence microscope;
(4) detecting expression of the recombinant protein: DF-1 cells 48h after transfection were collected for lysis, 12% SDS-PAGE gel electrophoresis, transferred to PVDF membrane, and developed with ECL using rabbit anti-RA OmpA prokaryotic expression recombinant protein serum as primary antibody and goat anti-rabbit HRP-IgG as secondary antibody.
5. The method for identifying the Riemerella anatipestifer DNA vaccine based on the RA OmpA gene according to claim 4, wherein the method comprises the following steps: in the step (2), the reaction system of the PCR amplification is as follows: 25. Mu.L, 2 XPCRMIX 12.5. Mu.L, template 2. Mu.L, 10 mol/. Mu.L of each of the upstream and downstream primers 1. Mu.L, ddH were taken 2 O is supplemented to 25 mu LThe method comprises the steps of carrying out a first treatment on the surface of the The PCR reaction conditions were: pre-denatured at 94℃for 3min, denatured at 94℃for 45s, annealed at 52℃for 1min, extended at 72℃for 1min, and after 30 cycles, extended at 72℃for 10min.
6. The method for identifying the Riemerella anatipestifer DNA vaccine based on the RA OmpA gene according to claim 4, wherein the method comprises the following steps: the nucleic acid dye is Gold View.
7. The method for identifying the Riemerella anatipestifer DNA vaccine based on the RA OmpA gene according to claim 4, wherein the method comprises the following steps: the solid-to-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1:500.
8. The method for identifying the Riemerella anatipestifer DNA vaccine based on the RA OmpA gene according to claim 4, wherein the method comprises the following steps: the solid-to-liquid ratio of the goat anti-rabbit HRP-IgG is 1:2000.
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| CN104046597A (en) * | 2014-06-05 | 2014-09-17 | 北京市农林科学院 | HVT (Herpesvirus of turkey) recombinant for expressing chlamydia psittaci pmpD-N gene and construction thereof |
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