CN112843225A - RA OmpA gene-based Riemerella anatipestifer DNA vaccine and preparation method and identification method thereof - Google Patents
RA OmpA gene-based Riemerella anatipestifer DNA vaccine and preparation method and identification method thereof Download PDFInfo
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Abstract
The invention discloses a RAOmpA gene-based Riemerella anatipestifer DNA vaccine, which comprises an RA OmpA gene eukaryotic expression plasmid pVAX1-OmpA, wherein when in inoculation, the neck of a duckling is subjected to subcutaneous immunization by using 100 mu g/recombinant eukaryotic expression plasmid PVAX1-OmpA, and a preparation method and an identification method are also disclosed. The application develops eukaryotic expression plasmid pVAX1-OmpA containing RAOmpA gene, and PVAX1-OmpA recombinant plasmid can induce to generate specific immune antibody after immunizing ducklings, can generate stronger immune protection effect, can be used as a candidate strain of novel DNA vaccine, and lays a foundation for further developing and researching RA novel genetic engineering vaccine. 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 RA OmpA gene, and a preparation method and an identification method thereof.
Background
Riemerella anatipestifer disease is a contact infectious disease caused by Riemerella Anatipestifer (RA), is also called as duck infectious serositis (infectious serositis), new duck disease, duck septicemia, duck plague syndrome, pasteurellosis and the like, is an acute or chronic contact infectious disease for various poultry such as infected ducks, geese and the like, is called goose influenza or goose exudative septicemia after goose infection, and has higher morbidity and mortality. Riemerella anatipestifer disease is popular in China, almost all duck breeding areas are covered, great loss is caused to the development of duck breeding industry, and particularly severe threat is brought to the brisk duck breeding industry in western areas.
At present, scholars at home and abroad are dedicated to researching a novel effective vaccine with protective effect on various serotype RA, wherein the DNA vaccine is a relatively ideal development direction. The DNA vaccine can induce humoral and cellular immune response at the same time, generate persistent immune response, can cross-protect different serotypes, and has the advantages of easy production, strong stability, convenient storage and transportation and the like. The key to the development of DNA vaccines is the selection of target gene sequences and expression vectors, and the selection of one or more cross-protective antigenic components provides more complete protection. OmpA is used as a main outer membrane protein of RA, the gene nucleic acid sequence of OmpA is highly conserved, the OmpA has good immunogenicity, not only can stimulate specific humoral immunity and cytotoxic reaction, assist the cross presentation of other antigens and improve the immune response level of an organism, but also can play a role in immune cross protection on different serotype RA, and is a preferred target gene for the development of RA nucleic acid vaccines. The document, "the construction of recombinant plasmid of co-expression RAOmpA and duck IL-2 and immune research" successfully constructs the eukaryotic expression plasmid pcDNA3.1(+) -OmpA of RAOmpA gene, and the result shows that the eukaryotic expression plasmid pcDNA3.1(+) -OmpA can stimulate the duck body to generate RA specific antibody by immunizing healthy ducklings and detecting duck body immune indexes, and has certain cross immune protection effect on both serum type 1 and type 2 RA.
The immune effect of the eukaryotic expression plasmid induced organism is positively correlated with the capability of the plasmid vector to express the antigen protein, so that the efficient plasmid expression vector is selected for constructing the DNA vaccine. pVAX1 is used as the expression vector for high-efficiency expression of target gene in mammalian cell expression system, has a size of 3.0kb, is a novel eukaryotic expression vector reconstructed on the basis of pcDNA3.1, uses Kanamycin to replace Ampicillin resistance screening gene, can effectively reduce the possibility of human genome recombination, and the pVAX1 vector is a non-fusion vector, and the generated protein is almost the same as the naturally-occurring protein in structure and function. Because of the advantages of BGHpolyA signal, strong human cytomegalovirus promoter (CMV), large expression capacity, small molecular weight and the like, pVAX1 has been used for constructing recombinant nucleic acid vaccines of various infectious diseases or parasitic diseases as a eukaryotic expression vector. The recombinant plasmids pVAX1-Cal-SAG1 and pVAX1-SAG1 are constructed in the literature 'influence of a molecular adjuvant Cal on a Toxoplasma SAG1 subunit vaccine', and after a mouse is immunized, the DNA vaccine can induce the body of the mouse to generate humoral and cellular immunity through an ELISA (enzyme-linked immuno sorbent assay), a CCK-8 method and a mouse insect attacking test. The results of experiments on immunized C57BL/6 mice show that Hsp65-Ag85B and Hsp65-ESAT6DNA vaccine strains can stimulate stronger immune response after the expression of recombinant plasmids PVAX1-Hsp65-Ag85B and PVAX1-Hsp65-ESAT6 is verified by in vitro transfection in the construction and immunogenicity research of fusion gene DNA vaccine strains of Mycobacterium tuberculosis Hsp65-Ag85B and Hsp65-ESAT 6.
Patent application CN201410098187.3 discloses a DNA vaccine of Riemerella anatipestifer based on OmpA gene, which is characterized in that: the eukaryotic expression plasmid pcDNA3.1(+) -OmpA containing the Riemerella anatipestifer OmpA gene has the plasmid concentration of 2 mg-5 mg/mL, contains the whole gene coding sequence of the Riemerella anatipestifer OmpA, has the length of 1164 nucleotides, the type of nucleotides and the full length of coding regions. The riemerella anatipestifer vaccine is safe and effective, can induce duck organisms to generate specific humoral immune response, and can effectively prevent occurrence and prevalence of riemerella anatipestifer.
However, the existing vaccine has insufficient immune protection effect, so the scheme of the application is researched to obtain more eukaryotic expression recombinant plasmids of the riemerella anatipestifer outer membrane protein A gene.
Disclosure of Invention
The invention provides a Riemerella anatipestifer DNA vaccine based on RA OmpA gene, a preparation method and an identification method thereof for solving the technical problems.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a Riemerella anatipestifer DNA vaccine based on RA OmpA gene 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 each vial of vaccine contained 10 ten thousand. mu.g of the recombinant plasmid PVAX 1-OmpA.
Furthermore, the RA OmpA gene-based Riemerella anatipestifer DNA vaccine is used for carrying out subcutaneous immunization on the neck of a duckling by using 100 mu g/recombinant eukaryotic expression plasmid PVAX1-OmpA during inoculation.
Further, the preparation method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) preparation of multiple antisera: constructing RA OmpA gene into pET32a + prokaryotic expression vector, converting recombinant expression plasmid pET32a + -RA-OmpA into competent cell BL21 for induction expression, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, filling a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni + column affinity purification on recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, immunizing 0.5 mg/time under skin, reinforcing immunization once by emulsifying recombinant protein with Freund's incomplete adjuvant every 14d, detecting the titer of the white rabbit antiserum to PA OPMA recombinant protein through indirect ELISA method, collecting blood of the white rabbits with titer of 1:12800 after 3 times of immunization and separating serum, thus obtaining rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal antibody;
(2) designing and synthesizing a primer: combining pVAX1 eukaryotic expression vector multiple cloning sites according to RAOmpA gene sequence in GenBank, designing specific primers of RA OmpA gene by using Primerpramier 5.0, and designing synthetic primers by using XhoI and BamHI as enzyme cutting sites;
(3) construction of eukaryotic expression plasmid of RA OmpA Gene: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double enzyme digestion by restriction endonucleases XhoI and BamHI, are connected and then transformed into DH5 alpha competent cells, are coated on an LB plate containing kanamycin resistance, are cultured overnight at 37 ℃, single white bacterial colony is selected from the transformed kanamycin LB plate, is inoculated into an LB liquid culture medium containing kanamycin and is cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, and double enzyme digestion identification and PCR identification are carried out by extracting recombinant plasmids to obtain the riemerella anatipestifer eukaryotic vaccine.
Further, in step (2), the specific primer used was synthesized by Shanghai Biopsis, and the upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', the XhoI enzyme recognition site is underlined; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', the BamHI enzyme recognition site is underlined.
Further, the identification method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
carrying out transfection: extracting the concentration and purity of eukaryotic expression plasmid PVAX1-OmpA and empty carrier plasmid pVAX1 which are identified correctly by using an endotoxin-free plasmid extraction kit, selecting a plasmid corresponding to the ratio of OD260 to OD280 of 1.8-2.0, transfecting DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, culturing the cells to grow to 80-100% of a monolayer by using DMEM nutrient solution containing 10% fetal calf serum, discarding supernatant, washing with PBS buffer solution for 2-3 times, transfecting the DF-1 cells by using recombinant plasmid PVAX1-OmpA and empty plasmid pVAX1 respectively according to the instruction of a liposome transfection reagent, and carrying out no treatment on a blank group at 37 ℃ and 5% CO at 5 DEG C2After culturing for 5 hours in the incubator, continuously culturing for 48 hours by replacing a new DMEM culture solution containing 5% fetal calf serum;
PCR amplification and electrophoresis detection: extracting total RNA of cells by adopting an RNA extraction kit, synthesizing cDNA according to the instruction of a HiFiScriptcDNA first strand synthesis kit, carrying out PCR amplification by taking the cDNA as a template, and carrying out electrophoresis detection on a 6 mu LPCR amplification product on 1.2% agarose gel containing a nucleic acid dye;
detecting the expression of RA OmpA gene in DF-1 cells: collecting the DF-1 cells after transfection for 48h, and washing for 3 times by using precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with precooled PBS for 3 times; adding 0.3% TritonX-100, permeabilizing at room temperature for 20min, discarding TritonX-100, and washing with precooled PBS 3 times; adding riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing for 3 times with precooled PBS; adding FITC labeled goat anti-rabbit IgG, incubating at 37 ℃ for 1h, discarding the FITC labeled goat anti-rabbit IgG, and washing with precooled PBS for 3 times; adding cell nucleus dye DAPI, acting at room temperature for 4min, washing with precooled PBS for 3 times; observing the result under a fluorescence microscope;
detecting the expression of the recombinant protein: collecting the transfected DF-1 cells for 48h, cracking, carrying out 12% SDS-PAGE gel electrophoresis, transferring to a PVDF membrane, taking rabbit anti-RA OmpA prokaryotic expression recombinant protein serum as a primary antibody, taking goat anti-rabbit HRP-IgG as a secondary antibody, and developing by ECL.
Further, in step two, the PCR reaction system is: 25 μ L, 2 XPCRMIX12.5 μ L, 2 μ L template, 1 μ L each of 10mol/L μ upstream and downstream primers, ddH2Supplementing O to 25 mu L; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 45s, annealing at 52 ℃ for 1min, extension at 72 ℃ for 1min, and extension at 72 ℃ for 10min after 30 cycles.
Further, the nucleic acid dye is GoldView.
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 Bionics; the pVAX1 eukaryotic expression vector is presented by Xiamei teacher of animal science institute of Guizhou university; DF-1 cells were purchased from Fenghui Bio Inc; DNA gel recovery kit and plasmid miniprep kit are purchased from OMEGA; DNALigationkit, DNAmakerIII, DNA restriction enzymes XhoI and BamHI were purchased from Takara; coli DH5 alpha, DL2000, HiFiScriptcDNA first strand synthesis kit, endotoxin-free plasmid extraction kit were purchased from Tiangen Biotechnology; goat anti-rabbit IgG-FITC was purchased from Beijing Boaosen. The trivalent inactivated vaccine for duck infectious serositis (type 1 ZJ01 strain + type 2 HN01 strain + type 7 YC03 strain) is purchased from Qiilu animal health products Co., Ltd. HRP-labeled goat anti-duck IgG was purchased from KPL, usa. The RA antibody negative ducklings are purchased from a duck farm of ecological food of Qianlishan, Guizhou, Ltd.
According to the application, pVAX1 is used as a eukaryotic expression vector, a eukaryotic expression plasmid pVAX1-OmpA of RA OmpA gene is constructed, plasmid PCR, double digestion and sequencing are used for identification, the identified positive plasmid pVAX1-OmpA is transfected to DF-1 cells, and RT-PCR, IFA and Westernblot tests are adopted to confirm that the eukaryotic expression plasmid pVAX1-OmpA can be transiently expressed in the DF-1 cells. Meanwhile, in the IFA test, pVAX1-OmpA eukaryotic expression protein can be identified by polyclonal antibody obtained by immunizing rabbits with OmpA gene prokaryotic expression protein, and combined with goat anti-rabbit IgG-FITC, green fluorescence is shown in cell cytoplasm, and a Westernblot result also shows that a band appears at about 41KD after recombinant plasmid transfects cells, which indicates that the eukaryotic expression protein has immunogenicity.
According to the application, PVAX1-OmpADNA is used for immunizing ducklings, the eukaryotic expression plasmid pVAX1-OmpA is successfully constructed, the specific expression of OmpA protein is detected by both methods, the recombinant plasmid PVAX1-OmpA test group is extremely obvious (P is less than 0.01) or obvious (P is less than 0.05) in 7d and 21d and is lower than that of an inactivated vaccine control group, the differences among 14d, 28d, 35d, 49d and 63d are not obvious, the immune protection rate of the ducklings reaches 100%, the result shows that the humoral immunity level induced by the recombinant plasmid PVAX1-OmpA group is equivalent to that of the inactivated vaccine group after 28d, and the result of a virus challenge experiment shows that the immune protection effect of the ducklings is higher than that of the inactivated vaccine group can be provided after the recombinant plasmid PVAX 1-OmpA. The result shows that the PVAX1-OmpA recombinant plasmid can induce and generate specific immune antibody after immunizing ducklings, can generate stronger immune protection effect and can be used as a candidate strain of a novel DNA vaccine.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
based on the fact that Omp A has high conservation among separated RA strains, cross immunogenicity exists among different serotype RA strain Omp A proteins, a multi-serotype RA vaccine can be prepared, the application develops a eukaryotic expression plasmid pVAX1-OmpA containing an RA OmpA gene, and a PVAX1-OmpA recombinant plasmid can induce generation of a specific immune antibody after being used for immunizing a duckling, can generate a strong immune protection effect, can be used as a candidate strain of a novel DNA vaccine, and lays a foundation for further developing and developing a novel RA genetic engineering vaccine. Compared with the prior art, the Riemerella anatipestifer DNA vaccine has better immune protection effect.
Drawings
In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some examples of the present invention, and for a person skilled in the art, without inventive step, other drawings can be obtained according to these drawings:
FIG. 1 is a diagram showing the result of PCR identification of the eukaryotic expression plasmid pVAX1-OmpA of the present application;
FIG. 2 is a diagram showing the results of double digestion of the eukaryotic expression plasmid pVAX1-OmpA of the present application;
FIG. 3 is a diagram showing the result of RT-PCR detection of the eukaryotic expression plasmid pVAX1-OmpA of the present application;
FIG. 4 is a diagram showing the result of IFA detection after DF-1 cells are transfected by recombinant eukaryotic expression plasmid PVAX1-OmpA for 48 h;
FIG. 5 is a graph showing the result of Merge-treatment IFA detection after DF-1 cells are transfected by recombinant eukaryotic expression plasmid PVAX1-OmpA for 48 h;
FIG. 6 is a diagram showing the results of Westernblot detection after transfection of the eukaryotic expression plasmid PVAX 1-OmpA;
FIG. 7 is a graph of the dynamic change of antibodies in different immune groups of the present application;
FIG. 8 is a graph showing the results of the challenge protection test of the PVAX1-OmpADNA vaccine of the present application against RA infected with duckling 21 d.
In the drawings: 1-PVAX 1-OmpA; 2-negative control; 3-PVAX1-OmpA double enzyme digestion; 4-PVAX 1; 5-normal cell control; 6-PVAX1-OmpA transfects DF-1 cells; transfecting DF-1 cells with PVAX1 empty vector; M-DL 2000; K-DL 5000; P-D2000; R-ProteinMarker.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A Riemerella anatipestifer DNA vaccine based on RA OmpA gene 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 vector, and each bottle of vaccine contains 10 ten thousand mug of recombinant plasmid PVAX 1-OmpA; when inoculating, the neck of the duckling is immunized subcutaneously by 100 mug/recombinant eukaryotic expression plasmid PVAX 1-OmpA.
A preparation method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) preparation of multiple antisera: constructing RA OmpA gene into pET32a + prokaryotic expression vector, converting recombinant expression plasmid pET32a + -RA-OmpA into competent cell BL21 for induction expression, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, filling a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni + column affinity purification on recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, immunizing 0.5 mg/time under skin, reinforcing immunization once by emulsifying recombinant protein with Freund's incomplete adjuvant every 14d, detecting the titer of the white rabbit antiserum to PA OPMA recombinant protein through indirect ELISA method, collecting blood of the white rabbits with titer of 1:12800 after 3 times of immunization and separating serum, thus obtaining rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal antibody;
(2) designing and synthesizing a primer: combining pVAX1 eukaryotic expression vector multiple cloning sites according to RA OmpA gene sequence in GenBank, designing specific primers of RA OmpA gene by using Primerpromier 5.0, and designing synthetic primers by using XhoI and BamHI as enzyme cutting sites;
the specific primer is synthesized by Shanghai bio-organisms, and the upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', the XhoI enzyme recognition site is underlined; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', the BamHI enzyme recognition site is underlined;
(3) construction of eukaryotic expression plasmid of RA OmpA Gene: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double enzyme digestion by restriction endonucleases XhoI and BamHI, are connected and then transformed into DH5 alpha competent cells, are coated on an LB plate containing kanamycin resistance, are cultured overnight at 37 ℃, single white bacterial colony is selected from the transformed kanamycin LB plate, is inoculated into an LB liquid culture medium containing kanamycin and is cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, and double enzyme digestion identification and PCR identification are carried out by extracting recombinant plasmids to obtain the riemerella anatipestifer eukaryotic vaccine.
The identification method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
carrying out transfection: extracting the concentration and purity of eukaryotic expression plasmid PVAX1-OmpA and empty carrier plasmid pVAX1 which are identified correctly by using an endotoxin-free plasmid extraction kit, selecting a plasmid corresponding to the ratio of OD260 to OD280 of 1.8-2.0, transfecting DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, culturing the cells to grow to 80-100% of a monolayer by using DMEM nutrient solution containing 10% fetal calf serum, discarding supernatant, washing with PBS buffer solution for 2-3 times, transfecting the DF-1 cells by using recombinant plasmid PVAX1-OmpA and empty plasmid pVAX1 respectively according to the instruction of a liposome transfection reagent, and carrying out no treatment on a blank group at 37 ℃ and 5% CO at 5 DEG C2After culturing for 5 hours in the incubator, continuously culturing for 48 hours by replacing a new DMEM culture solution containing 5% fetal calf serum;
PCR amplification and electrophoresis detection: extracting total RNA of cells by adopting an RNA extraction kit, synthesizing cDNA according to the instruction of a HiFiScriptcDNA first strand synthesis kit, carrying out PCR amplification by taking the cDNA as a template, and carrying out electrophoresis detection on a 6 mu LPCR amplification product on 1.2% agarose gel containing a nucleic acid dye;
detecting the expression of RA OmpA gene in DF-1 cells: collecting the DF-1 cells after transfection for 48h, and washing for 3 times by using precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with precooled PBS for 3 times; adding 0.3% TritonX-100, permeabilizing at room temperature for 20min, discarding TritonX-100, and washing with precooled PBS 3 times; adding riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing for 3 times with precooled PBS; adding FITC labeled goat anti-rabbit IgG, incubating at 37 ℃ for 1h, discarding the FITC labeled goat anti-rabbit IgG, and washing with precooled PBS for 3 times; adding cell nucleus dye DAPI, acting at room temperature for 4min, washing with precooled PBS for 3 times; observing the result under a fluorescence microscope;
detecting the expression of the recombinant protein: collecting the transfected DF-1 cells for 48h, cracking, carrying out 12% SDS-PAGE gel electrophoresis, transferring to a PVDF membrane, taking rabbit anti-RA OmpA prokaryotic expression recombinant protein serum as a primary antibody, taking goat anti-rabbit HRP-IgG as a secondary antibody, and developing by ECL.
Further, the PCR reaction system is as follows: 25 μ L, 2 XPCRMIX12.5 μ L, 2 μ L template, 1 μ L each of 10mol/L μ upstream and downstream primers, ddH2Supplementing O to 25 mu L; the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 45s, annealing at 52 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and extension at 72 deg.C for 10 min; the nucleic acid dye is GoldView; the solid-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1: 500; the solid-liquid ratio of the goat anti-rabbit HRP-IgG is 1: 2000.
Example 2
A Riemerella anatipestifer DNA vaccine based on RA OmpA gene 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 vector, and each bottle of vaccine contains 10 ten thousand mug of recombinant plasmid PVAX 1-OmpA; when inoculating, the neck of the duckling is immunized subcutaneously by 100 mug/recombinant eukaryotic expression plasmid PVAX 1-OmpA.
The eukaryotic expression plasmid pVAX1-OmpA of the RA OmpA gene is identified by means of PCR, double digestion and sequencing, the result shows that the OmpA gene is correctly connected with the PVAX1 vector, the size of the OmpA gene fragment is about 1149bp, the double digestion and sequencing result is identified as a positive recombinant eukaryotic expression plasmid which is named as PVAX1-OmpA, and the identification result is shown in figure 1 and figure 2.
A preparation method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
(1) preparation of multiple antisera: constructing RA OmpA gene into pET32a + prokaryotic expression vector, converting recombinant expression plasmid pET32a + -RA-OmpA into competent cell BL21 for induction expression, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, filling a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni + column affinity purification on recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, immunizing 0.5 mg/time under skin, reinforcing immunization once by emulsifying recombinant protein with Freund's incomplete adjuvant every 14d, detecting the titer of the white rabbit antiserum to PA OPMA recombinant protein through indirect ELISA method, collecting blood of the white rabbits with titer of 1:12800 after 3 times of immunization and separating serum, thus obtaining rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal antibody;
(2) designing and synthesizing a primer: combining pVAX1 eukaryotic expression vector multiple cloning sites according to RA OmpA gene sequence in GenBank, designing specific primers of RA OmpA gene by using Primerpromier 5.0, and designing synthetic primers by using XhoI and BamHI as enzyme cutting sites;
the specific primer is synthesized by Shanghai bio-organisms, and the upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', the XhoI enzyme recognition site is underlined; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', the BamHI enzyme recognition site is underlined;
(3) construction of eukaryotic expression plasmid of RA OmpA Gene: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double enzyme digestion by restriction endonucleases XhoI and BamHI, are connected and then transformed into DH5 alpha competent cells, are coated on an LB plate containing kanamycin resistance, are cultured overnight at 37 ℃, single white bacterial colony is selected from the transformed kanamycin LB plate, is inoculated into an LB liquid culture medium containing kanamycin and is cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, and double enzyme digestion identification and PCR identification are carried out by extracting recombinant plasmids to obtain the riemerella anatipestifer eukaryotic vaccine.
The identification method of the RA OmpA gene-based Riemerella anatipestifer DNA vaccine comprises the following steps:
carrying out transfection: extracting the concentration and purity of eukaryotic expression plasmid PVAX1-OmpA and empty carrier plasmid pVAX1 which are identified correctly by using an endotoxin-free plasmid extraction kit, selecting a plasmid corresponding to the ratio of OD260 to OD280 of 1.8-2.0, transfecting DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, culturing the cells to grow to 80-100% of a monolayer by using DMEM nutrient solution containing 10% fetal calf serum, discarding supernatant, washing with PBS buffer solution for 2-3 times, transfecting the DF-1 cells by using recombinant plasmid PVAX1-OmpA and empty plasmid pVAX1 respectively according to the instruction of a liposome transfection reagent, and carrying out no treatment on a blank group at 37 ℃ and 5% CO at 5 DEG C2After culturing for 5 hours in the incubator, continuously culturing for 48 hours by replacing a new DMEM culture solution containing 5% fetal calf serum;
PCR amplification and electrophoresis detection: extracting total RNA of cells by adopting an RNA extraction kit, synthesizing cDNA according to the instruction of a HiFiScriptcDNA first strand synthesis kit, carrying out PCR amplification by taking the cDNA as a template, and carrying out electrophoresis detection on a 6 mu LPCR amplification product on 1.2% agarose gel containing a nucleic acid dye;
detecting the expression of RAOmpA gene in DF-1 cell: collecting the DF-1 cells after transfection for 48h, and washing for 3 times by using precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with precooled PBS for 3 times; adding 0.3% TritonX-100, permeabilizing at room temperature for 20min, discarding TritonX-100, and washing with precooled PBS 3 times; adding riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing for 3 times with precooled PBS; adding FITC labeled goat anti-rabbit IgG, incubating at 37 ℃ for 1h, discarding the FITC labeled goat anti-rabbit IgG, and washing with precooled PBS for 3 times; adding cell nucleus dye DAPI, acting at room temperature for 4min, washing with precooled PBS for 3 times; observing the result under a fluorescence microscope;
detecting the expression of the recombinant protein: collecting the transfected DF-1 cells for 48h, cracking, carrying out 12% SDS-PAGE gel electrophoresis, transferring to a PVDF membrane, taking rabbit anti-RAOmpA prokaryotic expression recombinant protein serum as a primary antibody, taking goat anti-rabbit HRP-IgG as a secondary antibody, and developing by ECL.
Further, it is characterized byThe PCR reaction system is as follows: 25 μ L, 2 XPCRMIX12.5 μ L, 2 μ L template, 1 μ L each of 10mol/L μ upstream and downstream primers, ddH2Supplementing O to 25 mu L; the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 45s, annealing at 52 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and extension at 72 deg.C for 10 min; the nucleic acid dye is GoldView; the solid-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1: 500; the solid-liquid ratio of the goat anti-rabbit HRP-IgG is 1: 2000.
1. RT-PCR detection of transient expression result of eukaryotic expression plasmid
After DF-1 cells were transfected with eukaryotic expression plasmids PVAX1-OmpA and PVAX1 for 48h, total RNA was extracted from the cells, and expression of the desired gene was detected by RT-PCR, as shown in FIG. 3.
2. IFA detection of transient expression result of eukaryotic expression plasmid
IFA detection results show that after DF-1 cells are transfected by the eukaryotic expression plasmid PVAX1-OmpA for 48h, green fluorescence can be seen in cytoplasm of the cells, as shown in FIG. 4; carrying out Merge processing on the obtained protein fluorescence positioning picture and the corresponding cell nucleus fluorescence picture by using Photoshop CS3 software, and judging the subcellular positioning of the recombinant protein through fluorescence co-positioning, as shown in FIG. 5; no green fluorescence was observed in the PVAX1 empty vector group and the normal cell group, indicating that the field of observation was black.
3. Expression of recombinant protein detected by Westernblot
Western blot is carried out 48 hours after DF-1 cells are transfected by the recombinant eukaryotic expression plasmid pVAX1-OmpA, and the result shows that bands appear around 41KD when the recombinant plasmid is transfected by the cells and are consistent with the size of the predicted result, but the bands do not appear in an empty vector, as shown in figure 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 macroextraction kit, and the nucleic acid concentration was determined. 120 healthy ducklings with RA antibody negative of 1 day old are raised to 10 days old to adapt to the environment, and are randomly divided into 4 groups and 30 groups, namely a 100 mu g/PVAX 1-OmpA plasmid immunization group, a 100 mu g/PVAX 1 plasmid blank control group, a 0.2mL inactivated vaccine control group and a 0.2mL physiological saline control group. Performing neck subcutaneous immunization on the ducklings, and performing secondary immunization on the ducklings 7d after immunization. After 7d, 14d, 21d, 28d, 49d and 63d of first immunization, 10 ducklings in each group are randomly extracted, blood is collected, and serum is separated.
Detecting the RA antibody level of the immunized duckling by adopting an indirect ELISA method, coating 5 mu g of RNA AmpA prokaryotic expression recombinant protein on each hole of an ELISA plate, diluting the serum of the duckling at 1:100, incubating for 1h at 37 ℃, washing for 3 times by PBS (phosphate buffer solution), adding HRP (horse radish peroxidase) labeled goat anti-duck IgG (1:2000), incubating for 45min at 37 ℃, washing for 3 times by PBS, developing for 15min by TMB (tetramethylbenzidine) at room temperature, stopping reaction by 2M sulfuric acid, and determining the OD450nm value. As a result, the recombinant eukaryotic expression plasmid pVAX1-OmpA test group is extremely significant (P < 0.01) or significantly (P < 0.05) in 7d and 21d, and is lower than the inactivated vaccine control group, while the differences of 14d, 28d, 35d, 49d and 63d are not significant, as shown in FIG. 7.
In the challenge protection test, 10 test ducks after twice immunization are randomly extracted from each group, RA bacteria suspension with the bacteria liquid concentration of 4 multiplied by 107CFU/mL (2 times LD50 dosage) 1 is adopted, the challenge protection rate is determined through intramuscular injection of legs, and the morbidity or mortality of ducklings in 7 days after challenge is observed. As a result, it was found 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 normal saline control group show that: the 21d protection rates were all 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.
To further illustrate that the present invention can achieve the technical effects, the following experiments were performed:
selecting 90 young ducks which are equal in size, month age and physical sign and are all infected with riemerella anatipestifer, randomly and evenly dividing the young ducks into three groups, wherein each group is 30, the proportion of male ducks to female ducks is equal, respectively feeding the riemerella anatipestifer DNA vaccines prepared in the embodiment 1 and the comparative example 1 of the application to the young ducks for vaccine, observing the morbidity or mortality of each group of the young ducks, and recording whether the vaccines are effective, wherein the experimental results are shown in the following table 1.
TABLE 1
| Group of | Onset of disease (only) | Death (only) | Survival (only) | Effective rate of vaccine |
| Example 1 | 4 | 2 | 28 | 93.33％ |
| Example 2 | 3 | 1 | 29 | 96.67％ |
| Comparative example 1 | 12 | 8 | 22 | 73.33％ |
As can be seen from the experimental results in Table 1, compared with the prior art, the Riemerella anatipestifer DNA vaccine prepared by the method has better effect on the Riemerella anatipestifer, and can play a good vaccine effect.
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 attributes 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.
Claims (10)
1. A Riemerella anatipestifer DNA vaccine based on RA OmpA gene is characterized in that: eukaryotic expression plasmid pVAX1-OmpA comprising the RA OmpA gene.
2. The RA OmpA gene-based Riemerella anatipestifer DNA vaccine of claim 1, which is characterized in that: the eukaryotic expression plasmid pVAX1-OmpA is extracted by adopting escherichia coli, and is prepared by taking pVAX1 as a vector, and each bottle of vaccine contains 10 ten thousand mug of recombinant plasmid PVAX 1-OmpA.
3. The RA OmpA gene-based Riemerella anatipestifer DNA vaccine of claim 2, which is characterized in that: when inoculating, the neck of the duckling is immunized subcutaneously by 100 mug/recombinant eukaryotic expression plasmid PVAX 1-OmpA.
4. A method for preparing the RA OmpA gene-based Riemerella anatipestifer DNA vaccine according to any one of claims 1 to 3, which comprises the following steps:
(1) preparation of multiple antisera: constructing RA OmpA gene into pET32a + prokaryotic expression vector, converting recombinant expression plasmid pET32a + -RA-OmpA into competent cell BL21 for induction expression, after IPTG induced protein expression, determining target protein through 12% SDS-PAGE analysis, filling a recombinant protein urea solution into a dialysis bag for renaturation, carrying out Ni + column affinity purification on recombinant protein, mixing the purified recombinant protein with Freund's complete adjuvant, immunizing 3 New Zealand white rabbits with 2.0-2.5 kg, immunizing 0.5 mg/time under skin, reinforcing immunization once by emulsifying recombinant protein with Freund's incomplete adjuvant every 14d, detecting the titer of the white rabbit antiserum to PA OPMA recombinant protein through indirect ELISA method, collecting blood of the white rabbits with titer of 1:12800 after 3 times of immunization and separating serum, thus obtaining rabbit anti-RA OmpA gene prokaryotic recombinant protein polyclonal antibody;
(2) designing and synthesizing a primer: combining pVAX1 eukaryotic expression vector multiple cloning sites according to RA OmpA gene sequence in GenBank, designing specific primers of RA OmpA gene by using Primerpromier 5.0, and designing synthetic primers by using XhoI and BamHI as enzyme cutting sites;
(3) construction of eukaryotic expression plasmid of RA OmpA Gene: the positive plasmid PUC57-OmpA and the vector plasmid pVAX-1 are respectively subjected to double enzyme digestion by restriction endonucleases XhoI and BamHI, are connected and then transformed into DH5 alpha competent cells, are coated on an LB plate containing kanamycin resistance, are cultured overnight at 37 ℃, single white bacterial colony is selected from the transformed kanamycin LB plate, is inoculated into an LB liquid culture medium containing kanamycin and is cultured overnight, a small amount of plasmid extraction kit is adopted to extract plasmids, and double enzyme digestion identification and PCR identification are carried out by extracting recombinant plasmids to obtain the riemerella anatipestifer eukaryotic vaccine.
5. The method for preparing the RA OmpA gene-based Riemerella anatipestifer DNA vaccine according to claim 4, which is characterized in that: in step (2), the specific primer used was synthesized by Shanghai Biopsis, and the upstream primer: 5, -GGATCCATGTTGATGACTGGACTTGGT-3', the XhoI enzyme recognition site is underlined; a downstream primer: 5, -CTCGAGTTATTTTCTTTTCTTTTTTACTACT-3', the BamHI enzyme recognition site is underlined.
6. The method for identifying the RA OmpA gene-based Riemerella anatipestifer DNA vaccine of claim 5, which comprises the following steps:
carrying out transfection: extracting the concentrations and purities of the eukaryotic expression plasmid PVAX1-OmpA and the empty vector plasmid pVAX1 which are identified correctly by using an endotoxin-free plasmid extraction kit, and selecting OD260 and OD280 of 1.8-2.0, transfecting DF-1 cells, inoculating the DF-1 cells into a 6-hole cell culture plate, culturing the cells to grow to 80-100% of a monolayer by using DMEM nutrient solution containing 10% fetal calf serum, discarding the supernatant, washing the cells for 2-3 times by using PBS buffer solution, transfecting the DF-1 cells by using recombinant plasmid PVAX1-OmpA and empty plasmid pVAX1 respectively according to the instruction of a liposome transfection reagent, carrying out no treatment on a blank group, and carrying out treatment at 37 ℃ and 5% CO2After culturing for 5 hours in the incubator, continuously culturing for 48 hours by replacing a new DMEM culture solution containing 5% fetal calf serum;
PCR amplification and electrophoresis detection: extracting total RNA of cells by adopting an RNA extraction kit, synthesizing cDNA according to the instruction of a HiFiScriptcDNA first strand synthesis kit, carrying out PCR amplification by taking the cDNA as a template, and carrying out electrophoresis detection on a 6 mu LPCR amplification product on 1.2% agarose gel containing a nucleic acid dye;
detecting the expression of RA OmpA gene in DF-1 cells: collecting the DF-1 cells after transfection for 48h, and washing for 3 times by using precooled PBS; adding 4% paraformaldehyde, fixing at room temperature for 20min, discarding the fixing solution, and washing with precooled PBS for 3 times; adding 0.3% TritonX-100, permeabilizing at room temperature for 20min, discarding TritonX-100, and washing with precooled PBS 3 times; adding riemerella anatipestifer DNA vaccine, incubating for 1h at 37 ℃, discarding the riemerella anatipestifer DNA vaccine, and washing for 3 times with precooled PBS; adding FITC labeled goat anti-rabbit IgG, incubating at 37 ℃ for 1h, discarding the FITC labeled goat anti-rabbit IgG, and washing with precooled PBS for 3 times; adding cell nucleus dye DAPI, acting at room temperature for 4min, washing with precooled PBS for 3 times; observing the result under a fluorescence microscope;
detecting the expression of the recombinant protein: collecting the transfected DF-1 cells for 48h, cracking, carrying out 12% SDS-PAGE gel electrophoresis, transferring to a PVDF membrane, taking rabbit anti-RA OmpA prokaryotic expression recombinant protein serum as a primary antibody, taking goat anti-rabbit HRP-IgG as a secondary antibody, and developing by ECL.
7. The method for identifying the RA OmpA gene-based Riemerella anatipestifer DNA vaccine according to claim 6, which is characterized in that: in the second step, the PCR reaction system is: 25 μ L, 2 XPCRMIX12.5 μ L, 2 μ L template, 1 μ L each of 10mol/L μ upstream and downstream primers, ddH2O is supplemented to 25 muL; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 45s, annealing at 52 ℃ for 1min, extension at 72 ℃ for 1min, and extension at 72 ℃ for 10min after 30 cycles.
8. The method for identifying the RA OmpA gene-based Riemerella anatipestifer DNA vaccine according to claim 6, which is characterized in that: the nucleic acid dye is Gold View.
9. The method for identifying the RA OmpA gene-based Riemerella anatipestifer DNA vaccine according to claim 6, which is characterized in that: the solid-liquid ratio of the rabbit anti-RA OmpA prokaryotic expression recombinant protein serum is 1: 500.
10. The method for identifying the RA OmpA gene-based Riemerella anatipestifer DNA vaccine according to claim 6, which is characterized in that: the solid-liquid ratio of the goat anti-rabbit HRP-IgG is 1: 2000.
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