CN110845580A - Method for assembling porcine parvovirus-like particles and identifying immunogenicity thereof - Google Patents
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention belongs to the technical field of gene assembly and immunogenicity identification, and discloses a method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof, which comprises the steps of amplifying VP2 gene, optimizing and constructing recombinant donor plasmid; constructing and identifying VP2 recombinant shuttle plasmid; obtaining and identifying recombinant baculovirus; identifying the expression of the VP2 protein by Western-blot; indirect immunofluorescence identification of the VP2 protein; purifying and identifying PPV-AV30 virus; assembling and identifying virus-like particles. The invention carries out post-translational processing modification on the protein and displays the natural conformation of the target protein; the invention successfully expresses the protein VP2 of the PPV strain AV-30, correctly assembles VLPs, is similar to natural virion PPV-AV30, and lays a foundation for the next step of researching subunit vaccines based on VLPs or preparing chimeric VLPs.
Description
Technical Field
The invention belongs to the technical field of gene assembly and immunogenicity identification, and particularly relates to a method for assembling porcine parvovirus-like particles and identifying the immunogenicity of the porcine parvovirus-like particles.
Background
Currently, the closest prior art: porcine Parvovirus (PPV) can cause abortion, infertility, stillbirth, mummy, and weak piglets in primiparous sows and seronegative multiparous sows. The main cause of Postweaning Multisystemic Wasting Syndrome (PMWS) is mixed infection with Porcine Circovirus type 2 (PCV 2). PPV infectivity is strong, and the PPV has strong resistance to the external environment, and is not sensitive to pH and temperature. Once a healthy herd of pigs is infected with PPV, all pigs can be infected within three months. Furthermore, in pig farms with this disease, reproductive failures can last for years or even decades. The detection rate of the disease in the swinery is as high as 85%, which causes great economic loss to the pig industry in China.
PPV belongs to the family of parvoviridae, the genus parvovirus. The virus particles without the envelope are in icosahedral symmetry, and the diameter is 20-25 nm. The genome is single-stranded negative strand DNA with a size of about 5000bp, no Open Reading Frames (ORFs) on the negative strand, and 2 ORFs on the positive strand. The 5' end encodes non-structural proteins NS1, NS2, NS3, wherein NS1 is involved in gene replication, and the functions of the other two non-structural proteins are unknown. The 3' end encodes structural proteins VP1, VP2 and VP3, VP3 is generated by hydrolysis of VP2, virus replication is related to VP1, VP2 occupies the main part of the whole virus capsid and has 4 Loop rings, and the antigen site is mainly concentrated on the rings and is positioned on the protein surface. Many important linear epitopes are on VP2, and the present invention utilizes the self-assembly of VP2 protein into nucleic acid-free, self-replicating VLPs, which induce both humoral and cellular immunity.
At present, the PPV is mainly prevented by adopting vaccination, and the vaccination has two main categories of inactivated vaccine and attenuated vaccine. Although these vaccines are effective in controlling clinical PPV infection, inactivated vaccines risk incomplete inactivation, and attenuated vaccines risk virulent reversion. Therefore, the development of a safe novel PPV vaccine is of great significance for controlling and completely eradicating the disease.
In summary, the problems of the prior art are as follows:
the existing attenuated vaccine has the risk of returning strong toxicity, and the inactivated vaccine has the defects of incomplete virus inactivation, adverse reaction and even virus dispersion, thereby becoming a potential safety hazard.
The difficulty of solving the technical problems is as follows:
both attenuated vaccines and inactivated vaccines are composed of finished virus particles, and the existing finished virus genomes have insecurity problems, so that the problems cannot be solved fundamentally, and the occurrence of insecurity events can only be limited to the greatest extent by improving biosafety prevention and control measures. This will raise the production cost, and is not favorable to the popularization and production of vaccine.
The significance of solving the technical problems is as follows:
the vaccine prepared based on the virus-like particles does not contain virus genomes, and the risk of virus dispersion does not exist, so that the generation process of the vaccine can be effectively simplified, the production cost is reduced, the popularization and the use of the vaccine are improved, the occurrence of epidemic diseases is reduced, the production is promoted, and the agricultural income increase is guaranteed.
Furthermore, PPV VP 2-based VLPs are well-encapsulated, can accommodate foreign epitopes, and are ideal tools for the preparation of chimeric VLPs.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for assembling porcine parvovirus-like particles and identifying the immunogenicity of the porcine parvovirus-like particles.
The invention is realized in such a way that the method for assembling and identifying the immunogenicity of the porcine parvovirus-like particles comprises the following steps:
amplifying and optimizing VP2 gene and constructing recombinant donor plasmid;
step two, constructing and identifying VP2 recombinant shuttle plasmid;
step three, obtaining and identifying the recombinant baculovirus;
identifying the expression of the VP2 protein by Western-blot;
step five, performing indirect immunofluorescence identification on the VP2 protein;
step six, purifying and identifying PPV-AV30 virus;
and seventhly, assembling and identifying the virus-like particles.
Further, in the first step, the amplification, optimization and construction of the VP2 gene and the recombinant donor plasmid specifically comprise the following steps:
(1) according to the sequence of the PPV NADL-2 strain (GenBank accession number: KF913351.1), primers are designed by using bioinformatics software, and the PPV-AV30 is amplified to obtain the VP2 gene.
The upstream primer is SEQ ID NO: 1: p1: CGGGATCCATGAGTGAAAATGTGGAACAACAC; downstream primers P2: AACTGCAGCTAGTATAATTTTCTTGG (protective bases and cleavage sites: BamH I, Pst I, respectively, are underlined) and optimized to synthesize GS1814OP according to insect cell tropism.
(2) Designing a primer to amplify the VP2 gene, wherein an upstream primer is SEQ ID NO: 2: p3: CGGGATCCCTGGAACGCTACACTTTCAACCC; the downstream primer P4: AACTGCAGTTAGTACAGCTTTCTAGGGATCAGCTGGC (protected bases and cleavage sites BamHI, PstI are underlined).
(3) Identification of recombinant bacmids using universal primers: SEQ ID NO: 3: M13F: GTTTTTCCCAGTCACGAC, respectively; SEQ ID NO: 4: M13R: CAGGAAACAGCTATGAC, the primers were all synthesized by Xian engine corporation.
(4) Respectively using PPV-AV30 genome and GS1814OP plasmid as templates, amplifying the VP2 gene which is not optimized and optimized by PPV, and recovering and purifying target bands.
(5) Carrying out BamHI and Pst I double enzyme digestion on the target fragment and the pFastBac-Dual vector respectively, recovering, cleaning, connecting, chemically transforming DH5 α competent cells, screening positive clones, extracting unoptimized and optimized recombinant donor plasmid pFastVP2, and carrying out enzyme digestion and sequencing identification.
Further, in step two, the construction and identification method of the VP2 recombinant shuttle plasmid is as follows:
(1) the non-optimized and optimized pFastVP2 donor plasmid was transformed into DH10Bac competent cells containing AcBacmid and Helper plasmids, cultured for 48h on LB plates containing kanamycin (50g/m L), gentamicin (7g/m L), tetracycline (10g/m L), X-gal (100g/m L) and IPTG (40g/m L), screened in blue and white spots, and white colonies were selected.
(2) Extracting DNA, carrying out PCR detection by using an M13 universal primer, screening positive clone bacmid, namely, non-optimized and optimized rBacmid VP2, and extracting rBacmid VP2 recombinant bacmid for cell transfection.
Further, in step three, the recombinant baculovirus is obtained and identified as follows:
(1) according to the instruction of a liposome transfection kit Cellffectin @ II Reagent, non-optimized and optimized rBacmid VP2 is transfected into sf9 insect cells with good growth state, the cells are cultured in a constant-temperature incubator at 28 ℃ for 36-72 h, and after cytopathic effect occurs, the supernatant is collected to serve as P1 generation recombinant virus.
(2) Blind transferring the cell supernatant in sf9 insect cells for 3 generations, collecting the cell supernatant of the 3 rd generation, extracting virus DNA as a template, carrying out PCR detection by using primers P1, P2, P3 and P4 for amplifying VP2 gene, and screening to obtain a positive recombinant virus named as non-optimized and optimized rBacVP 2.
Further, in the fourth step, the expression method for identifying the VP2 protein by using Western-blot is as follows:
(1) and inoculating the recombinant virus which is not optimized and optimized rBacVP2P3 generation cytotoxicity to HF insect cells, infecting for 72h, and centrifuging to collect cell precipitates.
(2) And (3) cracking cells by using PMSF (cell lysate) and carrying out ultrasonic treatment, centrifuging at 10000r/min for 10min, and collecting supernatant for Western-blot analysis.
(3) The same procedure was used for normal HF insect cell protein samples as negative controls, with PPV positive serum diluted 1:100 at 5% Milk as the primary antibody and HRP-labeled anti-porcine IgG diluted 1:2000 at 5% Milk as the secondary antibody.
Further, in the fifth step, the indirect immunofluorescence identification method of the VP2 protein is as follows:
(1) the recombinant virus is not optimized and optimized rBacVP2P3 generation cell virus is inoculated to HF insect cells with good growth condition, and after 12h of infection, the HF insect cells are washed 3 times by PBS.
(2)4 ℃ precooled 4% paraformaldehyde is fixed for 15min at room temperature and then washed 3 times with PBS, and 0.1% Triton-100X permeates cell membranes for 10min and then washed 3 times with PBS.
(3) Blocking was performed using 5% NBS at 37 ℃ for 1 h. 5% NBS 1 PPV positive serum diluted 100 as primary antibody, incubated 1h at 37 ℃ and washed 5 times with PBST.
(4) 5% NBS 1 Anti-Swine IgG (H + L) diluted at 300: 5 was used as a secondary antibody, incubated at 37 ℃ for 1H, washed 5 times with PBST, placed under a fluorescence inverted microscope for observation, and HF cells were treated in the same manner as a negative control.
Further, in the sixth step, the PPV-AV30 virus purification and identification method is as follows:
(1) PK-15 cells grown to about 70-80% of the surface area of the bottom of the cell flask were removed from the 37 ℃ 5% CO2 incubator, and the old culture medium was discarded.
(2) The cells were rinsed twice with PBS, 1.5mL of PPV-AV30 cytotoxicity (-40 ℃ storage) was added directly to the rinsed PK-15 cell bottles, the cells were allowed to contact the virus solution sufficiently, and then incubated in a 37 ℃ 5% CO2 incubator for 1 h.
(3) After 1h of incubation, DMEM complete medium containing 10% FBS is added into a super clean bench, and when 70% of cells in the virus-inoculated group have typical lesions (most of the cells are exfoliated), the cells are repeatedly frozen and thawed for several times to obtain virus.
(4) Centrifuging at 10,000r/min for 30min to collect cell supernatant, adding 10% PEG6000 to concentrate virus while stirring, centrifuging at 10,000r/min for 60min, re-suspending the precipitate with PBS, centrifuging at 30,000r/min for 5h to obtain crude pure virus, dissolving the precipitate with PBS, purifying the virus by sucrose density gradient centrifugation, and absorbing negative stain to prepare an electron microscope.
Further, in step seven, the assembling and identifying method of the virus-like particles is as follows:
(1) HF cells are infected by P3 strain generation for recombinant protein expression, and shaking culture is carried out for 72h at 27 ℃ without a CO2 cell shaking table at 125 r/min.
(2) Centrifugally collecting cells, ultrasonically crushing to obtain non-optimized and optimized recombinant protein, centrifugally purifying by using sucrose density gradient to collect the fraction, carrying out phosphotungstic acid negative staining treatment, and observing the assembly condition of virus-like particles by using a Transmission Electron Microscope (TEM).
In summary, the advantages and positive effects of the invention are:
the invention utilizes an insect baculovirus expression system, an efficient mature foreign protein expression system, combines with the gene sequence analysis of a PPV-AV30 strain VP2 by an NCBI website, constructs and expresses the gene expression protein VP2 without optimization, optimizes the gene expression protein VP2, analyzes the difference between the two, and respectively analyzes the reactogenicity of the gene expression protein VP2, and lays a foundation for the research and development of novel PPV vaccines and the subsequent vaccine which uses PPV-VLPs as a carrier to be embedded with other virus neutralizing epitope so as to achieve the purpose of one-needle two-prevention.
The invention selects a eukaryotic expression system-insect baculovirus expression system Bac-to-Bac to construct baculovirus containing the optimized VP2 gene of porcine parvovirus strain AV-30, and infects insect cells to express the structural protein VP 2. Firstly, amplifying to obtain VP2 gene of porcine parvovirus PPV-AV30 virus strain, constructing recombinant donor plasmid pFastVP2, transforming DH10Bac competent cells, and obtaining recombinant shuttle vector rBacmindVP2 through blue-white spot screening and PCR detection. Recombinant baculovirus rbavp 2 was harvested using lipofection into sf9 insect cells in log phase growth. Western-blotting detection and indirect Immunofluorescence (IFA) experimental analysis show that the VP2 protein is correctly expressed in insect cells, the molecular weight is about 64KDa, and the insect cells have good reactogenicity. The VP2 protein is self-assembled into VLPs through TEM observation of an electron microscope, and a foundation is laid for further developing chimeric VP2 virus-like particles.
Drawings
FIG. 1 is a flow chart of the method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof according to the embodiment of the invention.
FIG. 2 is a schematic diagram of the amplification of an unoptimized VP2 sequence provided by an embodiment of the present invention;
in the figure: m represents a DNA molecular mass standard; 1-2 shows the amplification of the non-optimized VP2 gene.
FIG. 3 is a schematic diagram of the amplification of the optimized VP2 sequence provided by the embodiment of the present invention;
in the figure: m represents a DNA molecular mass standard; 1-2 shows the amplification of the non-optimized VP2 gene.
FIG. 4 is a schematic diagram illustrating the restriction identification of an unoptimized recombinant donor plasmid provided in an embodiment of the present invention;
in the figure: m1 and M2 are DNA molecular mass standards; 1 represents the double digestion product of the plasmid pFastVP2 which is not optimized; 2 denotes the non-optimized pFastVP2 plasmid.
FIG. 5 is an enzymatic identification of an unoptimized recombinant donor plasmid provided by an embodiment of the invention;
in the figure: m1 and M2 are DNA molecular mass standards; 1 represents the double digestion product of the optimized pFastVP2 plasmid; 2 denotes the optimized pFastVP2 plasmid.
FIG. 6 is a schematic representation of the PCR identification of an unoptimized recombinant shuttle plasmid provided in the examples herein;
in the figure: m represents a DNA molecular mass standard; 1-2 represent the PCR of the non-optimized recombinant shuttle plasmid.
FIG. 7 is a PCR identification of an optimized recombinant shuttle plasmid provided by an embodiment of the present invention;
in the figure: m represents a DNA molecular mass standard; 1-2 represent the PCR of the non-optimized recombinant shuttle plasmid.
FIG. 8 is a 400X schematic of CPE resulting from the non-optimized, optimized rBacVP2 infection of sf9 cells provided by an embodiment of the present invention;
in the figure: a indicates that the non-optimized rpbac vp2 infected sf9 cells; b indicates optimized rpac VP2 infection of sf9 cells; c indicates normal sf9 cells.
FIG. 9 is a Western-felt assay of VP2 protein expression provided in the examples of the present invention;
in the figure: 1 represents normal HF cells; 2 indicates non-optimized rpbac vp2 infected HF cells; 3 represents optimized rBacVP2 infected HF cells.
FIG. 10 is a 400X schematic diagram of the expression of the non-optimized, optimized VP2 protein in infected cells by IFA detection provided by the examples of the present invention;
in the figure: a represents normal HF cells; b represents non-optimized rback vp2 infected HF cells; c denotes optimized rbackvp 2 infected HF cells.
FIG. 11 is a schematic diagram of transmission electron microscopy of PPV whole virions (. times.25.0 k), non-optimized (. times.50.0 k), and optimized VP2 (. times.40.0 k) VLPs provided in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof provided by the embodiment of the invention comprises the following steps:
s101: amplifying VP2 gene, optimizing and constructing recombinant donor plasmid;
s102: constructing and identifying VP2 recombinant shuttle plasmid;
s103: obtaining and identifying recombinant baculovirus;
s104: identifying the expression of the VP2 protein by Western-blot;
s105: indirect immunofluorescence identification of the VP2 protein;
s106: purifying and identifying PPV-AV30 virus;
s107: assembling and identifying virus-like particles.
The technical solution of the present invention will be further described with reference to the following examples.
1. Materials and methods
1.1 materials
pFastBac Dual vector, PPV-AV30 strain, porcine tiny positive serum, PK-15 cells, Sf9 and HF insect cells were stored in the laboratory, various restriction enzymes, competent Escherichia coli DH5a and BL21(DE3) were purchased from TaKaRa (Dalian), insect cell culture medium Sf-900TM III SFM (1X) and transfection kit Cellffectin @ II Reagent were purchased from Gibco, GS1814OP optimized genes were synthesized and optimized in Nanjing King Kingsry, HRP-labeled porcine secondary antibody, Anti-Swine IgG (H + L) was purchased from Sigma.
1.2 amplification, optimization of VP2 Gene and construction of recombinant Donor plasmid
According to the sequence of a PPV NADL-2 strain (GenBank accession number: KF913351.1), bioinformatics software is used for designing primers, PPV-AV30 is amplified to obtain a VP2 gene, an upstream primer SEQ ID NO: 1: P1: CGGGATCCATGAGTGAAAATGTGGAACAACAC, a downstream primer P2: AACTGCAGCTAGTATAATTTTCTTGG (protective bases and restriction sites: BamH I and Pst I are underlined respectively), GS181OP 4 is synthesized according to insect cell tropism optimization, a VP2 gene is amplified, an upstream primer SEQ ID NO: 2: P3: CGGGATCCCTGGAACGCTACACTTTCAACCC and a downstream primer P4: AACTGCAGTTAGTACAGCTTTCTAGGGATCAGCTGGC (protective bases and restriction sites BamH I and Pst I are underlined) universal primers are used for identifying recombinant bacmid-plasmid, SEQ ID NO: 3: M13: GTTTTTCCCAGTCACGAC, SEQ ID NO: 4: M13: R: CAGGAAGCGAC, the primers are synthesized by Western Amphibianaceae, sequencing is synthesized by PPV-30, sequencing by a PPV-6866 company, digestion sites, sequencing is used for identifying a genome, digestion site, PCR gene is amplified, a plasmid of interest is cloned, a plasmid of Pagasac 2, a plasmid of a Pagasac is extracted, a plasmid of a.
1.3 construction and identification of the VP2 recombinant shuttle plasmid
The non-optimized and optimized pFastVP2 donor plasmid was transformed into DH10Bac competent cells containing AcBacmid and Helper plasmids, cultured for 48h on LB plates containing kanamycin (50g/m L), gentamicin (7g/m L), tetracycline (10g/m L), X-gal (100g/m L) and IPTG (40g/m L), screened in blue and white spots, and white colonies were selected. Extracting DNA, carrying out PCR detection by using an M13 universal primer, screening positive clone bacmid, namely, non-optimized and optimized rBacmid VP2, and extracting rBacmid VP2 recombinant bacmid for cell transfection.
1.4 acquisition and characterization of recombinant baculovirus
According to the instruction of a liposome transfection kit Cellffectin @ II Reagent, non-optimized and optimized rBacmid VP2 is transfected into sf9 insect cells with good growth state, the cells are cultured in a constant-temperature incubator at 28 ℃ for 36-72 h, and after cytopathic effect occurs, the supernatant is collected to serve as P1 generation recombinant virus. Blind transferring the cell supernatant in sf9 insect cells for 3 generations, collecting the cell supernatant of the 3 rd generation, extracting virus DNA as a template, carrying out PCR detection by using primers P1, P2, P3 and P4 for amplifying VP2 gene, and screening to obtain a positive recombinant virus named as non-optimized and optimized rBacVP 2.
1.5 Western-blot identification of VP2 protein expression
And inoculating the recombinant virus which is not optimized and optimized rBacVP2P3 generation cytotoxicity to HF insect cells, infecting for 72h, and centrifuging to collect cell precipitates. And (3) cracking cells by using PMSF (cell lysate) and carrying out ultrasonic treatment, centrifuging at 10000r/min for 10min, and collecting supernatant for Western-blot analysis. The same procedure was used for normal HF insect cell protein samples as negative controls, with PPV positive serum diluted 1:100 at 5% Milk as the primary antibody and HRP-labeled anti-porcine IgG diluted 1:2000 at 5% Milk as the secondary antibody.
1.6 Indirect immunofluorescence identification of VP2 protein
The recombinant virus is not optimized and optimized rBacVP2P3 generation cell virus is inoculated to HF insect cells with good growth condition, and after 12h of infection, the HF insect cells are washed 3 times by PBS. 4 ℃ precooled 4% paraformaldehyde is fixed for 15min at room temperature and then washed 3 times with PBS, and 0.1% Triton-100X permeates cell membranes for 10min and then washed 3 times with PBS. Blocking was performed using 5% NBS at 37 ℃ for 1 h. 5% NBS 1 PPV positive serum diluted 100 as primary antibody, incubated 1h at 37 ℃ and washed 5 times with PBST. 5% NBS 1 Anti-Swine IgG (H + L) diluted at 300: 5 was used as a secondary antibody, incubated at 37 ℃ for 1H, washed 5 times with PBST, placed under a fluorescence inverted microscope for observation, and HF cells were treated in the same manner as a negative control.
1.7 PPV-AV30 Virus purification and characterization
Taking out PK-15 cells growing to about 70-80% of the surface area of the bottom of the cell bottle from a 37 ℃ and 5% CO2 incubator, and discarding the old culture solution; the cells were rinsed twice with PBS, 1.5mL of PPV-AV30 cytotoxicity (-40 ℃ storage) was added directly to the rinsed PK-15 cell bottles, the cells were allowed to contact the virus solution sufficiently, and then incubated in a 37 ℃ 5% CO2 incubator for 1 h. After 1h of incubation, DMEM complete medium containing 10% FBS is added into a super clean bench, and when 70% of cells in the virus-inoculated group have typical lesions (most of the cells are exfoliated), the cells are repeatedly frozen and thawed for several times to obtain virus. Centrifuging at 10,000r/min for 30min to collect cell supernatant, adding 10% PEG6000 to concentrate virus while stirring, centrifuging at 10,000r/min for 60min, re-suspending the precipitate with PBS, centrifuging at 30,000r/min for 5h to obtain crude pure virus, dissolving the precipitate with PBS, purifying the virus by sucrose density gradient centrifugation, and absorbing negative stain to prepare an electron microscope.
1.8 Assembly and characterization of Virus-like particles
HF cells are infected by P3 strain generation for recombinant protein expression, and shaking culture is carried out for 72h at 27 ℃ without a CO2 cell shaking table at 125 r/min. Centrifugally collecting cells, ultrasonically crushing to obtain non-optimized and optimized recombinant protein, centrifugally purifying by using sucrose density gradient to collect the fraction, carrying out phosphotungstic acid negative staining treatment, and observing the assembly condition of virus-like particles by using a Transmission Electron Microscope (TEM).
2. Results
2.1 amplification of the VP2 Gene and identification of the recombinant donor plasmid pFastVP2
Using PPV-AV30 genome, plasmid GS1814OP as template, P1, P2, P3, P4 as primer to amplify non-optimized and optimized VP2 gene, we obtained 1740bp band (see FIG. 2 and FIG. 3), which is consistent with the expected size. Screening to obtain recombinant donor plasmid pFastVP2, carrying out double enzyme digestion verification on BamH I and Pst I, wherein the result (shown in figure 4 and figure 5) shows that 2 specific bands are formed after double enzyme digestion, wherein one band is about 1740bp of VP2 gene, and the other band is about 6000bp of vector fragment, which are consistent with the expected size. The pFastVP2 recombinant plasmid is further subjected to sequencing verification, and the result shows that the inserted sequence has no base mutation and is completely correct.
2.2 obtaining recombinant shuttle plasmids and baculoviruses
The un-optimized and optimized pFastVP2 recombinant donor plasmid is transformed into DH10Bac competent cells, and white colonies are screened for purification. The recombinant bacmid is extracted and PCR detection is carried out by M13 primer, so that about 4300bp of target band can be amplified, and the result is shown in FIG. 6 and FIG. 7. The non-optimized and non-optimized recombinant bacmid VP2 liposome method is used for transfecting sf9 cell insect cells, the insect cells are cultured at 28 ℃, the first generation has no obvious cytopathic effect, the obvious cytopathic effect appears after the insect cells are cultured for 72 hours in a blind mode from the 3 rd generation, the main symptoms are that the cells swell and become round, the cell nucleus is obvious, and the result is shown in figure 8.
2.3 Western-blot identification of recombinant protein expression
The recombinant protein was identified by Western-blot using the wet transfer method (see FIG. 9). The detection result shows that a specific band of about 64KDa exists in the sf9 insect cell sample infected by the recombinant virus, and the band does not appear in normal cells. The VP2 gene of PPV is successfully expressed in HF insect cells, and the expression product has certain reactogenicity.
2.4 Indirect immunofluorescence assay for recombinant protein expression
The non-optimized and non-optimized rBacVP2 baculovirus infects HF insect cells, and after immunofluorescence detection, observation results under a fluorescence microscope show that the non-optimized and non-optimized rBacVP2 infected HF cells show red fluorescence, while the normal HF cells do not show fluorescence, which indicates that the VP2 gene of PPV obtains correct expression in the HF insect cells (see figure 10).
2.5 Electron microscopy of recombinant proteins and purification of PPV-AV30 Virus
The purified protein and AV30 virions were observed by transmission electron microscopy, and the results showed that the diameter was about 25nm, and the morphology and size of the recombinant protein were similar to those of PPV whole virions (see FIG. 11), indicating that the VP2 gene of PPV was successfully expressed in HF insect cells and successfully assembled into VLPs.
3. Results
Virus-like particles (VLPs) are a safe and effective nanomaterial that has been studied for intense fire in recent years because they are free of viral nucleic acids, cannot replicate autonomously, are not infectious, but induce both humoral and cellular immune responses. Therefore, the vaccine has important clinical application value as a novel vaccine. The high infection rate of porcine parvovirus PPV has serious influence on the pig industry, and although vaccination controls the epidemic, certain biological safety risk exists, so that the development of novel and safe PPV vaccine is imperative. In recent years, VLPs have been formed by in vitro assembly of PPV-VP2 protein using different expression systems and high levels of specific antibodies have been produced in both guinea pigs and pigs.
The invention selects a eukaryotic expression system-insect baculovirus expression system, can carry out post-translational processing modification on protein, omits the complex processes of denaturation, renaturation and the like of a prokaryotic system, and displays the natural conformation of the target protein. The invention also carries out two treatment modes of non-optimization and optimization on the target gene, and researches show that: the insect expression system successfully expresses the protein VP2 of the PPV strain AV-30, and VLPs are correctly assembled, thereby laying a foundation for the next research on chimeric VLPs. Wherein, the virus particle capsid of PPV is composed of VP1, VP2 and VP3, VP2 accounts for the main proportion, and VLPs can be formed by in vitro assembly only needing VP2 protein.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
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Claims (8)
1. A method for assembling porcine parvovirus-like particles and identifying the immunogenicity of the porcine parvovirus-like particles is characterized by comprising the following steps:
amplifying and optimizing VP2 gene and constructing recombinant donor plasmid;
step two, constructing and identifying VP2 recombinant shuttle plasmid;
step three, obtaining and identifying the recombinant baculovirus;
identifying the expression of the VP2 protein by Western-blot;
step five, performing indirect immunofluorescence identification on the VP2 protein;
step six, purifying and identifying PPV-AV30 virus;
and seventhly, assembling and identifying the virus-like particles.
2. The method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof as recited in claim 1, wherein in the first step, the amplification, optimization and construction of the recombinant donor plasmid of VP2 gene specifically comprises the following steps:
(1) according to the sequence of the PPV NADL-2 strain, a bioinformatics software is used for designing a primer, and the PPV-AV30 is amplified to obtain a VP2 gene;
the upstream primer is as follows: p1: CGGGATCCATGAGTGAAAATGTGGAACAACAC; downstream primer P2: AACTGCAGCTAGTATAATTTTCTTGG, and GS1814OP is synthesized according to insect cell tropism optimization;
(2) designing a primer to amplify the gene VP2, wherein an upstream primer P3: CGGGATCCCTGGAACGCTACACTTTCAACCC; a downstream primer P4: AACTGCAGTTAGTACAGCTTTCTAGGGATCAGCTGGC;
(3) identification of recombinant bacmids using universal primers: M13F: GTTTTTCCCAGTCACGAC, respectively; M13R: CAGGAAACAGCTATGAC;
(4) respectively using PPV-AV30 genome and GS1814OP plasmid as templates, amplifying the VP2 gene which is not optimized and optimized by PPV, and recovering and purifying target bands;
(5) carrying out BamHI and Pst I double enzyme digestion on the target fragment and the pFastBac-Dual vector respectively, recovering, cleaning, connecting, chemically transforming DH5 α competent cells, screening positive clones, extracting unoptimized and optimized recombinant donor plasmid pFastVP2, and carrying out enzyme digestion and sequencing identification.
3. The method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof as recited in claim 1, wherein in step two, the VP2 recombinant shuttle plasmid is constructed and identified as follows:
(1) transforming an unoptimized and optimized pFastVP2 donor plasmid into a DH10Bac competent cell containing AcBacmid and Helper plasmids, culturing for 48h on an LB plate containing kanamycin 50g/m L, gentamicin 7g/m L, tetracycline 10g/m L, X-gal 100g/m L and IPTG 40g/m L, screening blue white spots, and selecting a white colony;
(2) extracting DNA, carrying out PCR detection by using an M13 universal primer, screening positive clone bacmid, namely, non-optimized and optimized rBacmid VP2, and extracting rBacmid VP2 recombinant bacmid for cell transfection.
4. The method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof as set forth in claim 1, wherein the method for obtaining and identifying the recombinant baculovirus in step three comprises:
(1) according to the instruction of a liposome transfection kit Cellffectin @ II Reagent, non-optimized and optimized rBacmid VP2 is transfected into sf9 insect cells with good growth state, the cells are cultured in a constant-temperature incubator at 28 ℃ for 36-72 h, and after cytopathic effect occurs, the supernatant is collected to serve as P1 generation recombinant virus;
(2) blind transferring the cell supernatant in sf9 insect cells for 3 generations, collecting the cell supernatant of the 3 rd generation, extracting virus DNA as a template, carrying out PCR detection by using primers P1, P2, P3 and P4 for amplifying VP2 gene, and screening to obtain a positive recombinant virus named as non-optimized and optimized rBacVP 2.
5. The method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof as recited in claim 1, wherein the expression method of VP2 protein is identified by Western-blot in step four as follows:
(1) inoculating the non-optimized and non-optimized rBacVP2P3 generation of recombinant virus into HF insect cells, infecting for 72h, and centrifugally collecting cell sediment;
(2) cell lysis solution PMSF is used for cell lysis and ultrasonic treatment, centrifugation is carried out at 10000r/min for 10min, and supernatant is collected and used for Western-blot analysis;
(3) PPV positive serum was diluted 1:100 with 5% Milk as the primary antibody, HRP-labeled anti-porcine IgG was diluted 1:2000 with 5% Milk as the secondary antibody, and a normal HF insect cell protein sample was used as the negative control.
6. The method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof as claimed in claim 1, wherein the indirect immunofluorescence identification method of VP2 protein in the fifth step is as follows:
(1) inoculating the non-optimized and non-optimized rBacVP2P3 generation cell virus of the recombinant virus to HF insect cells with good growth condition, and washing 3 times with PBS after infecting for 12 hours;
(2) fixing 4% paraformaldehyde precooled at 4 deg.C at room temperature for 15min, washing with PBS 3 times, penetrating cell membrane with 0.1% Triton-100X for 10min, and washing with PBS 3 times;
(3) blocking with 5% NBS at 37 ℃ for 1 h; 5% NBS 1 PPV positive serum was diluted 100 as primary antibody, incubated at 37 ℃ for 1h, washed 5 times with PBST;
(4) 5% NBS 1 Anti-Swine IgG (H + L) diluted at 300: 300 was used as a secondary antibody, incubated at 37 ℃ for 1H, washed 5 times with PBST, placed under a fluorescence inverted microscope for observation, and treated HF cells were used as a negative control.
7. The method for assembling porcine parvovirus-like particles and identifying the immunogenicity thereof as set forth in claim 1, wherein the PPV-AV30 virus is purified and identified in the sixth step as follows:
(1) taking out PK-15 cells growing to about 70-80% of the surface area of the bottom of the cell bottle from a 37 ℃ and 5% CO2 incubator, and discarding the old culture solution;
(2) rinsing the cells twice with PBS, collecting 1.5mL of PPV-AV30 cell toxin, preserving at-40 deg.C, directly adding into rinsed PK-15 cell bottle, contacting the cells with virus solution, placing at 37 deg.C and 5% CO2Incubating for 1h in an incubator;
(3) after incubation for 1h, adding a DMEM complete culture medium containing 10% FBS into a super clean bench, and repeatedly freezing and thawing for several times to collect the virus when 70% of cells of a virus-inoculated group have typical lesions;
(4) centrifuging at 10,000r/min for 30min to collect cell supernatant, adding 10% PEG6000 to concentrate virus while stirring, centrifuging at 10,000r/min for 60min, re-suspending the precipitate with PBS, centrifuging at 30,000r/min for 5h to obtain crude pure virus, dissolving the precipitate with PBS, purifying the virus by sucrose density gradient centrifugation, and absorbing negative stain to prepare an electron microscope.
8. The method for assembling porcine parvovirus-like particle and identifying the immunogenicity thereof as recited in claim 1, wherein the method for assembling and identifying the porcine parvovirus-like particle in the seventh step is as follows:
(1) HF cell is infected by P3 strain for recombinant protein expression, and CO is not generated at 27 DEG C2Shaking and culturing the cells for 72 hours at 125r/min by a cell shaking table;
(2) centrifugally collecting cells, ultrasonically crushing to obtain non-optimized and optimized recombinant protein, centrifugally purifying by using sucrose density gradient to collect the fraction, carrying out phosphotungstic acid negative staining treatment, and observing the assembly condition of virus-like particles by using a Transmission Electron Microscope (TEM).
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| CN113150081A (en) * | 2021-05-12 | 2021-07-23 | 河南省农业科学院动物免疫学重点实验室 | Construction method and application of porcine circovirus 2b subtype self-epitope chimeric virus-like particle in insect cell |
| CN114134180A (en) * | 2021-11-26 | 2022-03-04 | 山东滨州沃华生物工程有限公司 | Construction method of recombinant baculovirus expressing porcine reproductive and respiratory syndrome (GP) 5 protein |
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