CN112430625B - Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gD protein gene, virus, preparation method and application thereof - Google Patents
Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gD protein gene, virus, preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant adeno-associated virus transfer vector containing a gD protein gene of a variant porcine pseudorabies virus, a recombinant adeno-associated virus, and preparation and application thereof. The recombinant adeno-associated virus (rAAV) prepared by the invention is used as a vector to express the gD protein of the porcine pseudorabies virus, and the rAAV has multiple advantages of safety, effectiveness, specificity and the like. The gene engineering strain prepared by the invention expresses gD immunogenic protein of the epidemic variation porcine pseudorabies virus strain, and has good immune protection to multiple epidemic variation porcine pseudorabies virus strains. The mouse can be injected into the tibialis anterior muscle of the right lower limb once, so that the organism can be continuously induced to generate specific neutralizing antibody and cellular immunity at high level.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant adeno-associated virus transfer vector containing a variant porcine pseudorabies virus gD protein gene, a recombinant adeno-associated virus, preparation and application thereof.
Background
Pseudorabies (PR) is caused by Pseudorabies virus (PRV), and is also called Pseudorabies because clinical symptoms are similar to rabies, and fever, itching and encephalitis are the main symptoms after livestock (pigs, cattle, sheep) and wild animals suffer from diseases. PRV has a wide range of hosts in nature, with pigs being the primary natural host and storage host. PRV can infect pigs of different ages of day causing different clinical symptoms, especially the most severe damage after infection of pregnant sows and suckling piglets. PRV causes the reproduction disorder of pregnant sows, and the suckling piglets have nervous symptoms and high mortality rate, and the mortality rate of the 15-day-old piglets after infection reaches 100 percent; the adult pigs become latent infected pigs after being resistant to the infection, the latent infected pigs carry the toxin for the whole life and periodically expel the toxin, become PRV epidemic infectious agents, and increase the difficulty of eradicating the disease. Pseudorabies causes great economic loss to the pig industry in the world. At present, the vaccine is still the most economic and effective means for preventing and controlling the porcine pseudorabies.
Since 2011, pseudorabies caused by variant pseudorabies virus is outbreaked in northern China and northeast China, and rapidly spreads to other areas, and a plurality of pig farms immunized with classical strain vaccine Bartha-K61 are difficult to survive (An et al 2013).
Variant strains the gD protein presents an insertion of 2 amino acids (R and P) at positions 280 and 281 relative to classical strains (Wang et al 2014). The student carries out genetic evolution analysis on the gD gene of the variant strain and the gD gene of the classical strain, and compared with the classical strain, the gB protein of the variant strain has 1.1 percent of variation of amino acid of the gB protein and 0.4 percent of variation of nucleotide (Wang et al 2015), which shows that the gB protein has two less amino acid residues, the gB protein has 3 aa (S, P and G) deletions at the 94-77 positions, and 1 amino acid (G) insertion and 22 point mutation at the 94 position (Wang et al 2014). The variation in gB protein may be the reason why classical strains do not provide protection to the variant strain. The gD gene of the variant strain has 14 base mutation and 45 amino acid mutation (Song et al 2017), and the gD protein mutation is probably the reason that the classical strain can not provide protection for the variant strain. Ye et al genotyping PRV based on the genetic evolution of PRV gC gene shows that the current European and American strains and PRV strains prevalent in our country belong to different branches and have a relatively long affinity (Ye et al 2016). Since 2011, all Chinese newly isolated strains belong to genotype II, including classical Bartha-K61 strains, Buchares strains and the like in European and American strains belong to genotype I, and a reference basis is provided for poor protection of classical vaccines on current epidemic strains (He et al 2018).
The variant PRV and the classical strain have great difference in pathogenicity, virulence factor expression, propagation efficiency and the like. Classical vaccines have poor protection on variant strains, and variant pseudorabies viruses which are clinically separated are required to be used as gene samples to research and develop more effective vaccines to prevent and control pseudorabies, however, the preparation of inactivated vaccines and gene deletion attenuated vaccines by using whole viruses is complicated in operation and high in cost, so that DNA vaccines, subunit vaccines, live vector vaccines and the like are the main directions for researching and developing PRV vaccines.
The commercial vaccine adopted by the experimental contrast is a commercial porcine pseudorabies inactivated vaccine of a biological company before the department, plays an important role in the prevention and control of PR, but the loss of main antigenic determinants can occur in the inactivation process of the inactivated vaccine, so that the immune efficacy is poor, a larger inoculation dose is needed, multiple immunizations are needed, a better immune effect can be achieved, the components of the inactivated vaccine are complex, and anaphylactic reaction often occurs.
Disclosure of Invention
Aiming at the defects or the improvement requirements of the prior art, the invention aims to provide a recombinant adeno-associated virus transfer vector containing a variant porcine pseudorabies virus gD protein gene so as to realize the efficient, stable and long-term secretory expression of the variant porcine pseudorabies virus gD protein in an animal body.
The invention also aims to provide a recombinant adeno-associated virus capable of expressing the gD protein of the porcine pseudorabies virus and application of the recombinant adeno-associated virus in preparation of a vaccine for the variant porcine pseudorabies. The variant porcine pseudorabies vaccine prepared by the virus has high immunity, high safety and strong specificity.
In order to achieve the above object, the present invention provides a recombinant adeno-associated virus transfer vector containing a variant epidemic porcine pseudorabies virus gD protein gene, wherein the vector is a recombinant adeno-associated virus vector, and is a recombinant adeno-associated virus transfer vector obtained by inserting the variant epidemic porcine pseudorabies virus gD protein gene into coding regions between ITR sequences at two ends of the recombinant adeno-associated virus transfer vector.
Preferably, the gD protein gene has:
(1) a nucleotide sequence shown by SEQ ID No. 1; or
(2) A gD protein gene sequence with homology within 95 percent with the nucleotide sequence defined by the sequence SEQ ID No. 1; and the mutation region is located 820-837 after the initiation codon.
Preferably, the recombinant adeno-associated virus transfer vector is a recombinant adeno-associated virus transfer vector of serotype AAV 2/9.
Preferably, the recombinant adeno-associated virus transfer vector contains a CMV promoter sequence.
According to another aspect of the present invention, there is provided a method for constructing the recombinant adeno-associated virus transfer vector, comprising the steps of:
(1) taking the genome DNA of the epidemic variant strain as a template, and carrying out PCR amplification to obtain a gD protein gene fragment;
(2) carrying out double enzyme digestion on the recombinant adeno-associated virus vector, recovering a target vector fragment by using a glue recovery kit, and quantifying the target vector fragment, and marking the target vector fragment as a linear vector fragment;
(3) and carrying out recombination reaction on the gD protein gene segment and the linear vector segment by DNA polymerase to construct the recombinant adeno-associated virus transfer vector containing the variant porcine pseudorabies virus gD protein gene.
According to another aspect of the present invention, there is provided a recombinant adeno-associated virus capable of expressing the gD protein of porcine pseudorabies virus, which is a recombinant adeno-associated virus formed by inserting a variant gD protein gene of porcine pseudorabies virus into an adeno-associated virus vector.
Preferably, the serotype of the adeno-associated viral vector is AAV 2/9.
Preferably, the viral genome comprises a CMV promoter sequence.
According to another aspect of the invention, the application of the recombinant adeno-associated virus in preparing a variant porcine pseudorabies virus vaccine is provided.
Preferably, the recombinant adeno-associated virus is prepared as an intramuscular injection.
According to another aspect of the present invention, there is provided a method for preparing the recombinant adeno-associated virus, comprising the steps of:
(1) co-transfecting the recombinant adeno-associated virus transfer vector and the backbone plasmid into a host cell;
(2) culturing the host cell of step (1);
(3) harvesting the recombinant adeno-associated virus packaged in the cells of step (2);
(4) and (4) purifying the recombinant adeno-associated virus in the step (3).
Preferably, the host cell in step (2) is HEK-293T.
Through the technical scheme, compared with the prior art, the invention can obtain the following beneficial effects:
(1) the recombinant adeno-associated virus transfer vector containing the variant porcine pseudorabies virus gD protein gene is a recombinant adeno-associated virus vector, and the rAAV vector has multiple advantages of safety, effectiveness, specificity and the like.
(2) The gene engineering strain gD gene segment of the porcine pseudorabies virus expressed by the invention is an immunogenic segment of an epidemic variation porcine pseudorabies virus strain, and has good immune protection to multiple epidemic variation porcine pseudorabies virus strains. The injection is injected into the tibialis anterior muscle of the right lower limb once, so that the organism can be continuously induced to generate specific neutralizing antibody and cellular immunity at high level.
(3) After the traditional vaccine is immunized, multiple times of boosting immunization are needed, compared with the traditional vaccine, the live vector virus vaccine provided by the invention can obtain long-acting protection only by one time of immunization, the boosting immunization is not needed, and the operation is more convenient. Therefore, the vaccine prepared by the genetic engineering strain has wide market application prospect.
(4) The existing PRV commercial vaccines are mainly inactivated vaccines and attenuated vaccines. Inactivated viruses in the inactivated vaccines do not have the capability of replication and proliferation, cannot be infected latently and are dispersed, and the inactivated vaccines have the problem of safety, but the loss of main antigenic determinants can occur in the inactivation process of the inactivated vaccines, so that the immune efficacy is poor, a large inoculation dose is generally needed, or multiple immunizations are carried out, so that a good immune effect can be achieved, and the inactivated vaccines are complex in components and often cause anaphylactic reactions. Although the attenuated vaccine has good immunogenicity and low cost and plays an important role in the prevention and control of PRV, the attenuated vaccine can establish latent infection due to the possible problems of virulent return, variation and the like, the long-term virus scattering risk exists, and the country which purifies the porcine pseudorabies has banned the use of the attenuated vaccine. However, the vaccine for preparing the epidemic mutation porcine pseudorabies virus belongs to a live vector virus vaccine, the genome of the vaccine does not contain any AAV protein coding DNA, the virus vector can not be autonomously replicated, meets the biological safety requirement of the vaccine, and is suitable for purifying the porcine pseudorabies.
(5) The gene engineering strain gD gene fragment expressed by the porcine pseudorabies virus strain prepared in the preferred embodiment of the invention is an immunogenic fragment of the porcine pseudorabies virus strain PRV/JXFC/2015, and a cross neutralization test shows that the strain induces an organism to generate specific neutralizing antibodies and cellular immunity, and has good immune protection on PRV/JXFC/2015 strain and a wild strain CH-18 strain. Single injection of virus, blood was collected at 2, 12, 20, 24, 28 weeks post immunization. Detecting virus serum neutralizing antibodies of PRV/JXFC/2015 wild strains, wherein the neutralizing titer of the mixed serum samples of the negative control PBS group at all time points is 1:0, and the neutralizing titers of the mixed serum samples of the positive control commercial vaccine group are 1:16, 1:16 and 1:22.38 respectively; the neutralization titers of the mixed serum samples of the rAAV 2/9-PRV-gD-P2A-Mchery group are 1:5, 1:64, 1:32, 1:44, 1:39.8 and 1:56.2 respectively. For cross-neutralization detection of CH-18 wild strains on 28-week mixed serum samples, the rAAV 2/9-PRV-gD-P2A-Mchery group is more than or equal to 1:256, the positive control commercial vaccine group is 1:42, and the negative control PBS group is 1: 0. The porcine pseudorabies recombinant vaccine AAV2/9-PRV-gD group using the adeno-associated virus as a vector has a level of neutralizing antibody which is obviously higher than that of a commercial vaccine group aiming at an epidemic variant strain PRV/JXFC/2015, and simultaneously the AAV2/9-PRV-gD group also has a level of neutralizing antibody which is obviously higher than that of a commercial vaccine group aiming at a wild strain CH-18.
Drawings
FIG. 1 is a schematic diagram of the construction of the vector plasmids PT-1540 and pAAV-PRV-gD-Mchery used in the present invention;
FIG. 2 is a physical map of vector plasmids PT-1786 and pAAV-PRV-gB-P2A-EGFP used in the present invention;
FIG. 3 is a diagram showing PCR amplification of the present invention from a genomic template using primers for PRV gB gene, in which M is
Plus II DNA Marker; 1-6 are PCR amplification products of PRV gD genes;
FIG. 4 is a diagram showing PCR amplification of the present invention from a genomic template using primers for PRV gD gene, wherein M is
Plus II DNA Marker; 1-2 are PCR amplification products of PRV gD genes;
FIG. 5 is a diagram of a double-enzyme gel-cutting of the vector plasmid PT-1786/PT-1540 used in the present invention, in which M:
PlusⅡDNA Marker；1：PT-1540；2：PT-1786；
FIG. 6 is a colony identification map of plasmid pAAV-PRV-gB-P2A-EGFP constructed in the present invention, in which M:
plus II DNA Marker; 1: pAAV-PRV-gB-P2A-EGFP; 2-3 are negative;
FIG. 7 is a colony identification chart of plasmid pAAV-PRV-gD-P2A-Mchery constructed in the present invention, in which M is
Plus II DNA Marker; 1 is PT-PRV gD, and 2-5 is negative;
FIG. 8 shows a recombinant plasmid pAAV-PRV-gD-P2A-M constructed in the present inventionThe restriction enzyme identification map of cherry and pAAV-PRV-gB-P2A-EGFP; in the figure, M:
PlusⅡDNA Marker；1：pAAV-PRV-gD-P2A-Mcherry；2：pAAV-PRV-gB-P2A-EGFP；
FIG. 9 shows the morphology and fluorescence photographs of cell transfection packages of rAAV2/9-PRV-gB-P2A-EGFP and rAAV2/9-PRV-gD-P2A-Mcherry virus of 72 h; in the figure, A1: transfecting cells packaging rAAV 2/9-PRV-gD-P2A-Mchery with 3D red fluorescence; a2: 3D normal white light photographs of cells transfected with packaging rAAV 2/9-PRV-gD-P2A-Mchery; b1: transfecting cells packaging rAAV2/9-PRV-gB-P2A-EGFP with 3D green fluorescence; b1: 3D normal white light photographs of cells transfected with packaging rAAV 2/9-PRV-gB-P2A-EGFP; c, comparing a normal HEK-293T white light photo;
FIG. 10 is the SDS PAGE silver stained gel of rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery in the present invention, wherein M: marker CAS-26616; 1: rAAV 2/9-PRV-gD-P2A-Mcherry; 2: rAAV 2/9-PRV-gB-P2A-EGFP; 3: 2.5E +12vg/mL of control virus for cement titer;
FIG. 11 shows the electron microscope photographs of rAAV2/9-PRV-gB-P2A-EGFP and rAAV2/9-PRV-gD-P2A-Mcherry virus in the present invention, A: rAAV 2/9-PRV-gB-P2A-EGFP; b: rAAV 2/9-PRV-gD-P2A-Mchery;
FIG. 12 shows Western blotting detection results of rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery virus in the present invention;
FIG. 13 shows the results of detecting the level of neutralizing antibodies in mouse immunoassays for rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery virus of the present invention;
FIG. 14 shows the result of detecting cytokine IL-4 in mouse immunoassay for rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery virus in the present invention;
FIG. 15 shows the result of detecting the cytokine IFN-gamma in the mouse immunoassay of rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery virus in 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 described in further detail below with reference to the accompanying drawings and 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.
The invention provides a recombinant adeno-associated virus transfer vector containing gD protein gene of variant epidemic porcine pseudorabies virus, which is a recombinant adeno-associated virus vector, wherein the gD protein gene of the variant epidemic porcine pseudorabies virus is inserted into coding regions between ITR sequences at two ends of the recombinant adeno-associated virus transfer vector to obtain the recombinant adeno-associated virus transfer vector.
The gD protein of the variant epidemic porcine pseudorabies virus, namely gD (US6), belongs to type I glycoprotein, and has the relative molecular mass of about 44 KD. The gD protein is a receptor binding protein necessary for PRV to penetrate cells, and binding of a viral protein complex to cells is achieved by recognizing HVEM receptors on the cell surface. The gD protein is an immunogenic protein of PRV and can stimulate the body to produce specific neutralizing antibody and cell immune response.
The recombinant adeno-associated virus transfer vector provided by the invention is suitable for various variant epidemic porcine pseudorabies viruses, including the genotypes of I and II, and also can be variant epidemic porcine pseudorabies viruses containing gD sequences with homology within 95%; and the gD sequence mutation region is positioned at 820-837 th after the initiation codon. In the embodiment of the invention, a variant epidemic porcine pseudorabies virus (PRV/JXFC/2015) is taken as an example, a recombinant adeno-associated virus transfer vector containing a gD protein gene of the variant epidemic porcine pseudorabies virus (PRV/JXFC/2015) is provided, and the gD protein gene of the variant epidemic porcine pseudorabies virus (PRV/JXFC/2015) is inserted into a coding region between ITR sequences at two ends of the recombinant adeno-associated virus transfer vector to obtain the recombinant adeno-associated virus transfer vector. The nucleotide sequence of gD (US6) gene of variant pandemic porcine pseudorabies virus (PRV/JXFC/2015) is shown as SEQ ID No. 1.
It is understood that, in addition to the variant epidemic porcine pseudorabies virus (PRV/JXFC/2015), the recombinant adeno-associated virus transfer vector provided by the invention also comprises a recombinant adeno-associated virus transfer vector obtained by inserting gD protein genes of other variant epidemic porcine pseudorabies viruses into coding regions between ITR sequences at two ends of the recombinant adeno-associated virus transfer vector. The gD protein gene sequences of different variant epidemic porcine pseudorabies virus strains are slightly different. The researchers analyze the nucleotide sequence homology of the gD gene of 21 porcine pseudorabies viruses, find that the length of the nucleotide of the Open Reading Frame (ORF) of the 21 gD gene is between 1197nt and 1215nt, the nucleotide homology is between 97.3 percent and 100 percent, prove that the strains have high homology, and prove that the gene has strong conservation. Conserved genes generally play an important role in the growth cycle of viruses, and once a large variation occurs, the species risks being eliminated. The high homology (. gtoreq.95%) of the gD genes of different strains is an indication of the important function that the genes assume in the growth cycle of the virus. The largest difference between strains is the C (A) GGCCC repeated hypervariable region at position 820-837 after the initiation codon, and the mutation of other nucleic acids is mostly some point mutation because the base deletion or insertion of the repeated hypervariable region causes the variation of the ORF of PRV-gD between 1197nt and 1215nt, but the increase and decrease of the segment has a multiple of 3 bases, so the reading of the sequence behind the repeated hypervariable region is not influenced. (xu Yan hill et al 2006). Therefore, the gD protein gene sequence of the variant epidemic porcine pseudorabies virus can be not only the sequence shown in SEQ ID No.1, but also a gD sequence with homology within 95 percent; and the mutation region is located 820-837 after the initiation codon.
As a control group, a recombinant adeno-associated virus transfer vector containing a gB protein gene of a variant epidemic porcine pseudorabies virus (PRV/JXFC/2015) is prepared in the embodiment of the invention, and the gB protein gene of the variant epidemic porcine pseudorabies virus is inserted into a coding region between ITR sequences at two ends of the recombinant adeno-associated virus transfer vector to obtain the recombinant adeno-associated virus transfer vector. The gB (UL27) protein is the envelope protein of PRV, belongs to type I glycoprotein, and has a molecular weight of about 100 KD. gB is an important immunogenic protein of PRV and is the major glycoprotein that stimulates the body to produce neutralizing antibodies. The nucleotide sequence of the gB (UL27) gene is shown as SEQ ID No. 2.
Hair brushThe clinically epidemic PRV variant strain, PRV/JXFC/2015 variant strain, used in this study was isolated, identified and stored in 2015 in some pig farm with pseudorabies outbreaks in Jiangxi province. The laboratory has isolated 45 PRV variant strains in recent years and performed a series of tests to screen candidate strains for vaccines. The virus titer of PRV/JXFC/2015 is stable at 10 7.5 TCID50, PRV/JXFC/2015 is more lethal to mice than other PRV strains. The plaque area formed after PRV/JXFC/2015 infects PK-15 cells is obviously higher than that of a classical PRV/Ea strain and Bartha-K61 and that of other strains, and research shows that vaccines developed by strains with strong toxicity can provide better immunogenicity, so that a genome sample of the vaccine is subjected to new research on a pseudorabies gene engineering carrier live vaccine to construct a PRV vaccine strain based on rAAV, and the vaccine strain is expected to provide better immunogenicity.
Since 2011, the newly isolated Chinese strains all belong to genotype II and are in different branches with European and American strains, and PRV/JXFC/2015 also belongs to genotype II, so that a genome sample of the strain is subjected to new research on a pseudorabies genetic engineering vector live vaccine to construct rAAV for expressing gD and gB proteins of PRV, and the rAAV is used as a research on PRV vaccine strains, and is expected to have good immune protection on multiple epidemic variant porcine pseudorabies virus strains in China.
The rAAV is non-pathogenic, so that the safety is high, the immunogenicity is low, and the host for the rAAV to live is wide. rAAV is a virus vector capable of loading exogenous genes, has the characteristics of long-term and stable expression in animal bodies, and is applied to the fields of vaccines, interference screening, neural markers, animal disease model modeling, gene therapy and the like. The major disadvantage of rAAV is that the size of the virus particle is limited, the capability of carrying foreign gene is generally less than 4.7kb, otherwise the package titer is greatly reduced, the rate of empty shell of the virus package is increased, and even normal package is impossible.
In some embodiments, the method for constructing the recombinant adeno-associated virus transfer vector comprises the following steps:
(1) taking the genome DNA of the epidemic variant strain as a template, and carrying out PCR amplification to obtain a gD protein gene fragment;
(2) carrying out double enzyme digestion on the recombinant adeno-associated virus vector, recovering a target vector fragment by using a glue recovery kit, and quantifying the target vector fragment, and marking the target vector fragment as a linear vector fragment;
(3) and carrying out recombination reaction on the gD protein gene segment and the linear vector segment through DNA polymerase to construct the recombinant adeno-associated virus transfer vector containing the variant porcine pseudorabies virus gD protein gene.
In some embodiments, the construction of the recombinant adeno-associated virus transfer vector containing the variant porcine pseudorabies virus gD protein gene specifically comprises the following steps:
(1) taking the genomic DNA of the epidemic variant strain PRV/JXFC/2015 as a template, and carrying out PCR amplification to obtain a gD protein gene fragment;
(2) carrying out Sal I and Spe I double enzyme digestion on a recombinant adeno-associated virus Vector PT-1540(rAAV-CMV-SOD1-P2A-mcherry-WPRE-hGH poly), carrying out enzyme digestion to obtain two bands (468bp/6001bp), recycling 6001bp by using a gel recovery kit, and then quantifying, wherein the bands are marked as a linear Vector fragment Vector 2;
(3) and (3) carrying out recombination reaction on the gD protein gene segment and the linear Vector segment Vector2 through DNA polymerase to construct the recombinant adeno-associated virus transfer Vector containing the variant porcine pseudorabies virus gD protein gene.
In the embodiment, the construction of the recombinant adeno-associated virus transfer vector containing the variant porcine pseudorabies virus gB protein gene specifically comprises the following steps:
(1) taking the genomic DNA of the epidemic variant strain PRV/JXFC/2015 as a template, and carrying out PCR amplification to obtain a gB protein gene fragment;
(2) carrying out Xba I and Sal I double enzyme digestion on a recombinant adeno-associated virus Vector PT-1786(rAAV-CMV (584bp) -beta arrestin2-Flag-P2A-EGFP-WPREs) (1305bp/4742bp), recovering 4742bp by using a gel recovery kit, and then quantifying to obtain a linear Vector fragment Vector 1;
(3) and (3) carrying out recombination reaction on the gB protein gene segment and the linear Vector segment Vector1 through DNA polymerase to construct the recombinant adeno-associated virus transfer Vector containing the variant porcine pseudorabies virus gB protein gene.
The invention also provides a recombinant adeno-associated virus capable of expressing the gD protein of the porcine pseudorabies virus, which is a recombinant adeno-associated virus formed by inserting the gD protein antigen gene of the variant porcine pseudorabies virus into an adeno-associated virus vector. Similarly, the gD protein antigen gene herein may be a gD sequence having a homology of 95% or less, in addition to the above-mentioned SEQ ID No. 1; and the mutation region is located 820-837 after the initiation codon.
The recombinant adeno-associated virus of the present invention is preferably prepared as an intramuscular injection. In a preferred embodiment, the serotype of the adeno-associated virus vector is AAV2/9, and the AAV2/9 adeno-associated virus has a better affinity for muscle tissue.
The CMV promoter is a broad-spectrum promoter, and in preferred embodiments the viral genome comprises CMV promoter sequences.
The invention also provides a method for preparing the recombinant adeno-associated virus, which comprises the following steps:
(1) co-transfecting a plurality of host cells with the recombinant adeno-associated virus transfer vector and the backbone plasmid;
(2) culturing the host cell of step (1);
(3) harvesting the recombinant adeno-associated virus packaged in the cells of step (2);
(4) and (4) purifying the recombinant adeno-associated virus in the step (3).
In some embodiments, the backbone plasmids in step (1) are pRC-AAV2/9 plasmid of adeno-associated virus and pHelper plasmid of a viral packaging helper plasmid. Wherein the pRC-AAV2/9 plasmid contains a gene coding AAV Cap protein and a gene coding AAV Rep protein; the pHelper plasmid contains a gene for coding adenovirus E2a protein, a gene for coding adenovirus E4orf6 protein and a gene for coding adenovirus VA RNA protein. The recombinant adeno-associated virus transfer vector containing the genes of the gB and gD proteins of the variant porcine pseudorabies virus is cotransfected and packaged with pAAV2/9 plasmid of the adeno-associated virus and pHelper plasmid of the virus packaging auxiliary plasmid respectively to obtain the recombinant adeno-associated virus capable of expressing the gB protein of the porcine pseudorabies virus and the recombinant adeno-associated virus capable of expressing the gD protein of the porcine pseudorabies virus.
The recombinant adeno-associated virus for expressing the gD protein of the porcine pseudorabies virus can be prepared by adopting a conventional preparation method of rAAV in the prior art, and comprises plasmid transfection (the preparation method adopted by the embodiment of the invention), plasmid transfection combined with helper virus (such as adenovirus Adv or herpes simplex virus HSV) infection, or the helper virus infection of a mammal packaging cell line and the like, and can also construct an oneBac baculovirus expression system bacmid containing rAAV2/9-PRV-gB and rAAV2/9-PRV-gD, transfect sf9 cells with the bacmid, rescue and harvest Baculovirus Expression Vectors (BEV), and package the rAAV in a BEV amplification mode.
The invention also provides application of the recombinant adeno-associated virus in preparation of a vaccine for the pseudorabies of the variant pig, which is a live virus vaccine of a vector.
The gD (US6) protein of PRV belongs to type I glycoprotein, and is composed of 400 or 402 amino acids, and has a relative molecular mass of 44 KD. The gD protein is an important immunogenic protein of PRV, and can induce the organism to generate specific neutralizing antibody and cellular immunity (Zhang et al 2019). Thus, the gD protein is a major candidate antigen for PRV subunit vaccines (van Rooij et al 2010). The gB (UL27) protein is the envelope protein of PRV, belongs to type I glycoprotein, and has a molecular weight of about 100 KD. gB is an important immunogenic protein of PRV, the major glycoprotein that stimulates the body to produce neutralizing antibodies (van Rooij et al 2010). The genes encoding the gB and gD proteins of PRV are the genes of interest for the development of recombinant vector vaccines for PRV.
The invention is characterized in that the recombinant adeno-associated virus strain prepared by inserting different antigen protein genes into adeno-associated virus vector plasmids is screened from the traditional PRV immunogenic protein species, the immune effect of the vaccine is further prepared, and the gD protein gene is used as an antigen gene and matched with an adeno-associated virus vector, so that the prepared engineering vaccine has obviously better immune protection performance than the commercial vaccine on the mutant porcine pseudorabies virus strain. For example, the genetically engineered strain gD gene fragment expressed by the porcine pseudorabies virus prepared in some embodiments of the invention is an immunogenic fragment of porcine pseudorabies virus strain PRV/JXFC/2015, and has good immunoprotection property for porcine pseudorabies virus strain PRV/JXFC/2015. The injection is injected into the tibialis anterior muscle of the right lower limb once, so that the organism can be continuously induced to generate specific neutralizing antibody and cellular immunity at high level. Compared with the traditional vaccine, the long-acting protective force can be obtained only by one-time immunization, the immunization does not need to be strengthened, and the operation is more convenient. Therefore, the vaccine prepared by the genetic engineering strain has wide market application prospect.
The recombinant adeno-associated virus constructed by the invention is used for carrying out neutralizing antibody detection on a plurality of different epidemic variant strains, and the virus is found to have good immune effect on CH-18 wild strains belonging to the same genotype II, and is expected to have good immune effect when being used as a vaccine, particularly on branch epidemic variant strains belonging to the same genotype II.
The following are specific examples:
example 1
1. Primer design and PCR amplification
The genome of a porcine pseudorabies virus wild strain PRV/JXFC/2015 is used as a PCR template, and a primer is designed by using analysis software Oligo 6.0 to amplify gB and gD genes, wherein the sizes of amplified fragments are 2718bp and 1203bp respectively. The primer sequences are as follows:
table 1: PCR primer
a: the two ends are respectively designed with Sal I and Spe I enzyme cutting sites. The left end part of the primer sequence is a homologous arm sequence, the italic part is a Sal I and Spe I enzyme cutting site sequence, a transcription enhancing kozak sequence is arranged behind the enzyme cutting site, and the scribed part is a gD target gene primer sequence.
b: XbaI and Sal I enzyme cutting sites are respectively designed at two ends. The left end part of the primer sequence is a homologous arm sequence, the italic part is an XbaI and Sal I enzyme cutting site sequence, a transcription enhancing kozak sequence is arranged behind the enzyme cutting site, and the lined part is a gB target gene primer sequence.
2. Cloning of porcine pseudorabies gB and gD genes
The genome of the wild strain PRV/JXFC/2015 of the porcine pseudorabies virus is used as a PCR template, the gB and gD genes are amplified by using the primers gB-F/R, gD-F/R listed in the table 3-1, and a PCR amplification reaction system H 2 O40. mu.L, upstream and downstream primers (10pmol) each 4. mu. L, Template (50 ng/. mu.L), 2. mu.L 2xPHanta max master mix 50. mu.L, and PCR amplification program are shown in Table 2.
TABLE 2 reaction cycles for PCR amplification of the primers
The amplified PCR product was analyzed by 0.8% agarose Gel electrophoresis, and gB and gD gene fragments were recovered respectively using an Omega Gel Extraction Kit Gel recovery Kit.
3. Construction and identification of pAAV-PRV-gB-P2A-EGFP and pAAV-PRV-gD-P2A-Mchery
The adeno-associated virus commercial Vector gene circular plasmid PT-1786(rAAV-CMV (584bp) -beta arrestin2-Flag-P2A-EGFP-WPREs) is digested by Xba I and Sal I (purchased from Wuhan Pongsu biotechnology limited), two bands (1305bp/4742bp) are digested by enzyme, 4742bp is a linear Vector fragment, and 46 ng/mu L is quantified after 4742bp is recovered by an Omega Gel Extraction Kit Gel recovery Kit and is marked as Vector 1. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis using Sal I and Spe I (purchased from Wuhan Pongk Biotechnology Co., Ltd.) to double-cleave the adeno-associated virus commercial vector gene circular plasmid PT-1540(rAAV-CMV-SOD1-P2A-mcherry-WPRE-hGH poly), the sizes of the two amplified fragments were 468bp and 6001bp, 468bp was the cleaved SOD1 gene, and 6001bp was the linear vector fragment. After recovering the linear AAV Vector fragment (6001bp) using the Omega Gel Extraction Kit Gel recovery Kit, 43 ng/. mu.L was quantified and designated Vector 2.
The construction schematic diagram of the vector plasmids PT-1540 and pAAV-PRV-gD-Mchery is shown in FIG. 1; the physical maps of the vector plasmids PT-1786 and pAAV-PRV-gB-P2A-EGFP are shown in FIG. 2. FIG. 3 shows PCR amplification from genomic templates using PRV gB gene primers, where M is
Plus II DNA Marker; 1-6 are PCR amplification products of PRV gD genes; FIG. 4 shows PCR amplification from genomic templates using PRV gD gene primers, where M is
Plus II DNA Marker; 1-2 are PCR amplification products of PRV gD genes; FIG. 5 is a diagram of a double-enzyme gel-cutting of the vector plasmid used PT-1786/PT-1540, in which M:
PlusⅡDNA Marker；1：PT-1540；2：PT-1786。
the gB gene fragment and the Vector1 of the linear AAV Vector plasmid fragment were subjected to recombination reaction, and the recombination reaction system is shown in Table 3. The gD gene fragment and the Vector2 of the linear AAV Vector plasmid fragment were subjected to recombination reaction, and the recombination reaction system is shown in Table 4. After recombination, competent bacteria are transformed, positive single colonies are screened out, and colony PCR identification is carried out by using primers.
TABLE 3 PCR recovery of the product gB Gene and Linear vector recombination reaction System
TABLE 4 PCR recovery of the product gD Gene and Linear vector recombination reaction System
FIG. 6 is a colony identification map of plasmid pAAV-PRV-gB-P2A-EGFP constructed in this example, in which M:
plus II DNA Marker; 1: pAAV-PRV-gB-P2A-EGFP; 2-3 are negative; FIG. 7 is a colony identification chart of plasmid pAAV-PRV-gD-P2A-Mchery constructed in this example, in which M is
Plus II DNA Marker; 1 is PT-PRV gD, and 2-5 is negative.
FIG. 8 is a restriction enzyme identification diagram of recombinant plasmids pAAV-PRV-gD-P2A-Mchery and pAAV-PRV-gB-P2A-EGFP constructed in this example; in the figure, M:
plus II DNA Marker; 1: pAAV-PRV-gD-P2A-Mchery; 2: pAAV-PRV-gB-P2A-EGFP; FIG. 9 shows the morphology and fluorescence photographs of cell transfection packages of rAAV2/9-PRV-gB-P2A-EGFP and rAAV2/9-PRV-gD-P2A-Mcherry virus in 72h in this example; in the figure, A: rAAV 2/9-PRV-gD-P2A-Mchery; b: rAAV 2/9-PRV-gB-P2A-EGFP; c: control normal HEK-293T.
4. Preparation of rAAV expressing gD and gB proteins of PRV
And (3) preparing virus packages of recombinant adeno-associated virus strains rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery, referring to the specification of a transfection reagent, co-transfecting adherence HEK293T by using three plasmids, wherein the co-transfecting system of the three plasmids is shown in Table 5, and transfecting for 3d to obtain recombinant adeno-associated virus stock solution.
Table 5: three plasmid cotransfection system (10cm dish)
5. Purification of rAAV expressing gD and gB proteins of PRV
Adding collected cell mixture after transfection for 72h into 1/10 volumes of chloroform, shaking vigorously at 37 ℃ for 1h, adding NaCl until the final concentration is 1mol/L, shaking for dissolution, centrifuging at 4 ℃ and 12000rpm/min for 15min, taking out the upper aqueous phase, removing chloroform and precipitate, adding PEG8000 until the final concentration is 10% (W/V), shaking for dissolution, and standing in ice bath for 1 h. Centrifuging at 11000rpm/min for 15min, discarding the supernatant, blowing PBS with the volume of 1/10 primary cell mixed solution to elute, merging and resuspending, adding nuclease to the final concentration of 1 mug/mL, digesting at room temperature for 30min, adding chloroform with the same volume for extraction, centrifuging at 4 ℃ and 12000rpm/min for 5min, and taking out the water phase, thus completing the purification to the intermediate product. Further purifying the adeno-associated virus rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery into a finished product by adopting iodixanol centrifugation-dialysis-ultrafiltration harvest-aseptic filtration.
6. SDS-PAGE silver staining of rAAV expressing gD and gB proteins of PRV
Recombinant adeno-associated viruses generally comprise 3 capsid proteins, virus particles are boiled to cause the depolymerization of the capsid proteins, and 3 characteristic bands are formed after SDS-PAGE electrophoresis, wherein the sizes of the characteristic bands are 87KDa, 72KDa and 62KDa respectively, and the content ratio is 1:1: 10. The specific operation is as follows: a10. mu.L sample was taken, 2.5. mu.L of 5 XSDS-loading buffer was added, boiled at 100 ℃ for 10min, and the sample was collected by brief centrifugation. 10% polyacrylamide gel is prepared, AAV sample 10 uL/hole, loading amount is about 1E +11vg, protein marker 0.5 uL/hole, 120V electrophoresis is carried out for 1 h. And (3) carrying out silver staining decolorization by using a commercial silver staining kit according to the operation of the instruction, taking a picture in a gel imaging system, and carrying out gray scale analysis and comparison on the picture to determine the titer of the viral colloidal image. The SDS-PAGE silver gel map result of the finished recombinant adeno-associated virus is shown in FIG. 10, and the sizes of three protein bands in a sample lane show that the shells VP1, VP2 and VP3 of the adeno-associated virus are consistent and the content ratio is about 1:1:10, in proportion to the coat composition of the adeno-associated virus protein. Except three obvious protein bands of VP1, VP2 and VP3, no other foreign protein is obvious in the sample lane.
7. ITR primer quantitative detection of rAAV of gD and gB protein expressing PRV
The recombinant adeno-associated virus generally comprises ITR sequences (adeno-associated virus genome partial sequences) of the serotype 2 adeno-associated virus, and primers which are specifically combined with the ITR sequences of the serotype 2 recombinant adeno-associated virus are designed by a SYBR Green qPCR method, the copy number of the ITR sequences is detected, so that the copy number of the genome of the recombinant adeno-associated virus is quantified, and the QPCR titer of the recombinant adeno-associated virus is determined. And (3) extracting a genome from the purified finished virus according to a conventional method, detecting an ITR gene by QPCR, and determining the QPCR titer of the stock solution and the finished virus. The specific operation is as follows: diluting the original sample by 100 times, treating the original sample by nuclease and proteinase K, and diluting the original sample by 300 times to obtain the sample to be detected. The linearized standard quality particles were diluted to 1.0E +08 copies/. mu.L. mu.L of standard plasmid is added into 90. mu.L of diluent, and the dilution is carried out in a gradient way, so as to obtain a series of concentration gradient standards, which comprise: 1.0E +08 copies/mu L, 1.0E +07 copies/mu L, 1.0E +06 copies/mu L, 1.0E +05 copies/mu L, 1.0E +04 copies/mu L, and 1.0E +03 copies/mu L, namely the prepared standard Substance (STD). According to the sample treatment and standard substance preparation method, a sample for SYBY Green qPCR detection is treated and prepared, wherein each hole reaction system is 10 mu L, and the standard substance plasmid and the sample are both 2 mu L/hole. ITR amplification primer sequences:
forward primer ITR:5'-GGAACCCCTAGTGATGGAGTT-3'
Reverse primer ITR 5'-CGGCCTCAGTGAGCGA-3'
Table 6: SYBY Green qPCR detection reaction conditions
Table 7: SYBY Green qPCR detection reaction conditions
8. Identification of expression of gD and gB proteins in recombinant adeno-associated viruses
Finished adeno-associated virus strains rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery respectively infect PK-15 cells, 5% carbon dioxide is cultured at 37 ℃ for 3 days to collect the cells, the collected PK-15 cells are processed, the supernatant is centrifuged to be identified by Western blotting, and whether gB protein and gD protein are expressed or not is respectively identified. Monoclonal antibodies of gB and gD proteins are used as primary antibodies, commercial goat anti-mouse HRP-IgG is used as secondary antibodies, specific gB and gD protein bands of 100KD and 44KD are respectively seen through chemical development, and after the protein level proves that recombinant adeno-associated virus strains rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery respectively infect PK-15 cells, the expression of the gB and gD proteins in the PK-15 cells is respectively realized. Western blotting identification results of the recombinant adeno-associated virus strain rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery are shown in figure 12. PRV gB expression was lower and PRV gD expression was higher.
9. Electron microscopy of rAAV expressing gD and gB proteins of PRV
Copper sheet adsorption and ATP enzyme staining operation of virus samples: the titer is 10 12 And (2) selecting a sealing film to drip 10 mu L of virus sample liquid on the sealing film to soak the copper sheet for 2 minutes to complete the adsorption of the virus on the copper sheet, timely drying the filter paper, introducing 20 mu L of water, quickly soaking and cleaning, quickly drying the water absorbed by the filter paper, transferring the filter paper into 20 mu L of ATP enzyme staining solution to stain for 1 minute, quickly drying the water absorbed by the filter paper, placing the filter paper in a flat dish with a specified number, airing the filter paper overnight, and observing the copper sheet with a microscope on the second day. The titer is 10 13 And (2) selecting a sealing film with a vg/mL level, dripping 10 muL of virus sample liquid on the sealing film to soak the copper sheet for 1 minute to finish virus adsorption to the copper sheet, sucking dry the filter paper in time, introducing 20 muL of water, quickly soaking and cleaning, quickly sucking dry water by using the filter paper, transferring into 20 muL of ATP enzyme staining solution to stain for 1 minute, quickly sucking dry water by using the filter paper, placing in a flat dish paved with the filter paper, airing in an appointed numbering area overnight, and observing by using a microscope on the second day. The virus electron micrograph of the recombinant adeno-associated virus strain rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery is shown in figure 11. The results of a virus electron microscope show that the solid rate of rAAV2/9-PRV-gB-P2A-EGFP-Z20200103 is 6.34% (solid 43 hollow 635 pieces, totally 678), and the solid rate of rAAV2/9-PRV-gD- -P2A-mcherry-Z20200103 is 59.78% (solid 165 hollow 111 pieces, totally 276). The recombinant adeno-associated virus three-plasmid transient package can package the empty-shell virus, and the common purification mode can not completely separate the empty-shell virus from the solid-core virus. The proportion of the recombinant adeno-associated virus empty shell is related to the size of the target gene loaded by the recombinant adeno-associated virus vector and the gene itself. The rAAV2/9-PRV-gB-P2A-EGFP with higher rate of empty shells is probably the main reason for the ineffectiveness of the virus after immunizing mice.
10. Immunity test of rAAV expressing gD and gB proteins of PRV to C57 mouse
PRV vaccine strains of two recombinant adeno-associated virus vectors, namely rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery, are used for immunization by intramuscularly injecting the tibialis of the right lower limb of a mouseImmunization of mice in the test group at an immunization dose of 5X 10 12 VG/KG, the test groups are: a first group of gB (pAAV-PRV-gB-P2A-EGFP), a second group of gD (rAAV 2/9-PRV-gD-P2A-Mchery), a third group of gB + gD (rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery mixed at the same dose), and each group immunized 5C 57 mice; the positive control group is 5 mice immunized by porcine pseudorabies inactivated vaccine commercialized by the Pre-Council Bio-Inc., each immunization dose is 0.18mL, and the negative control group is 5 mice immunized by PBS. Blood is collected at 2 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks and 28 weeks of immunization respectively, serum is collected, the porcine pseudorabies virus wild virus PRV/JXFC/2015 strain is used for detecting the serum neutralization titer at 2 weeks, 12 weeks, 16 weeks, 20 weeks and 24 weeks of immunization, and PRV/JXFC/2015 strain and CH-18 strain cross neutralization experiment is carried out at 28 weeks. And detecting IFN-gamma and IL-4 antibody levels after 2 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks and 28 weeks of immunization, and the detection results are shown in FIG. 13.
TABLE 8 mouse immunogenicity test design
11. Mouse serum neutralizing antibody assay
After one immunization, serum was collected at each time point and the level of neutralizing antibodies was determined for each group of pooled serum samples at each time point. The results of the mouse serum neutralizing antibody experiment show that: detecting virus serum neutralizing antibodies of PRV/JXFC/2015 wild strains, wherein the neutralizing titer of the mixed serum samples of the negative control PBS group and the rAAV2/9-PRV-gB-P2A-EGFP group at all time points is 1:0, wherein the neutralizing titers of the mixed serum samples of the rAAV2/9-PRV-gB-P2A-EGFP and rAAV2/9-PRV-gD-P2A-Mcherry same-dose mixed injection groups are 1:8, 1:19.95, 1:22.9, 1:5.6, 1:5.01 and 1:31.6 respectively; the neutralization titers of the mixed serum samples of the positive control commercial vaccine group are 1:16, 1:22.38, respectively; the neutralization titers of the mixed serum samples of the rAAV 2/9-PRV-gD-P2A-Mchery group are 1:5, 1:64, 1:32, 1:44, 1:39.8 and 1:56.2 respectively. And performing cross-neutralization detection on the CH-18 wild strain on a 28-week mixed serum sample, wherein the rAAV2/9-PRV-gB-P2A-EGFP group is 1:0, the rAAV2/9-PRV-gD-P2A-Mcherry group is more than or equal to 1:256, the rAAV2/9-PRV-gB-P2A-EGFP and rAAV2/9-PRV-gD-P2A-Mcherry group is 1:89.125, the positive control commercial vaccine group is 1:42, and the negative control PBS group is 1: 0. The porcine pseudorabies recombinant vaccine AAV2/9-PRV-gD group using the adeno-associated virus as a vector has a level of neutralizing antibody which is obviously higher than that of a commercial vaccine group aiming at an epidemic variant strain PRV/JXFC/2015, and simultaneously the AAV2/9-PRV-gD group also has a level of neutralizing antibody which is obviously higher than that of a commercial vaccine group aiming at a wild strain CH-18.
TABLE 9 neutralizing antibody assay results (PRV/JXFC/2015 wild strain)
TABLE 10 results of cross-neutralizing antibody assay (28 week serum samples, PRV/JXFC/2015 wild strain, CH-18 wild strain)
12. Determination of IFN-gamma and IL-4 secretion amount in serum of immunized mouse
After the pseudorabies live vaccine is immunized, the humoral immunity can be stimulated, and a very high cellular immunity level can be induced. Th1 cell (I-type helper T cell) is CD4 positive cell, mainly secretes cytokines such as IL-2, IFN-gamma, TNF-beta and the like, and can reflect the cytokine level of Th1 by measuring the content of IFN-gamma; th2 cell (II type helper T cell) mainly secretes cytokines such as IL-4, IL-5 and IL-13, and IL-4 content measurement can reflect Th2(II type helper T cell) induced cytokine level. The results of measuring IFN-. gamma.and IL-4 cytokine levels in the mixed mouse serum samples at the respective time points of each group are shown in tables 11 and 12 and FIGS. 14 and 15. As can be seen, after the porcine pseudorabies recombinant vaccine strain rAAV 2/9-PRV-gD-P2A-P2A-Mchery which takes the adeno-associated virus as a vector is used for immunizing a mouse, the highest IFN-gamma level of 1973.65pg/mL and the highest IL-4 level of 161.01pg/mL are generated in the mouse, the highest IFN-gamma level of the commercial vaccine is 1959pg/mL, and the highest IL-4 level is 103.89 pg/mL. Cytokine assay results show that the rAAV 2/9-PRV-gD-P2A-Mchery group produces higher immunocytokine expression levels than the commercial vaccine group. The content of IFN-gamma and IL-4 in mouse serum is measured, the highest IFN-gamma level of 1973pg/mL and IL-4 level of 161.01pg/mL are generated in the mouse, and the result shows that the porcine pseudorabies recombinant vaccine strain AAV2/9-PRV-gD with the adeno-associated virus as a vector can stimulate the cellular immune response in the mouse.
Table 11: determination of IFN-gamma secretion from serum of immune mouse
Table 12: determination of IL-4 secretion amount in serum of immunized mouse
In the examples, rAAV2/9-PRV-gB-P2A-EGFP (rAAV-CMV (584bp) -gB-P2A-EGFP-WPREs) is prepared, considering that gB is 2718bp, and in order to avoid exceeding the limit of 4.7kb, the adopted truncated CMV promoter (584bp) is adopted, and the result of Western blotting identification shows that the starting effect of the truncated CMV promoter is weaker, the promoter is also one of the reasons that the experiment of the rAAV2/9-PRV-gB-P2A-EGFP group mice is invalid, the overall carried exogenous gene length is 3828bp, the carried exogenous gene is too long, the empty shell rate of the virus package is increased, and the solid rate of the rAAV2/9-PRV-gB-P2A-EGFP-Z20200103 is 6.34 percent and is also one of the reasons that the experiment of the rAAV2/9-PRV-gB-P2A-EGFP group mice is invalid. The rAAV 2/9-PRV-gD-P2A-Mchery (rAAV-CMV-gD-P2A-Mchery-WPRE-hGH poly), the adopted full-length CMV promoter, the whole length of the carried exogenous gene is 3375bp, the normal packaging range is suitable, the solid rate of the rAAV 2/9-PRV-gD-P2A-Mchery-Z20200103 is 59.78%, and most viruses can normally perform the function of stable expression of the target gene.
The same dose mixed group of rAAV2/9-PRV-gB-P2A-EGFP and rAAV 2/9-PRV-gD-P2A-Mchery (gB + gD group) produced lower levels of neutralizing antibodies than commercial vaccine groups and lower immune effects than the same dose of rAAV 2/9-PRV-gD-P2A-Mchery inoculated alone, because of the large amount of empty-shell viruses in rAAV2/9-PRV-gB-P2A-EGFP stimulated production in mice of large amounts of rAAV neutralizing antibodies against AAV serotype AAV2/9, which neutralized portions of rAAV 2/9-PRV-gD-P2A-Mchery, reduced the immune effects of the same dose of rAAV 2/9-PRV-gD-P2A-Mchery. In general, recombinant adeno-associated virus for expressing antigen genes of porcine pseudorabies virus variant strains is prepared by adopting recombinant adeno-associated virus vectors and is limited by rAAV loading, and suitable antigen genes are gD protein genes of the porcine pseudorabies virus variant strains.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<120> recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gD protein gene, virus, preparation and application thereof
<141> 2020-11-05
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1203
<212> DNA
<213> Artificial Sequence
<400> 1
ctgctcgcag cgctattggc ggcgctggtc gcccggacga cgctcggcgc ggacgtggac 60
gccgtgcccg cgccgacctt ccccccgccc gcgtacccgt acaccgagtc gtggcagctg 120
acgctgacga cggtcccctc gcccttcgtc ggccccgcgg acgtctacca cacgcgcccg 180
ctggaggacc cgtgcggggt ggtggcgctg atctccgacc cgcaggtgga ccggctgctg 240
aacgaggcgg tggcccaccg gcggcccacg taccgcgccc acgtggcctg gtaccgcatc 300
gcggacgggt gcgcgcacct gctgtacttt atcgagtacg ccgactgcga ccccaggcag 360
atctttgggc gctgccggcg ccgcaccacg ccgatgtggt ggaccccgtc cgcggactac 420
atgttcccca cggaggacga gctggggctg ctcatggtgg cccccgggcg gttcaacgag 480
ggccagtacc ggcgcctggt gtccgtcgac ggcgtgaaca tcctcaccga cttcatggtg 540
gcgctccccg aggggcaaga gtgcccgttc gcccgcgtgg accagcaccg cacgtacaag 600
ttcggcgcgt gctggagcga cgacagcttc aagcggggcg tggacgtgat gcgattcctg 660
acgccgttct accagcagcc cccgcaccgg gaggtggtga actactggta ccgcaagaac 720
ggccggacgc tcccgcgggc ctacgccgcc gccacgccgt acgccatcga ccccgcgcgg 780
ccctcggcgg gctcgccgag gcccaggccc cggccccggc cccggccccg gccgaagccc 840
gagcccaccc cggcgacgcc cgcgcccccc ggccgcctgc ccgagccggc gacgcgggac 900
cacgccgccg ggggccgccc cacgccgcga cccccgaggc ccgagacgcc gcaccgcccc 960
ttcgccccgc cggccgtcgt gcccagcggg tggccgcagc ccgcggagcc gttcccgccc 1020
cggaccaccg ccgcgccggg cgtctcgcgc caccgctcgg tgatcgtcgg cacgggcacc 1080
gcgatgggcg cgctcctggt gggcgtgtgc gtctacatct tcttcagcct gaggggggcg 1140
aaggggtatc gcctcctggg cggtcccgcg gacgccgacg agctaaaagc gcagcccggt 1200
ccg 1203
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<212> DNA
<213> Artificial Sequence
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atgcccgctg gtggcggtct ttggcgcggg ccccgcgggc atcggcccgg gcaccacggc 60
ggtgctggcc tcggacgtct ttggcctgct ccacaccacg ctgcagctgc gcggggcgcc 120
gtcgcgctag cgctgctgct gctggcgctc gccgcgaccc cgacgtgcgg cgcggcggcc 180
gtgacgcggg ccgcctcggc ctcgcccgcg cccgggacgg gcgccacccc agacggcttc 240
tccacggagg agtccctcga ggagatcgac ggggccgtct cccccggccc ctcggacgcc 300
cccgacggcg agtacggcga cctggacgcg cgcacggccg tgcgcgcggc cgcgaccgag 360
cgggaccgct tctacgtctg cccgccgccg tccggctcca cggtggtgcg cctggagccc 420
gagcaggcct gccccgagta ctcgcagggg cgcaacttca cggaggggat cgccgtgctc 480
ttcaaggaga acatcgcccc gcacaagttc aaggcccaca tctactacaa gaacgtcatc 540
gtcacgaccg tgtggtccgg gagcacgtac gcggccatca cgaaccgctt cacggaccgc 600
gtgcccgtcc ccgtgcagga gatcacggac gtgatcgacc gccgcggcaa gtgcgtctcc 660
aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc 720
gtcgaggtgg acctgcgccc ctcgcgcctg aacgcgctcg gcacccgcgg ctggcacacc 780
accaacgaca cctacaccaa gatcggcgcc gcgggcttct accacacggg cacctccgtc 840
aactgcatcg tcgaggaggt ggaggcgcgc tccgtgtacc cctacgactc cttcgccctg 900
tccacggggg acatcgtgta catgtccccc ttctacggcc tgcgcgaggg ggcccacggg 960
gagcacatcg gctacgcgcc cgggcgcttc cagcaggtgg agcactacta ccccatcgac 1020
ctggactcgc gcctccgcgc ctccgagagc gtgacgcgca actttctgcg cacgccgcac 1080
ttcacggtgg cctgggactg ggcccccaag acgcggcgcg tgtgcagcct ggccaagtgg 1140
cgcgaggccg aggagatgat ccgcgacgag acgcgcggcg ggtccttccg cttcacgtcg 1200
cgggccctgg gcgcctcctt cgtcagcgac gtcacgcagc tcgacctgca gcgcgtgcac 1260
ctgggcgact gcgtcctccg cgaggcctcg gaggccatcg acgccatcta ccggcggcgc 1320
tacaacaaca cgcacgtgct ggccggcgac aggcccgagg tgtacctcgc ccgcgggggc 1380
ttcgtggtgg ccttccgccc gctgatctcg aacgagctgg cgcagctgta cgcgcgcgag 1440
ctcgagcgcc tcggcctcgc cggcgtcgtg ggccccgcgt cccccgcggc cgcccgtcgg 1500
gcccggcgct cccccggccc ggcggggacg cccgagccgc cggccgtcaa cggcacgggg 1560
cacctgcgca tcaccacggg ctcggccgag tttgcgcgcc tgcagttcac ctacgaccac 1620
atccaggcgc acgtgaacga catgctgagc cgcatcgcgg ccgcctggtg cgagctgcat 1680
aacaaggacc gcaccctgtg gggcgagatg tcgcgcctga accccagcgc cgtggccacg 1740
gccgcgctgg gccagcgcgt ctcggcgcgc atgctcggcg acgtgatggc catctcgcgg 1800
tgcgtggagg tgcgcggcgg cgtgtacgtg cagaactcca tgcgcgtgcc cggcgagcgc 1860
ggcacgtgct acagccgccc gctggtgacc ttcgagcaca acggcacggg cgtgatcgag 1920
ggccagctcg gcgacgacaa cgagctcctc atctcgcgcg acctcatcga gccctgcacc 1980
ggcaaccacc ggcgctactt taagctgggc ggcgggtacg tgtactacga ggactacagc 2040
tacgtgcgca tggtggaggt gcccgagacg atcagcacgc gggtgaccct gaacctgacg 2100
ctgctcgagg accgcgagtt cctgcccctc gaggtgtaca cgcgcgagga gctcgccgac 2160
acgggcctcc tggactacag cgagatccag cgccgcaacc agccgcacac gctcaagttc 2220
tacgacattg accgcgtggt caaggtggac cacaacgtgg tgctgctgcg cggcatcgcc 2280
aacttcttcc agggcctcgg cgacgtgggc gccgccgtcg gcaaggtggt cctgggcgcc 2340
acgggggccg tgatctcggc cgtcggcggc atggtgtcct tcctgtccaa ccccttcggg 2400
gcgctcgcca tcgggctgct ggtgctggcc ggcctggtcg cggccttcct ggcctaccgg 2460
cacatctcgc gcctgcgccg caaccccatg aaggccctgt accccgtcac gacgaaggcg 2520
ctcaaggagg acggcgtcga agaggacgac gtggacgagg ccaagctgga ccaggcccgg 2580
gacatgatcc ggtacatgtc catcgtgtcg gccctcgagc agcaggagca caaggcgcgc 2640
aagaagaaca gcgggcccgc gctgctggcc agccgcgtcg gggtgatggc cacgcgccgc 2700
cggcactacc agcgcctc 2718
Claims (7)
1. A recombinant adeno-associated virus transfer vector containing variant epidemic porcine pseudorabies virus gD protein gene is characterized in that the vector is a recombinant adeno-associated virus vector, and the vector is a recombinant adeno-associated virus transfer vector obtained by inserting the variant epidemic porcine pseudorabies virus gD protein gene into coding regions between ITR sequences at two ends of the recombinant adeno-associated virus transfer vector;
the gD protein gene is a nucleotide sequence shown by SEQ ID No. 1;
the recombinant adeno-associated virus transfer vector is a serotype AAV2/9 recombinant adeno-associated virus transfer vector.
2. The method of claim 1, comprising the steps of:
(1) taking the genome DNA of the epidemic variant strain as a template, and carrying out PCR amplification to obtain a gD protein gene fragment;
(2) carrying out double enzyme digestion on the recombinant adeno-associated virus vector, recovering a target vector fragment by using a glue recovery kit, and quantifying the target vector fragment, and marking the target vector fragment as a linear vector fragment;
(3) carrying out recombination reaction on the gD protein gene segment and the linear vector segment to construct the recombinant adeno-associated virus transfer vector containing the variant porcine pseudorabies virus gD protein gene; the gD protein gene is a nucleotide sequence shown by SEQ ID No. 1; the recombinant adeno-associated virus transfer vector is a serotype AAV2/9 recombinant adeno-associated virus transfer vector.
3. A recombinant adeno-associated virus capable of expressing porcine pseudorabies virus gD protein is characterized in that the recombinant adeno-associated virus is formed by inserting variant porcine pseudorabies virus gD protein antigen gene into an adeno-associated virus vector; the gD protein antigen gene is a nucleotide sequence shown by SEQ ID No. 1; the serotype of the adeno-associated virus vector is AAV 2/9.
4. The recombinant adeno-associated virus of claim 3 wherein the viral genome comprises a CMV promoter sequence.
5. Use of the recombinant adeno-associated virus according to claim 3 or 4 for the preparation of a vaccine against pseudorabies virus in swine.
6. The use of claim 5, wherein the recombinant adeno-associated virus is prepared as an intramuscular injection.
7. A method for producing the recombinant adeno-associated virus according to claim 3 or 4, comprising the steps of:
(1) co-transfecting the recombinant adeno-associated virus transfer vector of claim 1 with a backbone plasmid into a host cell;
(2) culturing the host cell of step (1);
(3) harvesting the recombinant adeno-associated virus packaged in the cells of step (2);
(4) and (4) purifying the recombinant adeno-associated virus in the step (3).
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