WO2022007742A1 - 一种重组的伪狂犬病病毒及其疫苗组合物 - Google Patents

一种重组的伪狂犬病病毒及其疫苗组合物 Download PDF

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WO2022007742A1
WO2022007742A1 PCT/CN2021/104472 CN2021104472W WO2022007742A1 WO 2022007742 A1 WO2022007742 A1 WO 2022007742A1 CN 2021104472 W CN2021104472 W CN 2021104472W WO 2022007742 A1 WO2022007742 A1 WO 2022007742A1
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virus
protein
cd2v
nucleotide sequence
swine fever
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French (fr)
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张强
钱泓
吴有强
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浙江海隆生物科技有限公司
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Definitions

  • the invention belongs to the technical field of genetic engineering vaccines, and in particular relates to a recombinant pseudorabies virus rPRV and an African swine fever vaccine prepared by using the rPRV virus.
  • ASFV is an enveloped double-stranded DNA virus. Its genome is a single-molecule linear double-stranded DNA with a total length of about 170kb-190kb, a conserved region of about 125kb in the center, and variable regions at both ends, including terminal transversal regions. Transition repeats.
  • the ASFV genome encodes putative membrane proteins, secreted proteins, enzymes involved in nucleotide and nucleic acid metabolism (DNA repair), and protein modification.
  • the entire genome contains 151 ORFs that can encode 150-200 proteins.
  • a live attenuated vaccine refers to a vaccine prepared with a live virus of an attenuated ASFV strain.
  • Attenuated ASFV strains include: passaged attenuated strains, native attenuated strains, and recombinant attenuated strains.
  • Passage-attenuated strains refer to the strains obtained by gradually reducing the pathogenicity of ASFV in the process of passage of porcine bone marrow-derived cells, Vero and COS-1 cell lines. For example, when subcultured in Vero cells using the isolate ASFV-G, the virulence of the virus gradually decayed and was completely lost at passage 110.
  • Recombinant attenuated strains refer to strains obtained by knocking out viral functional genes, viral virulence genes or immunosuppressive genes by molecular biology methods. The recombination reduces virus virulence or increases the body's immune response to the virus, and can be used to develop genetically engineered live attenuated vaccines with better safety and higher efficacy than traditional attenuated vaccines.
  • ASFV virulence genes and immunosuppressive genes have been reported: TK (K196R), 9GL (B119L), CD2v (EP402R), DP148R, NL (DP71L), UK (DP96R) and multigene families 360 and 505 (MGF 360/505 ); and immune escape-related genes A238L, A179L, A224L, DP71L, MGF360/505, I329L, K205R, D96R, DP148R, A276R, D96R and EP153R, etc.
  • Zhang Yanyan of the Academy of Military Medical Sciences and others used genetic engineering to knock out the MGF and CD2V sequences on the SY18 strain isolated in my country.
  • Pseudorabies virus belongs to the Alphaherpesvirus subfamily of the family Herpesviridae and has a wide host range. It can infect pigs, dogs, cattle, sheep and other livestock and wild animals, but does not infect humans. It has strong proliferation ability and is easy to be amplified. It is a vaccine vector with great development potential. Its genetic background is clear and its genetic stability is strong; it contains many unnecessary genes for virus replication, and the capacity of foreign genes is large (40kb); recombination PRV has stable genetic traits, and foreign genes are not easily lost.
  • CN108504686A and CN108504687A provide recombinant adenoviral vectors expressing the EP153R and EP402R genes of ASFV, respectively.
  • Argilaguet JM et al. constructed a BacMam-sHAPQ based on baculovirus vector, which can induce specific T cell responses after immunizing pigs, and some pigs can resist the challenge of homologous sublethal strains. A large number of IFN- ⁇ secreting T cells were monitored.
  • HEK-purified ASFV protein can promote humoral immune response, but cellular immunity is weak, while MVA vector antigen can promote cellular immunity to produce IFN- ⁇ , but no challenge protection results have been reported.
  • Lynnette C et al. used adenovirus and poxvirus as vectors to express eight genes including B602L, B646L(p72), CP204L(P30), E183L(P54), E199L, EP153R, F317L, MGF505-5R, etc.
  • PRV pseudorabies virus
  • the present invention includes the following technical solutions.
  • rPRV pseudorabies virus
  • Nucleotide sequence B encoding an accessory protein used to facilitate the correct expression and folding of capsid protein P72 or a variant thereof, wherein the accessory protein amino acid sequence is altered by substitution, deletion or addition of one or more amino acid residues. formed, and has the function of promoting the correct expression and folding of capsid protein P72 or its variant; preferably, the amino acid sequence of the accessory protein variant has more than 90% homology, preferably more than 95% homology with the accessory protein more preferably more than 98% homology, more preferably more than 99% homology;
  • the above-mentioned accessory protein may be B602L protein derived from African swine fever virus.
  • exogenous gene may contain only nucleotide sequence A, nucleotide sequence B and nucleotide sequence C. That is, the recombinant pseudorabies virus expresses only three exogenous proteins, P72, B602L and CD2V, but does not express other exogenous proteins.
  • the recombinant pseudorabies virus (rPRV) described above is suitable for replicating and expressing nucleotide sequence A, nucleotide sequence B and nucleotide sequence C in cells selected from cells used for viral propagation such as mammalian cells , bird cells or insect cells.
  • useful cells include, for example, alveolar macrophages, porcine bone marrow-derived cells, Vero cells, COS-1 cells, human embryonic kidney 293 (HEK) cells, chick embryonic fibroblasts (CEFs), Chinese hamster ovary cells (CHO cells) ), baby hamster kidney cells (BHK), African green monkey kidney cells (VERO), cervical cancer cells (HELA), perC6 cells, sf9 cells, etc.
  • the proportion of p72 soluble in the above-mentioned cells accounts for more than 20% of the total amount of p72.
  • the genome of the above-mentioned recombinant pseudorabies virus can also comprise the following foreign genes:
  • Nucleotide sequence D encoding African swine fever virus-derived P49 or a variant thereof, wherein the P49 variant is formed by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence, and has the function of P49
  • the amino acid sequence of the P49 variant has more than 90% homology with P49, preferably more than 95% homology, more preferably more than 98% homology.
  • the genome of the above-mentioned recombinant pseudorabies virus may further comprise a nucleotide sequence D and a nucleotide sequence E selected from the group consisting of nucleotide sequence A, nucleotide sequence B and nucleotide sequence C. of foreign genes. That is, the recombinant pseudorabies virus can further express an exogenous protein selected from P49 in addition to the three exogenous proteins of P72, B602L and CD2V.
  • At least one replication non-essential region in the genome of the recombinant pseudorabies virus is deleted and/or replaced.
  • the above-mentioned replication non-essential region can be selected from one or more of the gC, gE, gG, gl, gM, TK, RR, and PK coding regions of pseudorabies virus.
  • nucleotide sequence A, nucleotide sequence B and nucleotide sequence C are respectively located in the replication non-essential regions of the genome.
  • These exogenous genes can be located in the same non-essential region for replication, or they can be located in different non-essential regions for replication.
  • the expression cassette of the above-mentioned B602L gene can be SV40-B602L-hGH, wherein SV40 is the SV40 promoter, B602L is the target gene, and hGH is the human growth hormone terminator. For the convenience of detection, HA tag was added to the 3 end of the target gene B602L.
  • the expression cassette of the above CD2V gene can be EF1 ⁇ -CD2V-HSV-TK, wherein EF1 ⁇ is the EF1 ⁇ promoter, CD2V is the target gene, and HSV-TK is the herpes virus TK gene terminator.
  • EF1 ⁇ is the EF1 ⁇ promoter
  • CD2V is the target gene
  • HSV-TK is the herpes virus TK gene terminator.
  • a His tag was added to the 3 end of the target gene CD2V.
  • a second aspect of the present invention provides a method for constructing the above-mentioned recombinant pseudorabies virus, comprising the steps of:
  • step (3) Replace the gG gene of PRV-BAC-P72-B602L- ⁇ TK obtained in step (2) with the codon-optimized expression cassette of African swine fever virus CD2V gene to obtain PRV-BAC-P72-B602L-CD2V- ⁇ TK- ⁇ gG;
  • nucleotides of the genes encoding p72, B602L, and CD2V proteins can also be replaced without affecting the encoding of amino acids, which can be understood by those skilled in the art.
  • step (1) can be realized by the following steps:
  • step b Transfer the target fragment obtained in step b into PRV-BAC/GS1783 electrotransformation competent cells (such as Escherichia coli GS1783 competent cells), and use Red/ET recombination to obtain PRV-BAC-P72-Kan- ⁇ TK; secondary recombination
  • PRV-BAC-P72-Kan- ⁇ TK was deleted to obtain PRV-BAC-P72- ⁇ TK strain.
  • B602L and CD2V can be inserted after P72 and at the gG site, respectively, using the same recombination method.
  • the codon-optimized African swine fever virus B602L gene is inserted into pSV40-kan to obtain Psv40-B602L-Kan, followed by amplification, subsequent ligation, transformation and other steps; the codon-optimized African swine fever virus CD2V
  • the gene was inserted into pEF1 ⁇ -kan to obtain pEF1 ⁇ -CD2V-kan, followed by amplification, subsequent ligation, transformation and other steps; finally, PRV-BAC-P72-B602L-CD2V- ⁇ TK- ⁇ gG strain was obtained.
  • the recombinant pseudorabies virus described above can be used as a live vector subunit vaccine.
  • the fourth aspect of the present invention provides an African swine fever vaccine or immune composition (or called immunogenic composition), which at least contains the above-mentioned recombinant pseudorabies virus as an immunogen.
  • pseudorabies virus composition of the present invention the following components may also be included: a pseudorabies virus comprising the nucleotide sequence A encoding the above-mentioned P72 or its variant and the nucleotide sequence B encoding the above-mentioned accessory protein such as B602L in the genome A rabies virus; and/or a pseudorabies virus comprising in its genome the nucleotide sequence C encoding CD2V or a variant thereof.
  • the above-mentioned African swine fever vaccine or immune composition may further contain a veterinary acceptable carrier, excipient, or adjuvant.
  • the above recombinant pseudorabies virus can replicate in pigs and express African swine fever immune antigens (ie, capsid protein P72 and outer envelope protein CD2V derived from African swine fever virus).
  • African swine fever immune antigens ie, capsid protein P72 and outer envelope protein CD2V derived from African swine fever virus.
  • Figure 1 is a schematic diagram of the structure of the eukaryotic expression plasmid pEE12.4-kan.
  • Figure 2 is a photo of agarose gel electrophoresis of recombinant PRV-BAC verified by enzyme digestion.
  • M DL15000 Marker
  • 1, 2, 3 recombinant PRV-BAC-CMV-P72-bGH- ⁇ TK
  • 4 PRV-BAC (HL) control.
  • Fig. 3 is an SDS-PAGE gel electrophoresis photograph of western-blot verifying p72 expression.
  • M Protein Marker
  • 1, 2, 3, 4 lysed cell supernatant of different screening strains of rPRV-P72--B602L-CD2V- ⁇ TK- ⁇ gG
  • 5 Negative control cell lysed supernatant.
  • Figure 4 is a photograph of SDS-PAGE gel electrophoresis of western-blot verification and CD2V expression.
  • M Protein Marker
  • 1 The positive protein control containing His tag is about 55kd
  • 2, 3, 4, 5 rPRV-P72--B602L-CD2V- ⁇ TK- ⁇ gG different screening strains lysed the cell supernatant.
  • pseudorabies virus is used as a carrier to express African swine fever immunogen, that is, ASFV-derived capsid protein P72, accessory protein B602L and outer envelope protein CD2V.
  • African swine fever immunogen that is, ASFV-derived capsid protein P72, accessory protein B602L and outer envelope protein CD2V.
  • the recombinant pseudorabies virus constructed in the present invention can replicate in pigs, and can express African swine fever immune antigens (ie, capsid protein P72 and outer envelope protein CD2V derived from African swine fever virus).
  • African swine fever immune antigens ie, capsid protein P72 and outer envelope protein CD2V derived from African swine fever virus.
  • African swine fever virus has a multi-layer structure, the exogenous protein CD2V is located in the outermost layer, and the exogenous protein P72 is located in the second layer.
  • the correct expression and correct folding of the foreign protein P72 to maintain the specific spatial structure of P72 is one of the key factors for the immunogenicity of recombinant pseudorabies virus to African swine fever.
  • B602L as a chaperone protein, can promote the correct expression and correct folding of the foreign protein P72.
  • the recombinant pseudorabies virus also allows the expression of other proteins derived from African swine fever virus ASFV, such as P49, but the variety and quantity of proteins allowed to be loaded are very limited, otherwise it will affect the exogenous protein P72. Correct folding seriously reduces the immunogenicity of recombinant pseudorabies virus, for example, loading of some proteins such as p12, p14(E120R), pE248R, p22, P32, P54, etc., will have adverse effects. Therefore, it is an optimal choice to load only the coding nucleic acid sequences A-C of P72, B602L and CD2V, such as SEQ ID NOs. 1-3, on the pseudorabies virus genome.
  • reducing the amount of exogenous protein is also beneficial to simplify the construction steps of recombinant pseudorabies virus, which is also advantageous. From this point of view, it is also a preferred solution to load only the coding nucleic acid sequences A and B of P72 and CD2V, such as SEQ ID NOs. 1-2, on the pseudorabies virus genome.
  • a certain protein such as P72 is sometimes mixed with the name of its encoding gene (DNA), and those skilled in the art should understand that they represent different substances in different description occasions.
  • DNA encoding gene
  • P72 when used to describe the capsid protein function or class, it refers to the protein; when described as a gene, it refers to the gene encoding the capsid protein P72.
  • codon optimization can be performed for specific expression hosts or vectors such as pseudorabies virus, host cells.
  • Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of the gene of interest. Different organisms often show a particular preference for one of several codons encoding the same amino acid due to mutational propensity and natural selection.
  • optimized codons reflect the composition of their respective genomic tRNA pools. Thus, in fast growing host cells, codons of low frequency for amino acids can be replaced with codons of high frequency for the same amino acid.
  • the gene sequences SEQ ID NOs: 1-3 provided herein are codon-optimized nucleotide sequences, but the P72, B602L and CD2V expressed genes of the present invention are not limited thereto.
  • Recombinant pseudorabies virus needs to be rescued in host cells to replicate and expand to express foreign proteins.
  • the term "rescue” refers to the use of a certain method to make the virus in the non-proliferative infection complete the process of replication and proliferation to produce progeny viruses.
  • the main methods of virus rescue include co-culture and co-infection.
  • immunogenic composition may also be referred to as “immunogenic composition” and refers to a composition comprising at least one antigen that elicits an immune response in a host to which the immunogenic composition is administered.
  • Such carriers are, for example, stabilizing salts, emulsifiers, solubilizers or osmotic pressure regulators, suspending agents, thickeners, redox compositions that maintain the physiological redox potential.
  • Preferred adjuvants include aluminum salts, microemulsions, lipid particles, and/or oligonucleotides used to increase the immune response.
  • carrier refers to diluents such as water, saline, dextrose, ethanol, glycerol, and phosphate buffered saline (PBS), excipients, or vehicles in which the composition can be administered.
  • diluents such as water, saline, dextrose, ethanol, glycerol, and phosphate buffered saline (PBS), excipients, or vehicles in which the composition can be administered.
  • carriers in pharmaceutical compositions may include: binders such as microcrystalline cellulose, polyvinylpyrrolidone (povidone or polyvinylpyrrolidone), tragacanth, gelatin, starch, lactose or monohydrate Lactose; decomposing agents such as alginic acid, corn starch and the like; lubricants or surfactants such as magnesium stearate or sodium lauryl sulfonate; glidants such as silica gel; sweeteners such as sucrose or saccharin; Stabilizers, for example, include, but are not limited to, albumin and alkali metal salts of EDTA.
  • binders such as microcrystalline cellulose, polyvinylpyrrolidone (povidone or polyvinylpyrrolidone), tragacanth, gelatin, starch, lactose or monohydrate Lactose
  • decomposing agents such as alginic acid, corn starch and the like
  • lubricants or surfactants
  • the vaccine or immune composition of the present invention can be used for the treatment or prevention of African swine fever virus ASFV infection.
  • treatment or prophylaxis generally relates to administering to an animal (primarily pigs) in need thereof an effective amount of a vaccine or immunological composition of the present invention.
  • treatment refers to the administration of an effective amount of a vaccine or immunizing composition after an individual animal or at least some animals of a population have been infected with ASFV and these animals have exhibited some clinical symptoms caused by or associated with ASFV infection.
  • prophylaxis refers to administering an effective amount of a vaccine or immunizing composition until the animal is not infected with ASFV or does not exhibit any clinical symptoms caused by or associated with ASFV infection.
  • PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, it can be adjusted by simple experimentation.
  • LB medium 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride, pH 7.2, sterilized at 121 °C for 20 min at high temperature and high pressure.
  • the U6 gene fragment was cloned into the pUC19 vector successively to construct pUC19-US2-US6; then the miniF fragment carrying the BAC element and green fluorescence (GFP) marker was inserted into pUC19-US2-US6 to construct the transfer vector miniF- US2-US6; BHK-21 cells were co-transfected with PRV(HL) genome and transfer vector miniF-US2-US6, plaques containing green fluorescent protein were selected, and PRV recombinant virus PRV-BAC(HL) carrying BAC vector was obtained, The recombinant virus PRV-BAC (HL) genome was extracted, electroporated into GS1783 competent cells, coated with LB agar plates containing 50 ⁇ g/ml kanamycin and 34 ⁇ g/ml chloramphenicol, and cultured at 32°C overnight. /ml kanamycin and 34 ⁇ g/ml chloramphenicol in LB liquid medium, cultured at 32°C overnight, namely PR
  • African swine fever virus structural protein CD2V is a glycosylated protein located in the outer envelope. It is encoded by the EP402R gene. It is predicted to have a transmembrane region at 207-229aa. Studies have shown that the CD2V protein can interact with red blood cells. It plays an important role in the process of proliferation and lymphocyte damage. It is well known to those skilled in the art that only the CD2V fragment located in the extracellular region has the function of interacting with host cells and is an ideal protective antigen. In order to facilitate the expression of CD2V, we selected the fragment with the transmembrane region removed.
  • African swine fever virus structural protein P72 is a polypeptide encoded by the B646L gene. The kinetics of P72 protein synthesis indicates that this protein is translated at the late stage of infection, accounting for about 32% of the viral protein. It is the main protein of the icosahedral structure of the virus, although P72 protein is reported to be expressed in many systems, but no report of expression in pseudorabies.
  • the African swine fever virus structural protein B602L is a polypeptide encoded by the B602L gene.
  • the B602L protein can promote the correct folding of the p72 protein. If the B602L protein is lacking, the expression of the p72 protein will be significantly reduced, although the protein is not viral Structural protein, but in the absence of this protein, the assembly of the virus is greatly altered, eventually leading to incorrect assembly into virus particles. Therefore, the pseudorabies vector was selected for simultaneous expression of these three proteins.
  • the synthesized sequence was subcloned into pUC57, named pUC57-OPTI-P72; the nucleotides of B602L were codon-optimized to obtain the OPTI-B602L sequence, as shown in SEQ ID NO.2, in order to facilitate subsequent detection , HA tag was added to the C-terminus; the nucleotides of CD2V were codon-optimized to obtain OPTI-CD2V as shown in SEQ ID NO.3. In order to facilitate detection, a His tag was added to the C-terminus, and the synthesized sequence It was cloned into pUC57 and named pUC57-OPTI-CD2V. The sequence synthesis work was entrusted to Nanjing GenScript Biotechnology Co., Ltd. to complete.
  • Double-enzyme digestion product gel recovery Take out the above double-enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein.
  • step (3) After completing the sample addition according to the table in step (2), place each 10 ⁇ l reaction system in a 16°C low-temperature cooling liquid circulator and water bath for 10-16 hours;
  • step (3) Take out the EP tube in step (3), place it in a 65°C water bath, and bath for 15min;
  • step (3) Add 250 ⁇ L of P2 buffer to the solution in step (3), immediately and gently invert the centrifuge tube 5-10 times to mix, and let stand for 2-4min at room temperature;
  • step (4) Add 350 ⁇ L of P3 buffer to the solution in step (4), immediately and gently invert the centrifuge tube for 5-10 times to mix; let stand for 2-4min at room temperature;
  • step (6) centrifuge the solution of step (5) at room temperature, 14,000rpm/min, 10min;
  • step (6) The supernatant solution in step (6) was moved to the center of the adsorption column, centrifuged at room temperature, 12,000rpm/min, 30s, and the liquid in the collection tube was poured out;
  • the recombination sequence with the TK homology arms of the PRV genome was PCR amplified.
  • PCR amplification program 95°C for 2 min; 30 cycles of 95°C for 30s, 55°C for 45s, 72°C for 1 min; 72°C for 10 min; hold at 8°C.
  • the PCR product is gel recovered, and the steps are as follows:
  • step (10) Take out the centrifuge tube in step (10) from the centrifuge, discard the middle adsorption column CB2, cover the centrifuge tube cap, and retain the DNA sample in the centrifuge tube;
  • step 11 (12) Store the DNA sample in step 11 at 4°C, and prepare agarose gel electrophoresis identification gel to recover DNA fragments.
  • step 3.1 The subcloned product obtained by PCR in step 3.1 was electroporated into GS1783 competent cells in step 3.2. Specific steps are as follows:
  • the enzyme digestion system is as follows:
  • step 3.5 (1) Pick the positive clones verified in step 3.5 into 1 ml of LB medium containing 30 ⁇ g/ml chloramphenicol, and culture at 32°C and 220 rpm for 1-2 hours until the bacterial liquid is cloudy and slightly cloudy;
  • a single colony of the positive clone in step 3.6 was correctly picked and placed in 6 ml of LB medium containing 30 ⁇ g/ml Chl, and cultured at 32°C for 24 hours;
  • the extracted rPRV-P72-B602L-CD2V- ⁇ TK- ⁇ gG plasmid was purified and the concentration was determined, and the recombinant virus was rescued in BHK-21 cells according to the lipofectamine LTX transfection method.
  • the operation steps are:
  • Lipofectamine LTX Dilute Lipofectamine LTX: add 9 ⁇ L Lipofectamine LTX to 125 ⁇ L OPTI-MEM, then add 2.5 ⁇ L plus, mix gently, and let stand at room temperature for 5 min.
  • step 1 and step 2 mixture Gently mix step 1 and step 2 mixture. It was placed at room temperature for 5 min, and then added dropwise to a six-well plate for even distribution.
  • the rPRV-P72-B602L-CD2V- ⁇ TK- ⁇ gG virus solution was propagated, centrifuged at 10,000 rpm/min for 5 min, the supernatant was collected, treated with the loading buffer, and electrophoresed on 10% SDS-PAGE gel by wet transfer (100 V , 90min) transferred to PVDF membrane, blocked with 5% nonfat milk powder for 2h, added 1:4000 times diluted mouse anti-flag monoclonal antibody (to detect p72) or 1:4000 times diluted mouse anti-His tag monoclonal antibody (Detect CD2V)), after 1h at room temperature, wash 3 times with PBST washing solution, add 1:5000-fold diluted HRP-labeled goat anti-mouse IgG secondary antibody, act at room temperature for 1h, add substrate chromogenic solution, protect from light After 5 minutes, a specific band at 70-90kDa was observed, which was consistent with the expected protein size and was p72 protein.
  • Recombinant p72 and CD2V proteins were used to coat the ELISA plate at a concentration of 0.5 ⁇ g/ml, and each antigen was coated in 12 wells (4 wells were added with immunized mouse serum samples, and 4 wells were free of negative Mouse serum, 4 wells plus blocking solution as control), 100 ⁇ l/well, 37°C for 1 h; PBST washing solution was washed 3 times, 5 min each; Act at 37°C for 1 h; wash 3 times with PBST washing solution for 5 min each time; add 100 ⁇ l/well of substrate for color development, incubate at 37 °C for 20 min, and finally add 2M H 2 SO 4 , 50 ⁇ l/well to terminate the reaction.
  • the results are shown in the following table:
  • the experimental results showed that the coated P72 protein could specifically bind to the serum after the second immunization, and the mean OD450 was 2.119; the coated CD2V protein could specifically bind to the serum after the second immunization, and the mean OD450 was 1.823. It can be clearly seen that the antibody concentrations detected by rPRV-P72-B602L-CD2V- ⁇ TK- ⁇ gG are all higher, indicating that the recombinant virus rPRV-P72--B602L-CD2V- ⁇ TK- ⁇ gG has better immunogenicity , can be used as a research ASFV-PRV live vector vaccine.

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Abstract

提供了一种重组的伪狂犬病病毒，其基因组包含编码非洲猪瘟病毒来源的衣壳蛋白P72、辅助蛋白B602L、外囊膜蛋白CD2V的核苷酸序列。该重组的伪狂犬病病毒能够用于制备活病毒载体疫苗来有效地治疗或预防非洲猪瘟，克服现有的灭活疫苗和减毒活疫苗的缺陷。

Description

一种重组的伪狂犬病病毒及其疫苗组合物 技术领域
本发明属于基因工程疫苗技术领域，具体地说，涉及一种重组的伪狂犬病病毒rPRV、以及使用该rPRV病毒制备的非洲猪瘟疫苗。
背景技术
非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的一种急性、烈性、高度接触性的传染病，其特点是发病过程短，最急性和急性感染死亡率高达100％，临床表现为发热(达40～42℃)，心跳加快，呼吸困难，部分咳嗽，眼、鼻有浆液性或粘液性脓性分泌物，皮肤发绀，***、肾、胃肠粘膜明显出血，非洲猪瘟临床症状与猪瘟症状相似，世界动物卫生组织(OIE)将其列为法定报告的动物疫病，中国也将其列为一类动物疫病。该病最早在1921年于非洲的肯尼亚首次发现，2007年以来，非洲猪瘟在全球多个国家发生、扩散、流行，特别是俄罗斯及其周边地区。2018年，我国已经发现了ASF疫情，带来了巨大的直接和间接经济损失。
ASFV是一种有囊膜的双链DNA病毒，其基因组是单分子线状双链DNA，全长为约170kb-190kb，中央有125kb左右的保守区，两端为可变区，包含末端反转重复序列。ASFV基因组编码假定膜蛋白、分泌性蛋白、参与核苷酸和核酸代谢(DNA修复)以及蛋白修饰的酶，整个基因组包含151个ORF，可以编码150-200种蛋白质。
ASFV能从被感染猪的血液、组织液、内脏、及其他***物中检测出来，低温暗室内存在血液中的病毒可生存六年，室温中可活数周，被病毒感染的血液在55℃加热30分钟或者在60℃加热10分钟，病毒才能被破坏，许多脂溶剂和消毒剂可以将其破坏。
疫苗是预防和治疗ASF的重要手段。目前的ASFV疫苗主要有灭活疫苗和减毒活疫苗。灭活疫苗是指通过物理或化学手段将ASFV灭活而获得的疫苗。灭活使其失去感染能力，但保留其抗原性。迄今为止，采用多种传统方法制备的ASF灭活疫苗均不能对强毒攻击提供有效的免疫保护，包括病毒接种肺泡巨噬细胞以及感染脾组织后匀浆制备的灭活疫苗。
减毒活疫苗是指用减毒的ASFV毒株的活病毒制备的疫苗。减毒的ASFV毒株包括： 传代减毒毒株、天然减毒毒株和重组减毒毒株。传代减毒毒株是指ASFV在猪骨髓来源细胞、Vero和COS-1等细胞系传代的过程中致病力逐渐下降而获得的毒株。例如，利用分离株ASFV-G在Vero细胞中进行传代培养，病毒毒力逐渐衰减，在传至第110代时完全丧失。然而，家猪接种传代减毒的ASFV-G毒株后并未获得相应保护力，以抵抗亲本病毒的攻击。此外，有报道接种传代减毒的ASFV毒株，家猪呈现出肺炎、流产和死亡等副作用，以及呈现ASF慢性感染临床症状。天然减毒毒株是指天然存在的减毒毒株，例如ASFV OURT88/3或NH/P68毒株。然而，天然减毒毒株免疫动物后可造成诸多副作用，包括肺炎、流产、死亡等。例如，以NH/P68毒株免疫后，25％～47％的猪呈现慢性感染；以OURT88/3免疫后，可导致发热、关节肿胀等症状。
重组减毒毒株是指采用分子生物学方法敲除病毒功能基因、病毒毒力基因或者免疫抑制基因获得的毒株。所述重组降低病毒毒力或增加机体对病毒的免疫应答，可以用于研制比传统弱毒疫苗安全性更好、且效力更高的基因工程减毒活疫苗。已经报道了ASFV毒力基因和免疫抑制基因：TK(K196R)、9GL(B119L)、CD2v(EP402R)、DP148R、NL(DP71L)、UK(DP96R)和多基因家族360和505(MGF 360/505)；以及免疫逃逸相关基因A238L、A179L、A224L、DP71L、MGF360/505、I329L、K205R、D96R、DP148R、A276R、D96R和EP153R等。2019年，军事医学科学院张艳艳等采用基因工程手段敲除我国分离的SY18毒株上的MGF和CD2V序列。初步试验结果表明，能够100％抵抗亲本毒株SY18的攻击，对照组全部死亡，但免疫组的猪出现发热等临床症状，因此长期使用其安全性还有待进一步验证。2019年底，美国科研人员研究发现，删除I177L基因后产生的弱毒苗可提供猪100％的保护，免疫后的猪也为未向未免疫猪传播病毒。但其安全性还需进一步验证。
活载体疫苗是指将病原体的蛋白质编码基因克隆到活病毒载体，然后用于免疫动物，在动物体内表达所述蛋白质，从而诱导针对所述蛋白质的免疫应答。与其他疫苗相比，活载体疫苗的优势体现在：(1)能主动感染靶组织或细胞，提高了外源基因进入细胞的效率；(2)载体自身有佐剂效应，能诱导细胞因子和趋化因子的产生；(3)多数能诱导长期的免疫应答。
伪狂犬病病毒(Pseudorabies virus，PRV)属疱疹病毒科α疱疹病毒亚科，具有广泛的宿主范围，可以感染猪、犬、牛、羊等多种家畜和野生动物，但不感染人，在宿主细胞内的增殖能力强，易于扩增，是一种极具开发潜力的疫苗载体，其基因背景清楚，遗传稳定性强；含有许多病毒复制的非必需基因，外源基因容量大(40kb)；重组PRV 遗传性状稳定，外源基因不易丢失。
现有技术报道了一些活载体疫苗。例如，CN108504686A和CN108504687A分别提供了表达ASFV的EP153R和EP402R基因的重组腺病毒载体。Argilaguet JM等基于杆状病毒载体构建了一种BacMam-sHAPQ，免疫猪后可诱导特异性T细胞应答，部分猪能抵御同源亚致死性毒株的攻击，在攻毒17天后在猪血液中监测出大量IFN-γ分泌型T细胞。Lokhandwala S等基于腺病毒载体将p32、p54、pp62和p72基因分别***复制缺陷型腺病毒载体中，重组腺病毒免疫猪后表明，能诱导产生高水平的非洲猪瘟特异性抗体、细胞免疫应答以及细胞毒性T淋巴细胞反应；2016年Lokhandwala S等将A151R、B119L、B602L、EP402RΔPRR、B438L、K205R和A104R分别重组进腺病毒载体，在重组腺病毒中添加佐剂免疫猪后，引发强烈的非洲猪瘟抗原特异性IgG应答以及IFN-γ。2017年，Loperamadrid J等采用Vaxign***筛选出五种ASFV的抗原，用人胚肾293(HEK)细胞表达出的p72、p54、p12抗原和三种MVA载体抗原(B646L、EP153R和EP402R)采用初免-加强免疫，用HEK纯化的ASFV蛋白接种可促进体液免疫应答，但细胞免疫较弱，而MVA载体抗原能促进细胞免疫产生IFN-γ，但未报道攻毒保护结果。2020年，Lynnette C等利用腺病毒、痘病毒作为载体表达B602L，B646L(p72)，CP204L(P30)，E183L(P54)，E199L，EP153R，F317L，MGF505-5R等八个基因，在高免疫剂量下，能够提供猪免受非洲猪瘟的完全保护，但有部分严重的副作用，依然出现非洲猪瘟的症状，因此不是很安全，并且免疫剂量大，需要表达的基因多，也不利于规模化生产和应用。因此，现行的活载体疫苗并未见到有效安全保护免疫猪抵抗非洲猪瘟的报道。这是因为非洲猪瘟病毒结构复杂，具有多层结构，单一组分很难起到有效保护。
发明内容
为了构建重组减毒毒株以便制备用于治疗和预防非洲猪瘟的活载体疫苗，我们对伪狂犬病病毒(PRV)作为非洲猪瘟病毒的活载体进行了深入研究。通过对免疫原蛋白进行大量筛选，意外发现PRV同时表达外囊膜CD2V、外层衣壳蛋白P72以及B602L组分，尤其是可溶性的p72占全p72蛋白总量的20％以上时可有效激活机体产生保护性抗体，有效安全保护猪免受致死剂量的攻击。该免疫原混合物包括最外层的囊膜CD2V和最外层的衣壳蛋白p72，同时为了确保P72蛋白能正确折叠表达，同时表达B602L，为开发新型重组活载体亚单位疫苗奠定了基础。具体而言，本发明包括如下技术方案。
一种重组的伪狂犬病病毒(rPRV)，其特征在于，其基因组包含下述外源基因：
编码非洲猪瘟病毒来源的衣壳蛋白P72或其变体的核苷酸序列A，所述变体是衣壳蛋白P72氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有衣壳蛋白P72的功能；优选地，该P72变体的氨基酸序列与P72有90％以上同源性、优选地95％以上同源性、更优地98％以上同源性、更优地99％以上同源性；
编码用于促使衣壳蛋白P72或其变体正确表达及折叠的辅助蛋白的核苷酸序列B，所述变体是辅助蛋白氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有促使衣壳蛋白P72或其变体正确表达及折叠的功能；优选地，该辅助蛋白变体的氨基酸序列与辅助蛋白有90％以上同源性、优选地95％以上同源性、更优地98％以上同源性、更优地99％以上同源性；
编码非洲猪瘟病毒来源的外囊膜蛋白CD2V或其变体的核苷酸序列C，所述变体是外囊膜蛋白CD2V氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有外囊膜蛋白CD2V的功能；优选地，该CD2V变体的氨基酸序列与CD2V有90％以上同源性、优选地95％以上同源性、更优地98％以上同源性、更优地99％以上同源性。
优选地，上述辅助蛋白可以是非洲猪瘟病毒来源的B602L蛋白。
上述外源基因可以仅包含核苷酸序列A、核苷酸序列B和核苷酸序列C。即，重组的伪狂犬病病毒仅表达P72、B602L和CD2V这三种外源蛋白，但不表达其他外源蛋白。
上述重组的伪狂犬病病毒(rPRV)适合于在细胞内复制并表达核苷酸序列A、核苷酸序列B和核苷酸序列C，所述细胞选自用于病毒增殖的细胞如哺乳动物细胞、鸟类细胞或昆虫细胞。例如，可用细胞的例子包括肺泡巨噬细胞、猪骨髓来源细胞、Vero细胞、COS-1细胞、人胚肾293(HEK)细胞、小鸡胚胎纤维原细胞(CEF)、中国仓鼠卵巢细胞(CHO)、幼仓鼠肾细胞(BHK)、非洲绿猴肾细胞(VERO)、***细胞(HELA)、perC6细胞、sf9细胞等。
优选地，P72可溶性的比例在上述细胞内占p72总量的20％以上。研究发现，可溶性的P72具有免疫原的作用，否则即便p72表达但不可溶的话，免疫效果不佳。
这些使用这些细胞内复制rPRV可以大量制备非洲猪瘟疫苗。
在一种优选的实施方式中，上述核苷酸序列A与SEQ ID NO:1的同源性≥90％，优选地≥95％，更优地≥98％，更优地≥99％；所述核苷酸序列B与SEQ ID NO:2的同源性≥90％，优选地≥95％，更优地≥98％，更优地≥99％；所述核苷酸序列C 与SEQ ID NO:3的同源性≥90％，优选地≥95％，更优地≥98％，更优地≥99％。
在另一种可选的实施方式中，上述重组的伪狂犬病病毒的基因组还可以包含下述外源基因：
编码非洲猪瘟病毒来源的P49或其变体的核苷酸序列D，所述P49变体是氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有P49的功能；优选地，该P49变体的氨基酸序列与P49有90％以上同源性、优选地95％以上同源性、更优地98％以上同源性。
换句话说，上述重组的伪狂犬病病毒的基因组除了包含核苷酸序列A、核苷酸序列B和核苷酸序列C外，还可以进一步包含选自核苷酸序列D和核苷酸序列E的外源基因。即，重组的伪狂犬病病毒除了表达P72、B602L和CD2V这三种外源蛋白外，还可以进一步表达选自P49的外源蛋白。
优选地，上述重组的伪狂犬病病毒的基因组中至少一个复制非必需区发生缺失和/或替代。
上述复制非必需区可以选自伪狂犬病病毒的gC、gE、gG、gI、gM、TK、RR、PK编码区中的一个或两个以上。
例如，PRV基因组的复制非必需区中的gE和/或gG编码区发生缺失。
优选地，上述重组的伪狂犬病病毒中，核苷酸序列A、核苷酸序列B和核苷酸序列C分别位于所述基因组的复制非必需区。这些外源基因可以位于同一个复制非必需区，也可以分别位于不同的复制非必需区。
上述重组的伪狂犬病病毒中，P72基因的表达框可以为CMV-P72-bGH，其中CMV是CMV启动子，p72是目的基因，bGH是牛生长激素终止子。为了便于检测，在目的基因p72的5端加入flag标签。
上述B602L基因的表达框可以为SV40-B602L-hGH，其中SV40是SV40启动子，B602L是目的基因，hGH是人生长激素终止子。为了便于检测，在目的基因B602L的3端加入HA标签。
上述CD2V基因的表达框可以为EF1α-CD2V-HSV-TK，其中EF1α是EF1α启动子，CD2V是目的基因，HSV-TK是疱疹病毒TK基因终止子。为了便于检测，在目的基因CD2V的3端加入His标签。
本发明的第二个方面提供了一种构建上述重组的伪狂犬病病毒的方法，其包含如下步骤：
(1)将伪狂犬病病毒株PRV-BAC(HL)基因组中的TK基因替换成密码子优化后的表达非洲猪瘟病毒p72蛋白的基因表达框，得到PRV-BAC-P72-△TK；
(2)将密码子优化后的非洲猪瘟病毒B602L基因表达框***到p72基因后，得到PRV-BAC-P72-B602L-△TK；
(3)用密码子优化后的非洲猪瘟病毒CD2V基因表达框替换步骤(2)中所得的PRV-BAC-P72-B602L-△TK的gG基因，得到PRV-BAC-P72-B602L-CD2V-△TK-△gG；
(4)用步骤(3)中所得的PRV-BAC-P72-B602L-CD2V-△TK-△gG转染BHK-21细胞，拯救得到同时表达p72、B602L和CD2V蛋白的重组伪狂犬病毒，命名为rPRV-P72-B602L-CD2V-△TK-△gG。
为了便于优化和表达，也可以将编码p72、B602L、CD2V蛋白的基因的核苷酸进行替换、但不影响氨基酸的编码，这是本领域技术人员能够理解的。
具体地，步骤(1)可以通过如下步骤实现：
a.将密码子优化后的非洲猪瘟病毒p72基因***到载体pEE12.4-kan上，得到pEE12.4-p72-kan；
b.以pEE12.4-p72-kan为模板，利用合适的引物对经PCR扩增得到目的片段，例如当p72编码序列为SEQ ID NO.1时，引物对为SEQ ID NO.4和SEQ ID NO.5：
正向引物(5’-3’)：
Figure PCTCN2021104472-appb-000001
反向引物(5’-3’)：
Figure PCTCN2021104472-appb-000002
c.将步骤b中所得的目的片段转入PRV-BAC/GS1783电转化感受态细胞(比如大肠杆菌GS1783感受态)，利用Red/ET重组得到PRV-BAC-P72-Kan-ΔTK；二次重组删除PRV-BAC-P72-Kan-ΔTK中Kan基因，得到PRV-BAC-P72–ΔTK菌株。
类似地，可以利用同样的重组方法将B602L和CD2V分别***P72之后和gG位点。例如，将密码子优化后非洲猪瘟病毒B602L基因***到pSV40-kan上，得到Psv40-B602L-Kan，再进行扩增、后续的连接、转化等步骤；将密码子优化后非洲猪瘟病毒CD2V基因***到pEF1α-kan上，得到pEF1α-CD2V-kan，再进行扩增、后续的连接、转化等步骤；最终得到PRV-BAC-P72-B602L-CD2V-ΔTK-ΔgG菌株。
本发明的第三个方面提供了上述重组的伪狂犬病病毒在制备用于预防和治疗非洲猪瘟的疫苗中的用途。
上述重组的伪狂犬病病毒可以作为活载体亚单位疫苗。
本发明的第四个方面提供了一种非洲猪瘟疫苗或免疫组合物(或称免疫原性组合物)，其至少含有上述的重组的伪狂犬病病毒作为免疫原。
在本发明的伪狂犬病毒组合物中，还可以包括下述组分：基因组中包含编码上述P72或其变体的核苷酸序列A及编码上述辅助蛋白比如B602L的核苷酸序列B的伪狂犬病病毒；以及/或者基因组中包含编码上述CD2V或其变体的核苷酸序列C的伪狂犬病病毒。
优选地，上述非洲猪瘟疫苗或免疫组合物还可以含有兽医学可接受的载体、赋形剂、或佐剂。
作为一种活病毒载体疫苗，上述重组的伪狂犬病病毒可在猪体内复制且能表达非洲猪瘟免疫抗原(即非洲猪瘟病毒来源的衣壳蛋白P72和外囊膜蛋白CD2V)。
当本发明构建的重组的伪狂犬病病毒作为活病毒载体疫苗免疫小鼠，能够从小鼠体内检测出p72、B602L、CD2V抗体，表明在动物中成功激发了针对ASFV的p72和CD2V抗体，为研制PRV-ASFV重组活载体疫苗奠定了基础，能够克服现有技术中灭活疫苗和减毒活疫苗的缺陷，显示出较好的工业化开发和应用前景。
附图说明
图1是真核表达质粒pEE12.4-kan的结构示意图。
图2是酶切验证重组PRV-BAC的琼脂糖凝胶电泳照片。M：DL15000 Marker；1、2、3：重组PRV-BAC-CMV-P72-bGH-△TK；4：PRV-BAC(HL)对照。
图3是western-blot验证p72表达的SDS-PAGE凝胶电泳照片。M：蛋白Marker；1、2、3、4：rPRV-P72--B602L-CD2V-△TK-△gG不同的筛选株裂解细胞上清；5：阴性对照细胞裂解上清。
图4是western-blot验证和CD2V表达的SDS-PAGE凝胶电泳照片。M：蛋白Marker；1：含His标签的阳性蛋白对照约为55kd；2、3、4、5：rPRV-P72--B602L-CD2V-△TK-△gG不同的筛选株裂解细胞上清。
具体实施方式
本发明将伪狂犬病病毒作为载体来表达非洲猪瘟免疫原，即ASFV来源的衣壳蛋白P72、辅助蛋白B602L和外囊膜蛋白CD2V，通过在合适的细胞内进行培养和复制，制备出用于预防和治疗非洲猪瘟的重组疫苗。
ASFV来源的衣壳蛋白P72的氨基酸序列为SEQ ID NO.6；ASFV来源的B602L的氨基酸序列为SEQ ID NO.7；ASFV来源的外囊膜蛋白CD2V的氨基酸序列为SEQ ID NO.8。
本发明构建的重组的伪狂犬病病毒可在猪体内复制、且能表达非洲猪瘟免疫抗原(即非洲猪瘟病毒来源的衣壳蛋白P72和外囊膜蛋白CD2V)。
非洲猪瘟病毒具有多层结构，外源蛋白CD2V位于最外层，而外源蛋白P72位于第二层。研究表明，外源蛋白P72的正确表达和正确折叠以便维持P72的特定空间结构是重组伪狂犬病病毒产生对非洲猪瘟的免疫原性的关键因素之一。在重组伪狂犬病病毒中，B602L作为伴侣蛋白能够促使外源蛋白P72进行正确表达和正确折叠。
进一步的研究和临床实验表明，重组伪狂犬病病毒还允许表达来源于非洲猪瘟病毒ASFV的其他蛋白比如P49，但允许加载的蛋白品种和数量是很有限的，否则就会影响外源蛋白P72的正确折叠，严重降低重组伪狂犬病病毒的免疫原性，例如，有些蛋白比如p12、p14(E120R)、pE248R、p22、P32、P54等的加载会产生不良影响。因此在伪狂犬病病毒基因组上仅加载P72、B602L和CD2V的编码核酸序列A-C比如SEQ ID NOs.1-3是一种最优选择。另一方面，减少外源蛋白的数量还有利于简化重组伪狂犬病病毒的构建步骤，这也是有利的。从这个角度讲，在伪狂犬病病毒基因组上仅加载P72和CD2V的编码核酸序列A和B比如SEQ ID NOs.1-2也是一种优选方案。
在本文中，为了描述简便，有时会将某种蛋白比如P72与其编码基因(DNA)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如，对于P72，用于描述衣壳蛋白功能或类别时，指的是蛋白质；在作为一种基因描述时，指的是编码该衣壳蛋白P72的基因。
在本发明中，术语“重组的伪狂犬病病毒”、“重组伪狂犬病病毒”、“重组病毒”、“重组PRV”和“rPRV”表示相同的意义，可以互换使用。术语“非洲猪瘟疫苗”、“PRV-ASFV疫苗”、“重组疫苗”表示相同的意义，都是指以伪狂犬病毒为载体制备的重组非洲猪瘟亚单位疫苗，可以互换使用。
在构建重组伪狂犬病病毒时，为了在不同表达宿主或载体中进行外源蛋白质的最佳表达，可以针对特定的表达宿主或载体比如伪狂犬病病毒、宿主细胞进行密码子优化。 密码子优化是可用于通过增加感兴趣基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常示出对于编码相同氨基酸的一些密码子之一的特殊偏好性。例如，在生长快速的宿主细胞中，优化密码子反映出其各自的基因组tRNA库的组成。因此，在生长快速的宿主细胞中，氨基酸的低频率密码子可以用用于相同氨基酸的但高频率的密码子置换。因此，优化的DNA序列的表达在快速生长的宿主细胞中得以改良。在本文中所提供的基因序列SEQ ID NO:1-3是经过密码子优化的核苷酸序列，但本发明的P72、B602L和CD2V表达基因不受限于此。
术语“宿主细胞”涵盖在本发明的构建体和载体构建过程中涉及的全部细胞，包括但不限于细菌、酵母、哺乳动物细胞。
重组伪狂犬病毒需要在宿主细胞中拯救才能复制扩增，从而表达外源蛋白。术语“拯救(rescue)”是指利用一定的方法使处于非增殖性感染中的病毒完成复制增殖过程，产生子代病毒。病毒拯救的主要方法包括协同培养、协同感染。
本领域技术人员容易理解，本发明的非洲猪瘟疫苗或免疫组合物中可以含有兽医学可接受的载体、赋形剂、或佐剂。
术语“免疫组合物”也可称为“免疫原性组合物”，是指包含至少一种抗原的组合物，所述抗原在施用免疫原性组合物的宿主中诱发免疫应答。
术语“兽医学可接受的载体”包括但不限于溶剂、分散介质、涂覆剂、稳定剂、稀释剂、防腐剂、抗细菌及抗真菌剂、等渗剂、佐剂、免疫攻击剂及其组合。
这些载体举例来说是稳定性盐、乳化剂、增溶剂或渗透压调节剂、悬浮剂、增稠剂、维持生理氧化还原电势的氧化还原剂组成。优选佐剂包括铝盐、微乳液、脂质颗粒、和/或被用来增加免疫应答寡核苷酸。
术语“载体”指的是稀释剂，例如水、盐、右旋糖、乙醇、甘油和磷酸盐缓冲盐水(PBS)、赋形剂或可以给药组合物的介质。作为固体成分，药物组合物中的载体可以包括：粘合剂，例如微晶纤维素、聚乙烯吡咯烷酮(聚维酮或聚乙烯吡咯酮)、黄蓍胶、白明胶、淀粉、乳糖或一水合乳糖；分解试剂，例如褐藻酸、玉米淀粉以及类似物；润滑剂或表面活性剂，例如硬脂酸镁或月桂基磺酸钠；助流剂，例如硅胶；甜味剂，例如蔗糖或糖精；稳定剂，例如包括但不限于白蛋白及乙二胺四乙酸的碱金属盐。
术语“佐剂”是指非特异性免疫增强剂，当与抗原一起注射或预先注入生物体时，可增强生物体对抗原的免疫应答或改变免疫应答类型。本发明的非洲猪瘟疫苗或免疫组合物可以包含佐剂；也可以不包含佐剂。
合适的佐剂可以选自：含铝佐剂(例如，氢氧化铝、磷酸铝、明矾)、脂多糖、弗氏完全佐剂、弗氏不完全佐剂、CpG寡核苷酸、矿物质胶、氢氧化铝、表面活性剂、溶血卵磷脂、普卢兰尼克多元醇类、聚阳离子或油乳剂例如油包水或水包油或其组合。当然，佐剂的选择取决于有目的的使用。例如，毒性可能取决于受试的生物体，可以从无毒至高毒性间变化。
本发明的疫苗或免疫组合物可以用于治疗或预防非洲猪瘟病毒ASFV感染。
术语“治疗或预防”通常涉及给有需要的动物(主要是猪)施用有效量的本发明的疫苗或免疫组合物。术语“治疗”是指在动物个体或群体的至少一些动物已感染ASFV且这些动物已展示一些由ASFV感染引起或与其有关的临床症状后，施用有效量的疫苗或免疫组合物。术语“预防”是指在动物没有受到ASFV感染或不展示任一由ASFV感染引起或与其有关的临床症状之前，施用有效量的疫苗或免疫组合物。
术语“有效量”包括但不限于诱发或能够诱发个体中的免疫应答的抗原量。所述有效量能够减小动物群体中ASFV感染的发病率或减小ASFV感染的临床症状的严重程度。
术语“临床症状”是指ASFV的感染体征。ASFV感染的临床症状的实例包含但不限于发热((达40-42℃)，心跳加快，呼吸困难，部分咳嗽，眼、鼻有浆液性或粘液性脓性分泌物，皮肤发绀，***、肾、胃肠粘膜明显出血。
在本发明中，“有效量”可以是10 5-10 9pfu，优选10 6-10 8pfu，更优选10 7pfu。在一些实施方案中，本发明的疫苗或免疫组合物是液体形式的组合物。在一些实施方案中，本发明的疫苗或免疫组合物的体积为0.5-5mL，优选1-4mL，更优选1.5-3mL，例如，1.5mL、2mL、2.5mL或3mL。
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
实施例
材料和方法
实施例中的全基因合成、引物合成及测序皆由南京金斯瑞生物科技有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、 培养基配制等等，主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB培养基：10g/L胰蛋白胨、5g/L酵母提取物、10g/L氯化钠，pH7.2，121℃高温高压灭菌20min。
实施例中所使用的重组PRV-BAC(HL株)GS1783菌株、真核表达质粒pEE12.4--kan(该质粒由pEE12.4(购自上海林渊生物科技有限公司)改造而来，添加了Kan抗性基因，便于整合到细菌人工染色体上筛选验证)(质粒图谱如图1所示)、BHK-21细胞为浙江海隆生物科技有限公司保存，任何单位和个人都可以获得该细胞和质粒用于验证本发明，但未经浙江海隆生物科技有限公司允许不得用作其他用途，包括开发利用、科学研究和教学。
其中，PRV-BAC(HL)细菌(大肠杆菌)人工染色体(BAC)的构建方法简要如下：首先，在浙江绍兴某猪场分离得到了毒株PRV(HL)，以PRV(HL)的US2和U6基因片段作为同源臂，先后克隆到pUC19载体，构建pUC19-US2-US6；再将携带BAC元件和绿色荧光(GFP)标记的miniF片段***到pUC19-US2-US6，构建了转移载体miniF-US2-US6；将PRV(HL)基因组与转移载体miniF-US2-US6共转染BHK-21细胞，挑选含有绿色荧光蛋白的噬斑，获得携带BAC载体的PRV重组病毒PRV-BAC(HL)，提取重组病毒PRV-BAC(HL)基因组，电转GS1783感受态细胞，涂含50μg/ml卡那霉素和34μg/ml氯霉素的LB琼脂平板，32℃过夜培养，挑单克隆菌落于含50μg/ml卡那霉素和34μg/ml氯霉素的LB液体培养基，32℃过夜培养，即为PRV-BAC(HL)GS1783菌株。
实施例1 非洲猪瘟病毒亚单位蛋白的选择和基因序列的优化
1.1非洲猪瘟病毒蛋白的选择
非洲猪瘟病毒结构蛋白CD2V是位于外囊膜的糖基化蛋白，由EP402R基因编码，预测在207-229aa处有一个跨膜区，已有研究表明，CD2V蛋白能够与红细胞相互作用，在病毒的扩散和淋巴细胞损伤的过程中具有很重要的作用。本领域技术人员公知，位于胞外区部分的CD2V片段，才具有和宿主细胞相互作用的功能，是理想的保护性抗原。为了便于CD2V表达，我们选择了去掉跨膜区的片段，同样地，本领域技术人员 为了便于CD2V表达，可选择CD2V胞外区片段、CD2V胞外区与其他片段如Fc或Cd3的融合片段或CD2V去掉跨膜区(207-229aa)片段等。非洲猪瘟病毒结构蛋白P72是由B646L基因编码的一段多肽，P72蛋白合成的动力学表明该蛋白在感染晚期被翻译，约占病毒蛋白量32％，是病毒二十面体结构的主要蛋白，尽管P72蛋白报道在多个***中存在表达，但未见在伪狂犬表达的报道。已有研究表明，非洲猪瘟病毒结构蛋白B602L是由B602L基因编码的一段多肽，B602L蛋白能够促进p72蛋白的正确折叠，若缺少B602L蛋白，p72蛋白的表达会显著减少，虽然该蛋白不是病毒的结构蛋白，但是缺少该蛋白，病毒的组装会发生很大的改变，最终导致不能正确组装成病毒颗粒。因此，在伪狂犬载体中选择同时表达这三种蛋白。
1.2基因合成
我们以2018年在中国报道流行的非洲猪瘟毒株亚型，参考Georgia 2007/1全基因序列(GenBank：FR682468.1)为模板，对编码非洲猪瘟P72蛋白的B646L的核苷酸序列进行密码子优化，得到OPTI-p72序列，如SEQ ID NO.1所示，为了便于后续构建和检测，在基因5’端加入HindIII酶切位点并添加Flag标签序列，在3’端加入EcoRI酶切位点，合成后序列亚克隆到pUC57，命名为pUC57-OPTI-P72；对B602L的核苷酸进行密码子优化，得到OPTI-B602L序列，如SEQ ID NO.2所示，为了便于后续检测，在C端添加了HA标签；对CD2V的核苷酸进行密码子优化，得到OPTI-CD2V如SEQ ID NO.3所示，为了便于检测，在C端添加了His标签，将合成的序列亚克隆到pUC57，命名为pUC57-OPTI-CD2V。该序列合成工作委托南京金斯瑞生物科技有限公司完成。
实施例2 中间载体pEE12.4-p72-kan的构建
2.1标记1.5ml EP管，将质粒pEE12.4-kan和实施例1中得到的pUC57-OPTI-P72分别用Hind III和EcoRI双酶切，酶切体系(50ul)如下所示。
Figure PCTCN2021104472-appb-000003
2.2将步骤2.1中的1.5mL EP管置于相应酶最适温度恒温水浴锅中，水浴2-3h。双酶切产物胶回收：取出上述双酶切体系，进行琼脂糖凝胶电泳以回收其中的DNA片段。
2.3连接反应
(1)准备洁净的1.5mL EP管若干，做好标记，置于EP管架上待用。
(2)在1.5mL EP管按照下表进行加样、混匀。
Figure PCTCN2021104472-appb-000004
(3)按照步骤(2)中表格完成加样后，将每个10μl反应体系置于16℃低温冷却液循环机中，水浴10-16h；
(4)取出步骤(3)中EP管，将其置于65℃水浴锅中，水浴15min；
(5)取出步骤(4)中的EP管，置于4℃保存。
2.4转化反应
(1)将10μL连接反应液快速加入100μL大肠杆菌JM109感受态细胞(购自Takara)中，并吹打混匀，冰浴30min；
(2)取出样品管，置于42℃水浴100s，然后立即冰浴2min；
(3)取出样品管，在超净工作台中，向样品管中加入600μL液体LB培养基，然后将样品管置于37℃恒温摇床，220rpm/min，培养1h；
(4)涂板：取出步骤(3)中样品管，室温离心8,000rpm/min，2min，去掉600μL上清液体，剩余上清液重悬管底部的菌体，将重悬的菌液放入相应的转化平板中心，用涂菌棒将转化平板中心的菌液均匀铺开。
(5)将转化步骤(4)平板正置于生化恒温培养箱中，37℃培养1h后，将转化平板倒置进行培养15h；
(6)观察转化结果。
2.5质粒抽提与双酶切鉴定
2.5.1质粒抽提(使用试剂盒DP6943，OMEGA)
(1)用10μL移液枪头从转化平板中挑取单克隆至5mL含氨苄抗性的LB液体培养基中，37℃，220rpm/min摇菌过夜；
(2)将菌液移至1.5mL EP管中，室温离心，12,000rpm/min，2min，弃上清；
(3)向步骤(2)的EP管中加入250μL质粒提取试剂P1 buffer，彻底悬浮菌体；
(4)向步骤(3)溶液中加入250μL P2 buffer，立即温和颠倒离心管5-10次混匀，室温静置2-4min；
(5)向步骤(4)溶液中加入350μL P3 buffer，立即温和颠倒离心管5-10次混匀；室温静置2-4min；
(6)将步骤(5)溶液，室温离心，14,000rpm/min，10min；
(7)将步骤(6)中上清溶液移至吸附柱中心，室温离心，12,000rpm/min，30s，倒掉收集管中液体；
(8)向吸附柱中心加入500μL buffer DW1，室温离心，12,000rpm/min，30s，倒掉收集管中液体；
(9)向吸附柱中心加入500μL洗液，室温离心，12,000rpm/min，30s，倒掉收集管中液体，重复一次；
(10)空吸附柱，室温离心，12,000rpm，2min。
(11)将吸附柱放入一个干净的1.5mL离心管中，向吸附膜中心加入30μL Elution buffer，室温静置5min，室温离心，12,000rpm，2min。保存管中DNA溶液。
2.5.2双酶切鉴定
(1)标记好需要用到的1.5mL EP管，按照下表进行加样：20μL反应体系
Figure PCTCN2021104472-appb-000005
(2)将步骤(1)中的EP管20μL反应体系置于37℃恒温水浴锅中，水浴2h。
(3)将步骤(2)中的双酶切体系样品进行琼脂糖凝胶电泳，检查***片段大小 是否正确；实验结果见图2。酶切鉴定构建正确。
(4)选择***片段正确的克隆送测序公司测序。证实构建出中间载体pEE12.4-p72-kan。
实施例3 亚克隆CMV-P72-bGH-kan序列
3.1通过PCR扩增带有CMV启动子、p72基因、I-sce I-Kan和TK同源臂的重组序列
(1)以实施例2中构建的pEE12.4-p72-kan为模板，其正向和反向引物为：
正向引物(5’-3’)：
Figure PCTCN2021104472-appb-000006
反向引物(5’-3’)：
Figure PCTCN2021104472-appb-000007
为了便于重组，PCR扩增带有PRV基因组TK同源臂的重组序列。
(2)加样体系50μL，如下表所示：
Figure PCTCN2021104472-appb-000008
PCR扩增程序：95℃2min；95℃30s，55℃45s，72℃1min，30个循环；72℃10min；8℃保持。
PCR产物进行胶回收，步骤如下：
(1)标记好样品收集EP管、吸附柱以及收集管；
(2)称取标记好的空的EP管重量，并记录数值；
(3)将单一的目的DNA条带在切胶仪上从琼脂糖凝胶中用手术刀小心切下放入干净的1.5mL离心管中；
(4)向步骤(3)中的1.5mL离心管中加入600μL PC buffer，50℃水浴放置5min左右，其间不断温和上下翻转离心管，以确保胶块充分溶解；
(5)柱平衡：向吸附柱CB2中(吸附柱预先放入收集管中)加入500μL平衡液BL，离心12,000rpm/min，1min，倒掉收集管中的废液，将吸附柱重新放回收集管中；
(6)将步骤(5)所得溶液加至吸附柱CB2中，静置2min，10,000rpm/min，离心30s，倒掉收集管中的废液，再将吸附柱CB2放入收集管中；
(7)向吸附柱中加入600μL漂洗液PW buffer，静置3min，离心10,000rpm/min，30s，倒掉收集管中的废液，将吸附柱CB2放入收集管中；
(8)重复步骤(7)；
(9)空吸附柱离心，12,000rpm/min，2min，尽量除去漂洗液，将吸附柱置于室温放置10min，彻底晾干；
(10)将吸附柱CB2放入收集管中，向吸附膜中间位置悬空滴加50μL Elution buffer(65℃预热)，静置3min，离心12,000rpm/min，2min；
(11)从离心机中取出步骤(10)中离心管，丢弃中间的吸附柱CB2，盖上离心管盖子，保留离心管中的DNA样品；
(12)将步骤11中的DNA样品置于4℃保存，准备琼脂糖凝胶电泳鉴定胶回收DNA片段。
3.2制备PRV-BAC(HL株)GS1783电转大肠杆菌GS1783感受态细胞
(1)取-80℃保存的含PRV-BAC(HL株)的GS1783菌液，在含有1％氯霉素(Chl)划单菌落，32℃，培养16h，挑单菌落接种5mL液体LB(30μg/ml Chl)，过夜培养12h，32℃；
(2)按照1:100的比例，接1ml的含PRV-BAC(HL株)的GS1783菌液GS1783菌液于100ml LB锥形瓶中，220rpm，32℃，摇菌2-4小时，至OD600在0.5-0.7之间；
(3)迅速拿至42℃水浴，220rpm摇菌15min；
(4)将锥形瓶冰浴20min，同时将配置好的10％甘油预冷；
(5)将锥形瓶中菌液倒至离心杯中，配平，5000rpm，4℃离心5min，倒掉上清；
(6)加入100ml 10％甘油洗涤，摇晃均匀，5000rpm 4℃，离心10min，倒掉上清；
(7)重复步骤(6)两次，最后倒掉上清，剩余约2-3ml。
(8)用Tips头吹打均匀，分装100μl至1.5ml EP管中，-80℃保存备用。
3.3电转及第一次同源重组制备PRV-BAC-CMV-P72-bGH-Kan-△TK
将步骤3.1中的PCR获得的亚克隆产物电转到步骤3.2中GS1783感受态细胞中。具体步骤如下：
(1)添加步骤3.1中100ng的PCR产物至步骤3.2中的重组电转感受态细胞；
(2)将DNA/菌体混合物转移至预冷的电转杯中。以15kV/cm(1mm电转杯为1.5kV)，6.0ms的条件进行电转；
(3)将电转后的样品转移至1.5ml EP管中，添加1ml无抗LB培养基，32℃培养1-2h；
(4)6000rpm离心1min后，涂布于含30μg/ml氯霉素和30μg/ml卡那霉素的LB固体平板，32℃培养24h；
(5)利用菌落PCR获得阳性克隆。
3.4异丙醇沉淀法提取第一次同源重组后的PRV-BAC-CMV-P72-bGH-Kan-△TK
将步骤3.3中PCR验证正确的阳性克隆挑取单菌落于6ml含30μg/ml Chl和30μg/ml Kan的LB培养基中，32℃培养24h；
(1)收集5mL菌液，室温5,000×g离心10min，4℃；
(2)倒去或吸取培养基并弃去；
注意：为保证所有的培养基都弃尽，用干净的吸水纸吸尽壁上多余的液体。
(3)加250μl SolutionI/RNaseA，涡旋和枪头吹打以重悬菌体，充分悬浮对获得上佳的产量至关重要；
注意：SolutionI在使用前必须先加入RNaseA。
(4)加入400μl SolutionII，轻轻温和颠倒并旋转离心管8-10次直至得到澄清的裂解液；该步可能需要在室温下孵育2-3分钟并间断颠倒混匀，总时长不超过5min。
注意：避免剧烈振荡，以免弄断染色体DNA，从而降低质粒的纯度。SolutionII使用后盖紧盖子且于室温保存，以免与空气中的CO 2反应；
(5)加入200μl预冷后的N3Buffer，轻轻颠倒离心管20次至出现絮状沉淀，冰浴10min；
注意：充分混匀对于获得高产至关重要，如果混合物看起来粘稠、棕色或未混匀，就继续混匀直到中和液体。
(6)4℃全速离心10min，使得细胞碎片沉淀完全；
(7)吸取700μl上清于干净的1.5ml离心管中，加入预冷的异丙醇，使体系终浓度为70％，轻轻颠倒旋转混匀几次，冰浴10min；
(8)4℃全速离心10min，使得质粒沉淀完全；
(9)弃上清，加入700μl的70％乙醇(现配)上下温和颠倒洗涤沉淀，4℃离心12000rpm，10min；
(10)重复步骤(9)；
(11)弃上清完全，室温超净台大风干燥，15min左右；
(12)用100μl Elution Buffer(65℃预热过)溶解沉淀；
(13)Nano仪测得质粒浓度(1000-2000ng/μl为宜)。
3.5第一次同源重组后PRV-BAC-CMV-P72-bGH-Kan-△TK验证
对3.4所提取的质粒进行BamHI酶切验证，同时设置阴性对照。酶切体系如下：
Figure PCTCN2021104472-appb-000009
于37℃，酶切1h后，跑胶验证，电泳条件为90v。
3.6第二次同源重组删除Kan基因
(1)挑取步骤3.5验证后的阳性克隆至1ml含30μg/ml氯霉素的LB培养基中，32℃，220rpm培养1-2h至菌液云状微浑；
(2)加入1ml含30μg/ml氯霉素和2％L-***糖且预热的LB培养基，32℃，220rpm培养1h；
(3)立即转移至42℃水浴摇床，220rpm，30min；
(4)转移至32℃，220rpm培养2-3h；
(5)吸取1ml菌液检测OD600值；
(6)当OD600≤0.5时，取适量菌液，稀释100倍；当OD600>0.5时，取适量菌液，稀释1000倍；吸取5-10μl的1：100稀释菌液涂布于含30μg/ml氯霉素和1％L-***糖LB固体平板；
(7)32℃培养1-2天直至菌体大小分明可见。
3.7异丙醇沉淀法提取第二次同源重组后的质粒PRV-BAC-CMV-P72-bGH-△TK验证
(1)将步骤3.6中的阳性克隆正确的挑取单菌落于6ml含30μg/ml Chl的LB培养基中，32℃培养24h；
其余步骤同步骤3.4和3.5，用BamHI酶切后验证，酶切图谱如图2所示，挑选正确的质粒，测序验证。
3.8用与上述同样的方法，很容易将CD2V整合到PRV-BAC-CMV-P72-bGH-△TK病毒上，构建得到包含P72和CD2V的病毒rPRV-P72-△TK-CD2V-△gG。
3.9用与上述同样的方法，很容易将B602L整合到rPRV-P72-CD2V-△TK-△gG病毒上，构建得到包含P72、B602L和CD2V的病毒rPRV-P72-B602L-CD2V-△TK-△gG。
需说明的是，P72、B602L和CD2V基因的整合顺序是可以颠倒的，可以根据需要和操作习惯进行任意调整。
实施例4 rPRV-P72-B602L-CD2V-△TK-△gG病毒的拯救
将提取的rPRV-P72-B602L-CD2V-△TK-△gG质粒纯化并测定浓度，根据lipofectamine LTX转染方法在BHK-21细胞中进行该重组病毒的拯救。操作步骤为：
①用OPTI-MEM稀释质粒，125μL OPTI-MEM中加入2.5μg质粒，然后加入2.5μL plus，混匀，室温静置5min。
②稀释Lipofectamine LTX：125μL OPTI-MEM中加入9μL Lipofectamine LTX，然后加入2.5μL plus，轻轻混匀，室温静置5min。
将步骤①和步骤②混合物轻轻混匀。室温放置5min，然后逐滴加入六孔板中均匀分布。将六孔板置于37℃，5％CO 2细胞培养箱中培养4-6h。换液：弃掉上清培养基，加入2mL DMEM/F12(含10％血清1％双抗)，将六孔板置于37℃，5％CO 2细胞培养箱中培养每天观察细胞状态及病毒噬斑形成情况，获得的病毒命名为rPRV-P72-B602L-CD2V-△TK-△gG。
实施例5 用western blot检测p72和CD2V蛋白的表达
扩繁rPRV-P72-B602L-CD2V-△TK-△gG病毒液，10000rpm/min，离心5min，收集上清，上样缓冲液处理后，用10％SDS-PAGE胶电泳，通过湿转(100V，90min)转印到PVDF膜上，用5％的脱脂奶粉封闭2h，加入1:4000倍稀释的鼠抗flag标签单抗(检测p72)或1:4000倍稀释的鼠抗His标签单抗(检测CD2V))，室温下作用1h后，PBST洗涤液洗涤3次，加入1：5000倍稀释的HRP标记的羊抗鼠IgG的二抗，室温下作用1h，加入底物显色液，避光5min后，观察在70-90kDa处有特异性条带与 预期蛋白大小一致为p72蛋白，结果如图3所示。观察在130kDa处有特异性条带与预期结果一致，由于CD2V是糖基化蛋白所以在SDS-PAGE上比实际分子量要偏大，如图4所示。图3和图4结果说明p72和CD2V蛋白都获得表达。
实施例6 重组病毒免疫小鼠试验
将8只Balb/c小白鼠(购买自浙江中医药大学)随机分为二组，每组4只，第一组免疫重组病毒rPRV-P72-B602L-CD2V-△TK-△gG，第二组注射生理盐水作为空白对照，通过肌肉注射分别进行免疫，共免疫两次，首免后21d进行二免，于二次免疫后的14d，小鼠采血通过ELISA测定抗体。具体方法：用重组p72和CD2V蛋白分别包被酶标板，包被浓度为0.5μg/ml，每种抗原均包被12孔(4孔加免疫后小鼠血清样品，4孔免加阴性小鼠血清，4孔加封闭液作为对照)，100μl/孔，37℃作用1h；PBST洗涤液洗涤3次，每次5min；加入1:5000稀释的HRP标记山羊抗鼠的IgG，100μl/孔，37℃作用1h；PBST洗涤液洗涤3次，每次5min；加入底物100μl/孔显色，37℃温育20min，最后加入2M H 2SO 4，50μl/孔，终止反应。结果如下表所示：
样品 包被p72蛋白OD450值 包被CD2V蛋白OD450值
二免后血清 2.105 1.878
二免后血清 1.984 1.763
二免后血清 1.853 1.696
二免后血清 2.532 1.954
空白对照阴性血清 0.232 0.12
空白对照阴性血清 0.354 0.136
空白对照阴性血清 0.221 0.327
空白对照阴性血清 0.246 0.225
封闭液 0.057 0.034
封闭液 0.06 0.042
封闭液 0.083 0.075
封闭液 0.075 0.051
实验结果表明，包被P72蛋白能与二免后血清特异性结合，OD450均值为2.119；包被CD2V蛋白能与二免后血清特异性结合，OD450均值为1.823。可明显看出，rPRV- P72-B602L-CD2V-△TK-△gG检测出的抗体浓度均较高，说明重组病毒rPRV-P72--B602L-CD2V-△TK-△gG的免疫原性较好，可以作为研究ASFV-PRV活载体疫苗。
本发明通过上面的实施例进行举例说明，但是，应当理解，本发明并不限于这里所描述的特殊实例和实施方案。在这里包含这些特殊实例和实施方案的目的在于帮助本领域中的技术人员实践本发明。任何本领域中的技术人员很容易在不脱离本发明精神和范围的情况下进行进一步的改进和完善，因此本发明只受到本发明权利要求的内容和范围的限制，其意图涵盖所有包括在由附录权利要求所限定的本发明精神和范围内的备选方案和等同方案。

Claims (10)

  1. 一种重组的伪狂犬病病毒，其特征在于，其基因组包含下述外源基因：
    编码非洲猪瘟病毒来源的衣壳蛋白P72或其变体的核苷酸序列A，所述变体是衣壳蛋白P72氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有衣壳蛋白P72的功能；
    编码用于促使衣壳蛋白P72或其变体正确表达及折叠的辅助蛋白的核苷酸序列B，所述变体是辅助蛋白氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有促使衣壳蛋白P72或其变体正确表达及折叠的功能；
    编码非洲猪瘟病毒来源的外囊膜蛋白CD2V或其变体的核苷酸序列C，所述变体是外囊膜蛋白CD2V氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有外囊膜蛋白CD2V的功能。
  2. 如权利要求1所述的重组的伪狂犬病病毒，其特征在于，所述辅助蛋白是非洲猪瘟病毒来源的B602L蛋白。
  3. 如权利要求1或2所述的重组的伪狂犬病病毒，其特征在于，所述外源基因仅包含核苷酸序列A、核苷酸序列B和核苷酸序列C。
  4. 如权利要求2所述的重组的伪狂犬病病毒，其特征在于，所述核苷酸序列A与SEQ ID NO:1的同源性≥90％，优选地≥95％，更优地≥98％；所述核苷酸序列B与SEQ ID NO:2的同源性≥90％，优选地≥95％，更优地≥98％；所述核苷酸序列C与SEQ ID NO:3的同源性≥90％，优选地≥95％，更优地≥98％。
  5. 如权利要求1所述的重组的伪狂犬病病毒，其特征在于，所述重组的伪狂犬病病毒适合于在细胞内复制并表达核苷酸序列A、核苷酸序列B和核苷酸序列C，所述细胞选自用于病毒增殖的细胞如哺乳动物细胞、鸟类细胞或昆虫细胞。
  6. 如权利要求1所述的重组的伪狂犬病病毒，其特征在于，其基因组还包含下述外源基因：
    编码非洲猪瘟病毒来源的P49或其变体的核苷酸序列D，所述P49变体是氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有P49的功能。
  7. 如权利要求1所述的重组的伪狂犬病病毒，其特征在于，所述基因组的至少一个复制非必需区发生缺失和/或替代，所述复制非必需区选自伪狂犬病病毒的gC、gE、gG、gI、gM、TK、RR、PK编码区中的一个以上。
  8. 一种构建如权利要求1所述重组的伪狂犬病病毒的方法，其包含如下步骤：
    (1)将伪狂犬病病毒株PRV-BAC(HL)基因组中的TK基因替换成密码子优化后的表达非洲猪瘟病毒p72蛋白的基因表达框，得到PRV-BAC-P72-△TK；
    (2)将密码子优化后的非洲猪瘟病毒B602L基因表达框***到p72基因后，得到PRV-BAC-P72-B602L-△TK；
    (3)用密码子优化后的非洲猪瘟病毒CD2V基因表达框替换步骤(2)中所得的PRV-BAC-P72-B602L-△TK的gG基因，得到PRV-BAC-P72-B602L-CD2V-△TK-△gG；
    (4)用步骤(3)中所得的PRV-BAC-P72-B602L-CD2V-△TK-△gG转染BHK-21细胞，拯救得到同时表达p72、B602L和CD2V蛋白的重组伪狂犬病毒，命名为rPRV-P72-B602L-CD2V-△TK-△gG。
  9. 如权利要求1-7中任一项所述的重组的伪狂犬病病毒在制备非洲猪瘟疫苗中的用途。
  10. 一种非洲猪瘟疫苗或免疫组合物，其特征在于，其至少含有权利要求1-7中任一项所述的重组的伪狂犬病病毒作为免疫原。
PCT/CN2021/104472 2020-07-10 2021-07-05 一种重组的伪狂犬病病毒及其疫苗组合物 WO2022007742A1 (zh)

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