WO2022007800A1 - 一种非洲猪瘟的重组腺病毒疫苗及其构建方法 - Google Patents

一种非洲猪瘟的重组腺病毒疫苗及其构建方法 Download PDF

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WO2022007800A1
WO2022007800A1 PCT/CN2021/104793 CN2021104793W WO2022007800A1 WO 2022007800 A1 WO2022007800 A1 WO 2022007800A1 CN 2021104793 W CN2021104793 W CN 2021104793W WO 2022007800 A1 WO2022007800 A1 WO 2022007800A1
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pad5lcl3
group
plasmid
vector
ires
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陈平
张婷婷
李娜
钟鑫涛
祝志刚
李楠
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嘉兴安宇生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/187—Hog cholera virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

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  • the invention relates to the technical field of genetic engineering and the field of immunology, in particular to a recombinant adenovirus vaccine of African swine fever virus and a construction method thereof.
  • African swine fever is a highly contagious viral disease of pigs. In domestic pigs it can result in a high mortality rate close to 100%.
  • ASF is caused by ASF virus (ASF Virus, ASFV).
  • ASFV is a large double-stranded DNA virus that replicates mainly in the cytoplasm of macrophages. 190kb, contains 151 open reading frames, can encode 150 to 200 kinds of proteins, double-stranded linear DNA virus with envelope.
  • the structural proteins that make up the ASFV virion include P30, P72, P49, P54, P220, P62, pB602L, CD2v protein, etc., based on one or two subunit vaccines so far have not been able to induce immunity strong enough to have a significant protective effect on vaccine recipients .
  • live vector vaccines are live vector vaccines.
  • the advantages of live vector vaccines are: (1) It can actively infect target tissues or cells, which improves the efficiency of foreign genes entering cells; (2) The vector itself has an adjuvant effect, which can induce cytokines and cells. The production of chemokines; (3) most of them can induce long-term immune response.
  • as many pathogen proteins as possible need to be delivered with as little live carrier as possible.
  • a live vector vaccine refers to the cloning of the protein-encoding gene of a pathogen into a live viral vector, which is then used to immunize animals, and the protein is expressed in the animal, thereby inducing an immune response against the protein.
  • Adenovirus type 5 has many advantages as a vector for expressing African swine fever antigenic proteins: 1 The adenovirus expression vector is replication-deficient and can only be produced in its unique complementary cell line, and the adenovirus does not need to be integrated into the host cell genome Among them, the target gene is expressed in the free state outside the host cell genome, the integration mutation is less likely to cause cancer, the genotoxicity is low, and the vaccine preparation safety is good; 2 the recombinant adenovirus vector can obtain a higher titer, which is conducive to large-scale production, High factory efficiency and low production cost; 3 At present, the research on the structure, characteristics and function of adenovirus type 5 is relatively in-depth, and the adenovirus
  • ASFV p32, p54, p72 and pp62 genes were recombined into the human adenovirus Ad5 vector for "cocktail" immunization, and a good antigen-specific CTL response was obtained;
  • a total of seven ASFV antigen genes of B438L and K205R-A104R were recombined into a replication-deficient adenovirus vector, which could induce strong humoral and cellular immune responses after a "cocktail” mixed immunization.
  • each ASFV antigen gene must be recombined into a replication-deficient adenoviral vector, so the number of vectors required is very large, and there is a risk of an immune response against the adenoviral vector during the immunization process.
  • CN108504686A and CN108504687A provide recombinant adenovirus vectors expressing the EP153R and EP402R genes of ASFV, respectively.
  • CN109652449A discloses a recombinant adenovirus vector co-expressing EP153R and EP402R antigen genes
  • CN109735567A discloses a recombinant adenovirus vector co-expressing EP153R and P54 antigen genes.
  • CN110269932A discloses that 5-7 antigen genes of ASFV, such as A104R, A151R, B119L, B602L, CD2v, K205R, P49, etc., are fused together based on adenovirus vector for preparing live vector vaccine.
  • ASFV ASFV
  • the fusion of multiple antigen genes has the risk of reducing immunogenicity and may lead to immune failure. Therefore, to improve vaccine activity, it is necessary to express completely independent antigen genes on each adenovirus vector.
  • the P30 protein of ASFV is a very important structural protein encoded by the CP204L gene. Studies have found that P30 can induce host cells to produce neutralizing antibodies that inhibit cellular internalization, thereby delaying disease onset or even protecting cells against viral infection. Therefore, P30 plays an important role in blocking the interaction between viruses and cells. As an early protein of the virus, P30 is mainly distributed in the cytoplasm after infection of cells, and can be detected in the cytoplasm 4 hours after infection; P30 is also one of the most antigenic ASFV proteins, with strong immunogenicity. In infected animals, it can induce the body to produce virus-neutralizing antibodies, so it is usually used as a diagnostic antigen.
  • the P54 protein of ASFV is encoded by the E183L gene, and its antibody has a certain ability to neutralize the virus.
  • the P30 protein and the P54 protein interact with two different receptors or binding sites on susceptible cells, which can alleviate the course of the disease.
  • P72 protein is one of the main detection antigens of ASFV, about 75ku in size. Good stability and small variation. Using the P72 protein as an antigen, a series of detection products have been developed.
  • the pB602L protein, encoded by the B602L gene stimulates the substrate to produce higher levels of antibodies.
  • the present invention provides an African swine fever virus vaccine, which is obtained by constructing a recombinant adenovirus vector co-expressing four African swine fever virus antigen genes, and packaging it in 293TD37 cells.
  • five groups of four antigen genes of African swine fever virus have been designed, and any group of four antigen genes can be used to prepare a recombinant adenovirus vector co-expressing the four antigen genes of African swine fever virus, namely African swine fever virus vaccine.
  • the invention can greatly increase the capacity of the adenovirus vector vaccine, and enhance the specific immune response to the African swine fever virus by simultaneously expressing four independent antigens of African swine fever on one adenovirus vector.
  • antigenic genes of African swine fever virus There are more than 160 antigenic genes of African swine fever virus.
  • the inventors selected 20 antigenic genes with stronger immune effects, namely: P72, B602L, P30, P54, CP129R, MGF5L6L, CP312R , MGF110-4L, L8L, I215L, I73R, E146L, EP402R, EP153R, I177L, K205R, F317L, A151R, P34, pp62; these 20 antigen genes are divided into five groups according to the size of the gene fragment and protein structure.
  • the four antigen genes can be co-expressed in the recombinant adenovirus vector pAd5LCL3 provided by the present invention, that is, the four antigen genes can be completely and independently expressed in the same vector.
  • These five groups of antigen gene vaccines (including five recombinant adenovirus vectors pAd5LCL3) constitute a complete African swine fever virus vaccine, which has achieved very good immune effects.
  • the four antigen genes of each group can be well matched and assembled in the same recombinant adenovirus vector, so that the four antigen genes can be expressed completely independently.
  • the present invention provides a recombinant adenoviral vector pAd5LCL3 capable of expressing multiple antigen genes simultaneously.
  • the E1 region and the E4 region of the exogenous antigen gene; the antigen gene can be an antigen gene of suitable size from any source.
  • E1 region and the E4 region of the recombinant adenovirus vector pAd5LCL3 can express four antigen genes from different or the same origin.
  • ORF1-ORF7 is deleted in the E4 region of the recombinant adenovirus vector pAd5LCL3.
  • E2a region also known as DNA Binding Protein, DBP
  • DBP DNA Binding Protein
  • E4 promoter, ORF6, ORF7 and polyA sequences of the E4 region of the recombinant adenovirus vector pAd5LCL3 are placed at the E2a position.
  • E1 region of the recombinant adenovirus vector pAd5LCL3 is preset with a SwaI restriction site.
  • E4 region of the recombinant adenovirus vector pAd5LCL3 is preset with an I-scel restriction site.
  • the present invention conducts sequence analysis on the E4 gene, finds out the basic elements of the promoter, ORF6/7 and polyA of E4, integrates them into a complete expression frame, and constructs it at the sequence position where the E2a gene has been knocked out. , the ORF6 and ORF7 genes were expressed normally, and finally the replication-defective adenovirus type 5 vector pAd5LCL3, which knocked out E1, E3, E4, and E2a, and placed the E4 expression box at the position of E2a, was able to express in 293TD37 cells containing DBP sequences. Copy packaging.
  • ORF6/7 needs to be replenished.
  • ORF6/7 needs to be expressed at E2a, so as to obtain an adenovirus vector with a larger capacity and better expression effect.
  • the recombinant adenovirus vector pAd5LCL3 that can simultaneously express four antigen genes must pass through the 293TD37 cell constructed by pcDNA3.1+(hyg)-ORF6-IRES-DBP to realize recombinant adenovirus packaging, and the cell line of 293TD37 cell
  • the preservation number is: CCTCC NO: C201996, which is preserved in the China Center for Type Culture Collection.
  • Ordinary 293 cells contain the E1 gene of adenovirus type 5. Adenoviruses knocking out E1 and E3 can replicate in this cell line, but adenoviruses knocking out E4 and E2a genes cannot replicate in 293 cells.
  • the 293TD37 cell line was invented by the present invention group, and has applied for an invention patent CN201911033247.2, which was deposited with the China Type Culture Collection on May 8, 2019.
  • the deposit number is CCTCC NO: C201996
  • the classification name is human embryonic kidney transformed cell AY293 -TD-37
  • this cell line contains E2a-DBP gene and E4-ORF6/7 gene of adenovirus, which can be used to package second-generation adenovirus lacking E2a-DBP gene and E4 gene to form a complete infectious
  • the second-generation adenovirus has a significantly lower probability of RCA, which lays the foundation for the preparation of live vector vaccines, and due to the simultaneous deletion of E2a-DBP and E4 genes, the packaging capacity is similar.
  • the invention provides a method for constructing a recombinant adenovirus vector pAd5LCL3, which mainly uses CRISPR/cas9 to knock out the E1, E3, E4 and E2a genes of the adenovirus circular vector plasmid, and puts the ORF6/7 expression box of the E4 region in the knockout Sequence position of the E2a region.
  • the construction method of the recombinant adenovirus vector pAd5LCL3 capable of simultaneously expressing four antigen genes comprises the following steps:
  • knocking out the E4 gene can increase the capacity of the adenovirus vector and reduce its immunogenicity.
  • foreign genes can be inserted into the E4 region, and the foreign genes can be expressed in large quantities at the E4 position. , without affecting the packaging of the adenopathy vector.
  • Expressing exogenous genes at the E1 and E4 genes can avoid the mutual interference of multiple exogenous genes expressed in the same region, which is more conducive to expression, while reducing unnecessary E4-related genes, reducing the immunogenicity of adenovirus, making Adenovirus can exist in the host cell for a long time, so that the foreign gene can be expressed for a long time.
  • the E4 region gene plays a key role in immunogenicity.
  • the expression of a large number of E4 region genes will make the host produce a stronger immune response and induce the production of antibodies, which is not conducive to the long-term expression of the target protein by the adenovirus vector in the host.
  • Knockout of unnecessary genes in the E4 region can reduce the immunogenicity of the adenoviral vector, allowing the vector to be expressed over a longer period of time.
  • the upstream Fiber gene in the E4 region and the E4 gene were knocked out using the CRISPR/cas9 method, and PCR was used to amplify part of the fiber and introduce I- sceI single-enzyme cleavage site, and then use Gibson's seamless cloning method to ligate the excess excised fragment to the vector to regain the E4 knockout vector plasmid with the introduction of the I-sceI single-enzyme cleavage site.
  • the vector plasmid was linearized with I-scel to construct a shuttle plasmid in the E4 region, so that the exogenous gene was recombined into the E4 region and expressed in large quantities in the E4 region.
  • the sequence of ORF1-ORF5 in the E4 region is knocked out, and the E4 promoter, ORF6, ORF7, and polyA sequences are retained, and they are inserted into the E2a position, so the E4 position can express foreign genes.
  • the DBP sequence in the E2a region was also knocked out.
  • the adenovirus E2a gene is a DNA binding protein that is associated with adenovirus replication. Knockout of this gene does not affect adenovirus structural proteins and adenovirus packaging. Deletion of DBP can prevent or greatly reduce back mutations. Knockout of the E2a and E4 partial sequences increased the vector capacity by about 3 kb.
  • adenoviral vector construction generally uses shuttle plasmids, which need to find a single enzyme cleavage site.
  • the present invention creatively uses CRISPR/cas9 to construct a recombinant adenovirus vector, selects appropriate E1, E3, E4, E2a knockout sites through comparison, and knocks out genes according to the positions of the E1, E3, E4, E2a sequences The number of bases, the CRISPR site was selected, and the optimal gRNA was designed to complete the construction of the recombinant adenovirus vector.
  • the present invention provides a recombinant adenovirus vaccine, which comprises a target gene in the E1 region and the E4 region of pAd5LCL3.
  • E3 gene Since the E3 gene is related to replication, it needs to be knocked out to make its replication defective; the role of E3 is related to the immune escape of adenovirus; knocking out the E3 region can increase the capacity of adenoviral vectors; and enable adenoviral vectors to be packaged normally.
  • the target gene is a virus, bacteria, tumor gene or gene fragment.
  • the target gene is an African swine fever virus gene.
  • the present invention provides an African swine fever virus vaccine, characterized in that, the vaccine is a recombinant adenovirus vector co-expressed by constructing four antigen genes of African swine fever virus, and then packaged in 293TD37 cells and obtained.
  • the recombinant adenovirus vector co-expressed by the four antigen genes of the African swine fever virus needs to be packaged by the 293TD37 cell constructed by pcDNA3.1+(hyg)-ORF6-IRES-DBP, and the 293TD37 cell
  • the cell line preservation number is: CCTCC NO: C201996, which is deposited in the China Center for Type Culture Collection.
  • the four antigen genes are any one of the following five groups of antigen genes: the first group: P72, B602L, P30 and P54; the second group: C129Rubiqutin, MGF5L6L, CP312R and MGF110-4L; the third group Group: L8Lubiqutin, I215L, I73Rhbsag and E146L; fourth group: EP402R, EP153R, I177L and K205 Rubiqutin; fifth group: F317L, A151R, P34 and pp62. 23.
  • the vaccine of claim 12 wherein the In the first group, P72 and B602L are expressed in the E1 region, and P30 and P54 are expressed in the E4 region, constituting a recombinant adenovirus vector pAd5LCL3-P72-B602L-P30-P54 that co-expresses four antigen genes; in the second group , CP129Rubiqutin is obtained by adding molecular adjuvant ubiqutin to CP129R, CP129Rubiqutin and MGF5L6L are expressed in the E1 region, and CP312R and MGF110-4L are expressed in the E4 region, constituting a recombinant adenovirus vector pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R that co-expresses four antigen genes -MGF110-4L; in the third group, L8Lubiqutin is obtained by adding molecular adjuvant
  • the inventors selected 20 antigen genes with stronger immune effect from more than 160 antigen genes of African swine fever virus. These 20 antigen genes were divided into five groups according to the size of gene fragments and protein structure. , the four antigen genes of each group can be co-expressed in the recombinant adenovirus vector pAd5LCL3 provided by the present invention, that is, the four antigen genes can be completely independently expressed in the same vector.
  • the four antigen genes of African swine fever virus in each group are 1, P72, B602L, P30 and P54; 2, C129Rubiqutin, MGF5L6L, CP312R and MGF110-4L; 3, L8Lubiqutin, I215L, I73Rhbsag and E146L; 4, EP402R, EP153R, I177L and K205 Rubiqutin; 5, F317L, A151R, P34 and pp62.
  • P72, B602L, P30, P54 and pAd5LCL3-P72-B602L-P30-P54 respectively have Seq ID NO.1, Seq ID NO.2, Seq ID NO.3, Seq ID NO. 4.
  • the nucleotide sequence shown in Seq ID NO.6; the CP129R, ubiqutin, MGF5L6L, CP312R, MGF110-4L, pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L respectively have the Seq ID NO. 14.
  • the present invention provides a method for constructing an African swine fever virus vaccine as described above, which mainly comprises the following steps:
  • pS5E1 Constructing a shuttle plasmid in the E1 region of adenovirus, pS5E1 is respectively combined with P72, IRES, B602L of the first group, or CP129 Rubiqutin, IRES, MGF5L6L of the second group, or L8Lubiqutin, IRES, I215L, or The EP402R, IRES, EP153R of the fourth group, or the F317L, IRES, A151R gene fragments of the fifth group were connected to construct a shuttle plasmid in the E1 region of the African swine fever adenovirus type 5 vector, respectively the first group: pS5E1-P72-IRES- B602L; second group: pS5E1-CP129Rubiqutin-IRES-MGF5L6L; third group: pS5E1-L8Lubiqutin-IRES-I215L; fourth group: pS5E1-EP40
  • the E1 region will be shuttled plasmid pS5E1-P72-IRES-B602L, or pS5E1-CP129Rubiqutin-IRES-MGF5L6L, or pS5E1-L8Lubiqutin-IRES-I215L, or pS5E1-EP402R-IRES-EP153R, or pS5E1-F317L-IRES, respectively -A151R was homologously recombined with the adenovirus vector plasmid pAd5LCL3 to obtain the first group of adenovirus vector plasmids: pAd5LCL3-P72-IRES-B602L; the second group pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L; the third group: pAd5LCL3-L8Lubiqutin-IRES- I215L; fourth panel: p
  • the adenovirus circular vector plasmid described in step 1) is derived from amplifying the wild-type human adenovirus type 5 virus in A549 cells, collecting and concentrating the virus liquid, and using the HirtViral DNA Extract method to extract the adenovirus type 5 genome,
  • the linear adenovirus type 5 genome was constructed into a circular adenovirus circular vector plasmid using the cosmid method.
  • the ORF6/7 expression box gene described in step 3) has the nucleotide sequence shown in Seq ID NO.7 in the sequence listing; the IRES described in step 4) has Seq ID NO.8 in the sequence listing The nucleotide sequence shown; 2A described in step 5) has the nucleotide sequence shown in Seq ID NO.9 in the sequence listing.
  • step 4) described shuttle plasmid pS5E1 skeleton adopts puc origin, amp basic element, Ad5 left arm ITR partial sequence, right arm PIX, PIVa2 partial sequence, and CMV-MCS SV40 early polyA; step 5) described
  • the backbone of the shuttle plasmid pS5E4-EGFP in the E4 region adopts the basic elements of puc origin and amp, the ITR sequence of the left arm of the Ad5E4 region, the fiber gene sequence of the right arm part, and the EF1 ⁇ -EGFP-HBV polyA gene;
  • the basic elements of puc origin and amp have the following sequence
  • the nucleotide sequence shown in Seq ID NO.10 in the list can be seen, and the EF1 ⁇ -EGFP-HBV polyA gene has the nucleotide sequence shown in Seq ID NO.11 in the sequence table.
  • the skeleton of the shuttle plasmid pS5E1 was synthesized by Beijing Bomed Gene Technology Co., Ltd., and the synthesis adopted basic elements such as puc origin and amp (2796bp), Ad5 left arm ITR partial sequence (400bp), right arm PIX, PIVa2 partial sequence (2100bp), and CMV-MCS (944bp) SV40 early polyA (160bp). After PCR amplification and gene fragment purification, seamless cloning and ligation were carried out. The ligated products were transformed into competent cells and coated with ampicillin-resistant plates. After culturing, positive clones were selected for restriction digestion verification, and the shuttle plasmid pS5E1 in the adenovirus E1 region was obtained.
  • basic elements such as puc origin and amp (2796bp), Ad5 left arm ITR partial sequence (400bp), right arm PIX, PIVa2 partial sequence (2100bp), and CMV-MCS (944bp) SV40 early
  • the backbone of the shuttle plasmid pS5E4 adopts basic elements such as puc origin and amp, the left arm ITR sequence of the Ad5E4 region (370bp), the right arm part of the fiber gene sequence (1746bp), and the EF1 ⁇ -EGFP-HBV polyA gene. After PCR amplification and gene fragment purification, seamless cloning and ligation were carried out, the ligation products were transformed into competent cells, and ampicillin resistance plates were coated. EGFP.
  • the homologous recombination of the shuttle plasmid in the E1 region and the adenovirus vector plasmid pAd5LCL3 in step 6) is that the shuttle plasmid and the adenovirus vector plasmid pAd5LCL3 are digested by PacI and SwaI, and the enzyme digested product is dephosphorylated, OMEGA The Ultra-Sep Gel Extraction Kit was used for gel recovery of the vector and fragments, the transformation product was spread on a plate, and the colonies were picked for verification by XhoI digestion.
  • the homologous recombination of the E4 region shuttle plasmid and the adenovirus vector plasmid described in step 7) is that the E4 region shuttle plasmid and the adenovirus vector plasmid are digested by PacI and I-sceI, and the digested product is dephosphorylated, OMEGA The Ultra-Sep Gel Extraction Kit was used for gel recovery of the vector and fragments, the transformation product was spread on a plate, and the colonies were picked for verification by XhoI digestion.
  • the present invention provides a packaging method for recombinant adenovirus vector, the main steps are as follows: the first group of the recombinant adenovirus vaccine: pAd5LCL3-P72-B602L-P30-P54; the second group: pAd5LCL3 -CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L; third group: pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L; fourth group: pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin; fifth group: pAd5LCL3-F317L-A151 -pp62; digested with PacI, and the linearized plasmid was used for transfection; 293TD37 cells constructed from pcDNA3.1+(hyg)-ORF6-IRES-DB
  • the 293TD37 cell line was deposited in the China Center for Type Culture Collection on May 8, 2019.
  • the deposit number is CCTCC NO: C201996, and the classification name is human embryonic kidney transformed cell AY293-TD37.
  • This cell line contains adenovirus E2a and The E4-ORF6/7 gene is obtained by genetic engineering of HEK293 cells, and can be used to package the second-generation recombinant adenovirus lacking E2a and E4 genes to form infectious second-generation adenovirus particles.
  • packaging method of the recombinant adenovirus vector is mainly prepared by the following steps:
  • the first group of the recombinant adenovirus vaccine pAd5LCL3-P72-B602L-P30-P54; the second group: pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L; I215L-I73Rhbsag-E146L; fourth group: pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin; fifth group: pAd5LCL3-F317L-A151R-P34-pp62; digested with PacI, and the linearized plasmid was used for transfection; using 293TD37 cells were transfected with PEI transfection reagent;
  • the transfected 293TD37 cells were cultured in a 37°C, 5% CO 2 incubator for 72-96 hours, and the cell suspension was collected, which was the TP0 generation adenovirus;
  • 293TD37 cells were infected with TP0 adenovirus and cultured in a 37°C, 5% CO 2 incubator for 72 hours, and the cell suspension, ie TP1 adenovirus, was collected;
  • the present invention provides the use of 293TD37 cells for packaging recombinant adenovirus vectors co-expressed with four antigen genes of African swine fever virus, the four antigen genes are respectively the first group: P72, B602L, P30 and P54 ; second group: C129Rubiqutin, MGF5L6L, CP312R and MGF110-4L; third group: L8Lubiqutin, I215L, I73Rhbsag and E146L; fourth group: EP402R, EP153R, I177L and K205Rubiqutin; fifth group: F317L, A151R, P34 and pp62 ;
  • P72 and B602L are expressed in the E1 region
  • P30 and P54 are expressed in the E4 region, constituting a recombinant adenovirus vector pAd5LCL3-P72-B602L-P30-P54 that co-expresses
  • the 293TD37 cell is constructed from pcDNA3.1+(hyg)-ORF6-IRES-DBP, and the cell line preservation number is: CCTCC NO: C201996, which is preserved in the China Center for Type Culture Collection.
  • the present invention provides an African swine fever virus vaccine, which is obtained by constructing a recombinant adenovirus vector co-expressing four African swine fever virus antigen genes, and packaging it in 293TD37 cells.
  • an African swine fever virus vaccine which is obtained by constructing a recombinant adenovirus vector co-expressing four African swine fever virus antigen genes, and packaging it in 293TD37 cells.
  • five groups of four antigen genes of African swine fever virus have been designed, and any group of four antigen genes can be used to prepare a recombinant adenovirus vector co-expressing the four antigen genes of African swine fever virus, namely African swine fever virus vaccine.
  • the construction of the recombinant adenovirus vector co-expressing the four antigen genes of African swine fever virus mainly by knocking out the E1, E3, E2a and E4 genes of the adenovirus vector through CRISPR/cas9, and constructing the shuttle plasmids of the E1 and E4 regions, respectively using to express four antigen genes, thereby obtaining a new adenovirus vector.
  • the beneficial effects of the present invention are mainly reflected in:
  • E4 region gene plays a key role in immunogenicity
  • the expression of a large number of E4 region genes will cause the host to produce a relatively strong immune response and induce the production of antibodies, which is not conducive to the long-term expression of the target protein by the adenovirus vector in the host.
  • the invention found that knocking out unnecessary genes in the E4 region can reduce the immunogenicity of the adenovirus vector, so that the vector can be expressed over a longer period of time.
  • the present invention knocks out the ORF1-ORF5 sequences in the E4 region, retains the E4 promoter, ORF6, ORF7, and polyA sequences, and inserts them into the E2a position, so that the E4 position can express foreign genes.
  • the present invention further knocks out the DBP (E2a) sequence, and DBP deletion can prevent or greatly reduce back mutation.
  • the knockout of the partial sequences of E2a and E4 increases the vector capacity by about 3 kb compared with the first generation vector.
  • E2a and E4 of adenovirus vector Knock out E2a and E4 of adenovirus vector, and put E4promoter-ORF6/7-polyA in the E2a region, so that it can be rescued by a cell line complementary to E2a (DBP sequence).
  • the E4 region is expressed simultaneously without mutual interference.
  • the adenovirus vaccine has been rescued in the complementary cell line constructed by our company, the 293TD37 cell line, which can permanently express the DBP protein.
  • the present invention constructs shuttle plasmids in the E1 and E4 regions, which are used for the expression of exogenous genes in the E1 and E4 regions.
  • the titer of the recombinant adenovirus prepared by packaging the 293TD37 cell line of the present invention is relatively high.
  • the present invention can greatly increase the capacity of the adenovirus vector vaccine, and enhance the specific immune response to the African swine fever virus by simultaneously expressing four independent African swine fever antigens on one adenovirus vector. Make domestic pigs get better immune protection.
  • Fig. 1 is the schematic diagram of Ad5-E4-up-gRNA cleavage site and PAM site of Example 2
  • Fig. 2 is the schematic diagram of Ad5-E4-down-gRNA cleavage site and PAM site of Example 2
  • Figure 3 shows the results of electrophoresis detection of Ad5-E4-up-gRNA, Ad5-E4-down-gRNA and cas9 "double-enzyme digestion" vector plasmids of Example 2, wherein lane 1 is Ad5-E4-up-gRNA, Ad5-E4 -down-gRNA and cas9 "double digestion", M is maker
  • Fig. 4 is the result of electrophoresis detection of fiber and ITR fragments containing partial knockout in Example 2, wherein lane 1 is the amplification result of fiber partial fragment, swimming lane 2 is the amplification result of ITR partial fragment, and M is maker
  • Fig. 5 is the electrophoresis detection result of Fiber-ITR fusion fragment of embodiment 2, wherein swimming lane 1 is Fiber-ITR fusion fragment, and M is maker
  • Fig. 6 is the colony PCR verification electrophoresis detection result of embodiment 2, wherein swimming lane 1-24 is colony, M is maker
  • Fig. 7 is the electrophoresis detection result that embodiment 2 carries out BamHI, XhoI restriction enzyme digestion to the positive clone colony plasmid of Fig. 6, wherein 1-5 is BamHI restriction enzyme digestion, 6-10 is XhoI restriction restriction enzyme, 1,10 are pAd5 control ( is the true E4 gene), M is maker
  • Fig. 8 is the schematic diagram of 100k-gRNA cleavage site and PAM site of Example 3
  • Figure 9 is a schematic diagram of the protease-gRNA cleavage site and PAM site of Example 3.
  • Figure 10 is the result of electrophoresis detection of 100k-gRNA, protease-gRNA and cas9 "double digestion" vector plasmid of Example 3, lane 1 is the result of 100k-gRNA, protease-gRNA and cas9 "double digestion” vector plasmid, M is maker
  • Fig. 11 is the 100k, E4 ORF6/7 expression frame, protease PCR amplification electrophoresis detection result of embodiment 3, wherein swimming lane 1 is E4ORF6/7 expression frame, swimming lane 2 is 100k, M is Marker
  • Fig. 12 is the fusion PCR electrophoresis detection result of 100k, E4 ORF6/7 expression cassette, Protease fragment of embodiment 3, wherein swimming lane 1 is fragment 100k, E4 ORF6/7 expression cassette, protease fusion PCR product, M is Marker
  • Figure 13 is the colony PCR verification electrophoresis detection result of Example 3, wherein the lanes 1-24 are colonies, and M is maker
  • Fig. 14 is the electrophoresis detection result that the positive clone colony of No. 9, 18, 21, 24 of Fig. 13 is picked and verified by XhoI digestion in Example 3, wherein swimming lane 1 is the XhoI digestion of No. 9 positive clone, and swimming lane 2 is 18 No. 21 positive clone XhoI digested, lane 3 is No. 21 positive clone XhoI digested, lane 4 is No. 24 positive clone XhoI digested, lane 5 is the control plasmid pAd5LCL3 digested by XhoI, M is maker
  • Figure 15 is the CMV-MCS and SV40 earlypolyA fragment amplification electrophoresis detection results of Example 4, wherein swimming lane 1 is the CMV-MCS fragment, swimming lane 2 is the SV40 earlypolyA fragment, and M is 2000Marker
  • Fig. 16 is the CMV-MCS-SV40 earlypolyA, PUC, Ad5 right arm, Ad5 left arm amplification electrophoresis detection result of embodiment 4, wherein swimming lane 1 is CMV-MCS-SV40 earlypolyA fusion fragment, swimming lane 2 is PUC, and swimming lane 3 is Ad5 right arm, lane 4 is Ad5 left arm, M is 2000Marker
  • Fig. 17 is the pS5E1 skeleton of embodiment 4, Ad5 left arm, Ad5 right arm, CMV-MCS-SV40 earlypolyA four fragment ligation products transform competent cell colony PCR to verify electrophoresis detection result, wherein swimming lane 1-6 is colony, and M is Marker
  • Fig. 18 is the electrophoresis detection result of selecting No. 1-6 colony in Fig. 17 for restriction enzyme digestion of Example 4, wherein left 1-6 are plasmid pS5E1 NcoI single restriction digestion, right 1-6 are plasmid pS5E1 PacI single restriction restriction digestion, M is 15000bp Marker
  • Fig. 19 is the IRES fragment PCR amplification electrophoresis detection result of embodiment 4, wherein swimming lane 1, 2 are IRES fragment PCR amplification products, M is 15000bp Marker
  • Fig. 20 is the fragment IRES of embodiment 4 and pS5E1 carrier enzyme digestion electrophoresis detection result, wherein swimming lane 1 is fragment IRES EcoRV, NotI digestion, swimming lane 2 is pS5E1 EcoRV, NotI digestion, M is 15000bp Marker
  • Figure 21 is the result of PCR verification electrophoresis detection of colonies transformed into competent cells transformed by the pS5E1 vector and the IRES fragment ligation product of Example 4, wherein Nos. 1-9 are colonies, and M is Marker
  • Figure 22 is the verification of pS5E1-IRES plasmid NotI and EcoRV digestion and electrophoresis detection of Example 4, select 2 and 6 of Figure 21 to carry out plasmid extraction, and digestion verification, wherein No. 1 swimming lane is No. 2 plasmid NotI, EcoRV digestion identification, Lane 2 is identification of plasmid No. 6 NotI and EcoRV digestion
  • Figure 23 is the P72 and pS5E1-IRES vector restriction electrophoresis detection result of embodiment 4, wherein swimming lane 1 is fragment pS5E1-IRES, NotI digestion, swimming lane 2 is P72, NotI digestion, M is 15000bp Marker
  • Figure 24 is the result of PCR verification electrophoresis detection of colony transformation of competent cells transformed by P72 and pS5E1-IRES ligation product of Example 4, wherein Nos. 1-10 are colonies, and M is Marker
  • Figure 25 is the pS5E1-P72-IRES plasmid digestion and electrophoresis detection verification of Example 4. Select colonies 2 and 5 in Figure 24 to carry out plasmid extraction and digestion verification, wherein the No. 2 swimming lane is the No. 2 plasmid digestion verification, 5 Lane No. 5 is the digestion verification of plasmid No. 5, M is Marker
  • Figure 26 is the result of electrophoresis detection of fragment B602L and pS5E1-P72-IRES vector digested products of Example 4, wherein lane 1 is pS5E1-P72-IRES, digested by NotI and XhoI, and lane 2 is fragment B602L, digested by NotI and XhoI , M is 15000bp Marker
  • Fig. 27 is the result of PCR verification electrophoresis detection of colony transformation of competent cells transformed by B602L and pS5E1-P72-IRES ligation product of Example 4, wherein Nos. 1-7 are colonies, and M is Marker
  • Fig. 28 is the pS5E1-P72-IRES-B602L plasmid digestion electrophoresis detection verification of Example 4, wherein No. 1, 2, 4, 6 swimming lanes are selected from No. 1, 2, 4, 6 colony plasmids NotI, XhoI in Fig. 27 Enzyme digestion identification, M is 15000bp Marker
  • Figure 29 is the result of electrophoresis detection of backbone amplification of the pS5E4-EGFP shuttle plasmid left arm, pS5E4-EGFP shuttle plasmid right arm, EF1a-EGFP-HBV, pS5E4-EGFP shuttle plasmid of Example 5, wherein lane 1 is the pS5E4-EGFP shuttle plasmid Left arm, lane 2 is pS5E4-EGFP shuttle plasmid right arm, lane 3 is EF1 ⁇ -EGFP-HBV, lane 4 is pS5E4-EGFP shuttle plasmid backbone, M is 2000Marker
  • Figure 30 is the result of PCR verification electrophoresis detection of the four fragments of the pS5E4-EGFP shuttle plasmid left arm, pS5E4-EGFP shuttle plasmid right arm, EF1 ⁇ -EGFP-HBV, and pS5E4-EGFP shuttle plasmid backbone products transformed into competent cells of Example 5. , where lanes 1-20 are colonies, and M is Marker
  • Figure 31 is the electrophoresis detection result of selecting No. 3, 4, 5, and 6 colonies in Figure 30 to verify the digestion of Example 5, wherein 1-4 are positive clones No. 3, 4, 5, and No. 6 Pad single digestion, 5 -8 is the positive clone No. 3, 4, 5, 6 HindIII single digestion, M1, M3 is 15000bp Marker, M2 is 2000bp Marker
  • Figure 32 is the PCR amplification electrophoresis detection results of the fragments P30, P54, 2A of Example 5, wherein the swimming lane 1 is the P30 amplified fragment, the swimming lane 2 is the P54 amplified fragment, the swimming lane 3 is the 2A amplified fragment, and M1 and M2 are 2000bp Marker.
  • Figure 33 is the fragment P30-2A-P54 fusion PCR amplification electrophoresis detection result of Example 5, wherein lane 1 is the P30-2A-P54 fragment, and M is Maker
  • Figure 34 shows the detection results of fragment P30-2A-P54 and pS5E4-EGFP vector enzyme digestion electrophoresis in Example 5, wherein lanes 1 and 2 are pS5E4-EGFP, BamHI, XhoI double-enzyme digestion gel recovery, lanes 3, 4: fragment P30 -2A-P54 BamHI, XhoI double digestion gel recovery, M is 15000bp Marker
  • Fig. 35 is the pS5E4 and P30-2A-P54 fragment ligation product of embodiment 5 transformed competent cell colony PCR verification electrophoresis detection result, wherein No. 1-20 is colony, M is 2000bp Marker
  • Figure 36 is the electrophoresis detection result of picking the positive clones No. 2 and No. 19 of Figure 35 and extracting plasmids for verification by BamHI and XhoI double digestion in Example 5, wherein swimming lane 2 is the verification of No. 2 positive clone BamHI and XhoI double digestion, and swimming lane 19 It was verified by double digestion of BamHI and XhoI for the positive clone No. 19, and M is 15000bp Marker
  • Fig. 37 shows the detection results of pAd5LCL3 and pS5E1-P72-IRES-B602L agarose gel verification electrophoresis in Example 6, wherein lane 1 is pAd5LCL3, and lane 2 is pS5E1-P72-IRES-B602L
  • Figure 38 shows the electrophoresis detection results of the plasmid pAd5LCL3-P72-IRES-B602L obtained by homologous recombination of the shuttle plasmid pS5E1-P72-IRES-B602L of Example 6 and the adenovirus vector plasmid pAd5LCL3, wherein lanes 1-7 are pAd5LCL3-P72-IRES -B602L clone, M is 15000bp Marker
  • Figure 39 shows that the positive plasmid No. 1 in Figure 38 was picked and transformed into competent cells in Example 6, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 1 swimming lane was pAd5LCL3-P72-IRES-B602L plasmid XhoI digestion, No. 2 swimming lane For pAd5LCL3-P72-IRES-B602L plasmid PacI digestion, M is 15000bp Marker
  • Figure 40 is the result of verification electrophoresis of the shuttle plasmid pS5E4-P30-2A-P54 and the adenovirus vector plasmid pAd5LCL3-P72-IRES-B602L agarose gel in Example 6, wherein the lane 1 is pS5E4-P30-2A-P54, and the lane 2 is pAd5LCL3-P72-IRES-B602L, M is 15000bp Marker
  • Figure 41 shows the electrophoresis detection results of the recombinant adenovirus vector pAd5LCL3-P72-B602L-P30-P54 plasmid obtained by homologous recombination of the shuttle plasmid pS5E4-P30-2A-P54 of Example 6 and the adenovirus vector plasmid pAd5LCL3-P72-IRES-B602L , in which lanes 1-8 are colonies, and M is 15000bp Marker
  • Fig. 42 shows that the positive plasmid No. 4 of Fig. 41 was picked and transformed into competent cells in Example 6, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 1 swimming lane was pAd5LCL3-P72-B602L-P30-P54 plasmid XhoI digestion, Lane 2 is pAd5LCL3-P72-B602L-P30-P54 plasmid PacI digestion, M is 15000bp Marker
  • Figure 43 is a photo of cells 72 hours after transfection of pAd5LCL3-P72-B602L-P30-P54 plasmid TPO with 293TD37 of Example 7
  • Figure 44 is a photo of TP1 cells after 293TD37 of Example 7 was infected with TP0
  • Figure 45 is a photo of TP2 cells after 293TD37 of Example 7 was infected with TP1
  • Figure 46 is a photo of TP3 cells after 293TD37 of Example 7 was infected with TP2
  • Figure 47 is a photograph of TP4293TD37 cell lesions caused by TP4 of Example 7.
  • Figure 48 is a schematic diagram of the results of Western Blot detection of P30 protein in the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-P72-B602L-P30-P54 in Example 11
  • Figure 49 is a comparison of the cytotoxic T cell (CTL) killing experiments induced by the African swine fever multi-antigen recombinant adenovirus pAd5LCL3-P72-B602L-P30-P54, the non-related antigen pAd5-FMDO adenovirus group and the normal saline group in Example 12 result graph
  • Figure 50 is the vector map of pAd5LCL3
  • Figure 51 is the vector map of pS5E1
  • Figure 52 is the vector map of pS5E1-P72-IRES-B602L
  • Figure 53 is the vector map of pS5E4-EGFP
  • Figure 54 is the vector map of pS5E4-P30-2A-P54
  • Figure 55 is a vector map of pAd5LCL3-P72-B602L-P30-P54
  • Figure 56 is a schematic diagram of the expression results of P54 and P72 in the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-P72-B602L-P30-P54 detected by Western Blot in Example 11, wherein M, pre-stained with Makker; lane 1, P54 antibody serum; Lane 2, P72 antibody serum; Lane 3: 293TD37 cell control
  • Figure 57 is a schematic diagram of the titer of IgG antibodies against African swine fever target proteins P72 and P30 in serum detected by indirect ELISA in Example 12 (ns, P ⁇ 0.05; *, P ⁇ 0.05; **, P ⁇ 0.01; * **,P ⁇ 0.001;****,P ⁇ 0.0001), the left picture is the IgG antibody titer of protein P72, the right picture is the IgG antibody titer of P30
  • Figure 58 is a schematic diagram of the CD8+ T cell response induced by the African swine fever recombinant adenovirus vaccine Ad5LCL3-P72-B602L-P30-P54 in Example 12
  • Figure 59 is a schematic diagram of the CD4+ T cell response induced by the African swine fever recombinant adenovirus vaccine Ad5LCL3-P72-B602L-P30-P54 in Example 12
  • Figure 60 is a representative graph of the cellular immune response induced by the African swine fever recombinant adenovirus vaccine Ad5LCL3-P72-B602L-P30-P54 in Example 12
  • Figure 61 is a representative graph of the immune response of the blank control in Example 12
  • Fig. 62 is the IRES fragment PCR amplification electrophoresis detection result of embodiment 14, wherein swimming lane 1, 2 are IRES fragment PCR amplification products, M is 15000bp Marker
  • Figure 63 is the fragment IRES and pS5E1 vector restriction enzyme digestion electrophoresis detection results of Example 14, wherein swimming lane 1 is fragment IRES EcoRV, NotI digestion, swimming lane 2 is pS5E1 EcoRV, NotI digestion, M is 15000bp Marker
  • Figure 64 is the result of PCR verification electrophoresis detection of colony transformation of competent cells transformed by pS5E1 vector and IRES fragment ligation product of Example 14, wherein Nos. 1-9 are colonies, and M is Marker
  • Figure 65 shows the verification of pS5E1-IRES plasmids NotI and EcoRV enzyme digestion and electrophoresis in Example 14. Select 2 and 6 of Figure 64 to carry out plasmid extraction, and enzyme digestion verification, wherein No. 1 swimming lane is No. 2 plasmid NotI and EcoRV enzyme digestion identification, Lane 2 is identification of plasmid No. 6 NotI and EcoRV digestion
  • Figure 66 shows the detection results of MGF5L6L and pS5E1-IRES vector digestion electrophoresis in Example 14, wherein lane 1 is pS5E1-IRES, NotI and XhoI double digestion, lane 2 is fragment MGF5L6L, NotI and XhoI double digestion, M is 15000bp Marker
  • Figure 67 is the result of PCR verification electrophoresis of the transformed competent cell colony of MGF5L6L and pS5E1-IRES ligation product of Example 14, wherein Nos. 1-12 are colonies, and M is a 2000bp Marker
  • Figure 68 is the pS5E1-IRES-MGF5L6L plasmid digestion and electrophoresis detection verification of Example 14. Select colonies 2, 9, and 11 in Figure 67 for plasmid extraction and digestion verification, wherein the No. 2 swimming lane is the No. 2 plasmid digestion verification. , swimming lane 9 is the digestion verification of plasmid No. 9, lane 11 is the digestion verification of plasmid No. 11, M is Marker
  • Figure 69 shows the electrophoresis detection results of the fragment CP129Rubiqutin of Example 14 and the digested product of the pS5E1-IRES-MGF5L6L vector, wherein lane 1 is the pS5E1-IRES-MGF5L6L plasmid, digested by EcoRV and BamHI, and lane 2 is the C129 Rubiqutin fragment, EcoRV and BamHI enzymes Cut, M is 15000bp Marker, 2000bp Marker
  • Figure 70 is the result of PCR electrophoresis verification of the transformation of the ligation product of pS5E1-IRES-MGF5L6L and CP129 Rubiqutin into competent cells of Example 14, wherein Nos. 1-5 are colonies, and M is a 2000bp Marker
  • Figure 71 is the verification of pS5E1-CP129Rubiqutin-IRES-MGF5L6L plasmid digestion and electrophoresis in Example 14, wherein the No. 1 and No. 2 swimming lanes are selected from the No. 1 and No. 2 colony plasmids BamHI and EcoRV digestion and identification in Figure 70, and M is 2000bp Marker
  • Figure 72 shows the results of electrophoresis detection of backbone amplification of the pS5E4-EGFP shuttle plasmid left arm, pS5E4-EGFP shuttle plasmid right arm, EF1 ⁇ -EGFP-HBV and pS5E4-EGFP shuttle plasmids of Example 15, wherein lane 1 is the pS5E4-EGFP shuttle plasmid Left arm, lane 2 is pS5E4-EGFP shuttle plasmid right arm, lane 3 is EF1 ⁇ -EGFP-HBV, lane 4 is pS5E4-EGFP shuttle plasmid backbone, M is 2000Marker
  • Figure 73 shows the results of PCR verification electrophoresis detection of the four fragments of the pS5E4-EGFP shuttle plasmid left arm, pS5E4-EGFP shuttle plasmid right arm, EF1 ⁇ -EGFP-HBV, and pS5E4-EGFP shuttle plasmid backbone products transformed into competent cells of Example 15. , where lanes 1-20 are colonies, and M is Marker
  • Fig. 74 is the electrophoresis detection result of selecting No. 3, 4, 5, and 6 colonies in Fig. 30 in Example 15 and verified by enzyme digestion, wherein 1-4 are positive clones 3, 4, 5, and 6 Pad single-enzyme digestion, and 5 -8 is the positive clone No. 3, 4, 5, 6 HindIII single digestion, M1, M3 is 15000bp Marker, M2 is 2000bp Marker
  • Figure 75 shows the PCR amplification electrophoresis detection results of fragments CP312R, MGF110-4L and 2A of Example 15, wherein lane 1 is the amplified fragment of CP312R, lane 2 is the amplified fragment of 2A, and lane 3 is the amplified fragment of MGF110-4L, M is 2000bp Marker
  • Figure 76 is the result of electrophoresis detection of fragment CP312R-2A-MGF110-4L fusion PCR amplification of Example 15, wherein lane 1 is CP312R-2A-MGF110-4L fragment, M is 2000bp Marker
  • Figure 77 shows the results of electrophoresis detection of fragment CP312R-2A-MGF110-4L and pS5E4-EGFP vector of Example 15, wherein lane 1 is gel recovery of fragment CP312R-2A-MGF110-4L, lane 2 is fragment pS5E4-EGFP, BamHI And XhoI double enzyme digestion gel recovery, M is 15000bp Marker
  • Figure 78 is the result of PCR verification electrophoresis detection of the transformed competent cell colony of pS5E4 and CP312R-2A-MGF110-4L fragment ligation product of Example 15, wherein Nos. 1-12 are colonies, and M is a 15000bp Marker
  • Figure 79 is the electrophoresis detection result of picking positive clones No. 1, 2, 3, and 4 of Figure 78 to extract plasmids and verifying double digestion with BamHI and XhoI in Example 15. Lanes 1, 2, 3, and 4 are respectively 1 and 2. , 3, 4 positive clones BamHI, XhoI double digestion verification, M is 15000bp Marker
  • Figure 80 is the result of electrophoresis verification of pAd5LCL3 and pS5E1-CP129Rubiqutin-IRES-MGF5L6L agarose gel of Example 16, wherein lane 1 is pS5E1-CP129Rubiqutin-IRES-MGF5L6L, and lane 2 is pAd5LCL3
  • Figure 81 shows the electrophoresis detection results of the plasmid pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L obtained by homologous recombination of the shuttle plasmid pS5E1-C129Rubiqutin-IRES-MGF5L6L and the adenovirus vector plasmid pAd5LCL3 in Example 16, wherein lanes 1-5 are pAd5LCL3-C129Rubiqutin-IRES -MGF5L6L clone, M is 15000bp Marker
  • Figure 82 shows that in Example 16, the positive plasmid No. 4 in Figure 81 was picked and transformed into competent cells, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 1 and No. 2 swimming lanes were pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L plasmid XhoI digestion, M is 15000bp Marker
  • Figure 83 is the result of electrophoresis verification of the shuttle plasmid pS5E4-CP312R-2A-MGF110-4L and the adenovirus vector plasmid pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L agarose gel in Example 16, wherein lane 1 is pS5E4-CP312R-2A-MGF110 -4L, lane 2 is pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L, M is 15000bp Marker
  • Figure 84 shows the homologous recombination of the shuttle plasmid pS5E4-CP312R-2A-MGF110-4L and the adenovirus vector plasmid pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L of Example 16 to obtain the recombinant adenovirus vector pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L plasmid
  • Fig. 85 shows that the positive plasmid No. 3 in Fig. 84 was picked and transformed into competent cells in Example 16, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 1 swimming lane was pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L plasmid XhoI enzyme Cut, lane 2 is pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L plasmid PacI digestion, M is 15000bp Marker
  • Figure 86 is a photograph of 293TD37 cells induced by TPO of Example 17
  • Figure 87 is a photograph of 293TD37 cells induced by TP1 of Example 17
  • Figure 88 is a photograph of 293TD37 cells induced by TP2 of Example 17
  • Figure 89 is a photograph of 293TD37 cells induced by TP3 of Example 17
  • Figure 91 is a schematic diagram of the results of Western Blot detection of the CP312R protein in the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L in Example 21
  • Figure 92 is a vector map of pS5E1-C129Rubiqutin-IRES-MGF5L6L
  • Figure 93 is the vector map of pS5E4-EGFP
  • Figure 94 is the vector map of pS5E4-CP312R-2A-MGF110-4L
  • Figure 95 is a vector map of pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L
  • Figure 96 is a schematic diagram showing the results of CD8+ T cell response induced by pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L of Example 22
  • Figure 97 is a schematic diagram showing the results of CD4+ T cell response induced by pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L of Example 22
  • Figure 98 is a representative graph of the cellular immune response after intramuscular injection of pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L in Example 22
  • Figure 99 is a representative graph of the blank control immune response of Example 22
  • Figure 100 is the detection result of I215L and pS5E1-IRES vector enzyme digestion electrophoresis of Example 23, wherein the lane vector is pS5E1-IRES, NotI and XhoI double digestion, the swimming lane I215L is fragment I215L, NotI and XhoI double digestion, M is 15000bp , 2000bp Marker
  • Figure 101 is the result of PCR electrophoresis detection of the transformed competent cell colony of I215L and pS5E1-IRES ligation product of Example 23, wherein Nos. 1-11 are colonies, and M is a 2000bp Marker
  • Figure 102 is the pS5E1-IRES-I215L plasmid enzyme digestion and electrophoresis detection verification of Example 23, selecting colonies 5, 6, 7, and 8 in Figure 101 to carry out plasmid extraction, and enzyme digestion verification, wherein the No. 2 swimming lane is the No. 2 plasmid enzyme Digestion verification, lane 9 is plasmid digestion verification, lane 11 is plasmid digestion verification, M is Marker
  • Figure 103 is the electrophoresis detection result of the L8Lubiqutin fragment after fusion of L8L and ubiqutin of Example 23 and purified by the gel recovery and purification kit, wherein lane 1 is the L8Lubiqutin fragment, and M is Marker
  • Figure 104 is the electrophoresis detection result of the fragment L8Lubiqutin of Example 23 and the pS5E1-IRES-I215L vector digestion product, wherein lane 1 is the pS5E1-IRES-I215L plasmid; lane 2 is the pS5E1-IRES-I215L plasmid EcoRV and BamHI digestion; 3 is L8Lubiqutin fragment EcoRV and BamHI digestion, M is 15000bp Marker
  • Figure 105 is the result of PCR electrophoresis detection of the transformed competent cell colony with the pS5E1-IRES-I215L vector of Example 23 and the L8Lubiqutin ligation product, wherein Nos. 1-24 are colonies, and M is a 2000bp Marker
  • Fig. 106 is the pS5E1-L8Lubiqutin-IRES-I215L plasmid digestion electrophoresis detection verification of Example 23, wherein No. 4, 6, 9, 14, 17, 18 lanes are selected from 4, 6, 9, 14, 17 in Fig. 105 , No. 18 colony plasmids BamHI, EcoRV digestion identification, M is 15000bp Marker
  • Figure 107 is the electrophoresis detection result of the fusion PCR amplification of I73Rhbsag and 2A-E146L fragment of Example 24, wherein swimming lane 1 is the I73Rhbsag fragment, swimming lane 2 is the 2A-E146L fragment, and M is the 2000bp Marker
  • Figure 108 is the detection result of fragment pS5E4-EGFP vector restriction electrophoresis of Example 24, wherein lane 1 is fragment pS5E4-EGFP, BamHI and XhoI double restriction gel recovery, M is 15000bp Marker
  • Figure 109 is the pS5E4-EGFP gel recovery vector of Example 24 and the I73Rhbsag fragment, 2A-E146L seamless clone connection and transformed competent cell colony PCR verification electrophoresis detection results, wherein Nos. 1-12 are colonies, M is 15000bp Marker
  • Figure 110 is the electrophoresis detection result of picking the positive clones No. 1, 2, and 3 of Figure 109 to extract plasmids and verifying double digestion with BamHI and XhoI in Example 24. Lanes 1, 2, and 3 are positive for No. 1, 2, and 3, respectively. Cloning BamHI, XhoI double digestion verification, M is 15000bp Marker
  • Figure 111 is the result of electrophoresis verification of pAd5LCL3 and pS5E1-L8Lubiqutin-IRES-I215L agarose gel of Example 25, wherein lane 1 is pAd5LCL3, and lane 2 is pS5E1-L8Lubiqutin-IRES-I215L
  • Figure 112 shows the electrophoresis detection results of the plasmid pAd5LCL3-L8Lubiqutin-IRES-I215L obtained by homologous recombination of the shuttle plasmid pS5E1-L8Lubiqutin-IRES-I215L and the adenovirus vector plasmid pAd5LCL3 in Example 25, wherein lanes 1-7 are pAd5LCL3-L8Lubiqutin-IRES -I215L clone, M is 15000bp Marker
  • Figure 113 shows that the positive plasmid No. 6 in Figure 112 was picked and transformed into competent cells in Example 25, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 1 swimming lane was pAd5LCL3-P72-IRES-B602L plasmid XhoI digestion, and M was 15000bp Marker
  • Figure 114 is the result of electrophoresis verification of the shuttle plasmid pS5E4-I73Rhbsag-2A-E146L and the adenovirus vector plasmid pAd5LCL3-L8Lubiqutin-IRES-I215L agarose gel in Example 25, wherein lane 1 is pS5E4-I73Rhbsag-2A-E146L, lane 2 is pAd5LCL3-L8Lubiqutin-IRES-I215L, M is 15000bp Marker
  • Figure 115 is the electrophoresis detection result of the recombinant adenovirus vector pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L plasmid obtained by homologous recombination of the shuttle plasmid pS5E4-I73Rhbsag-2A-E146L of Example 25 and the adenovirus vector plasmid pAd5LCL3-L8Lubiqutin-IRES-I215L , in which lanes 1-8 are plasmids, and M is 15000bp Marker
  • Fig. 116 shows that the positive plasmid No. 2 of Fig. 115 was picked and transformed into competent cells in Example 25, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 2 swimming lane was pAd5LCL3-L8Lubiqutin-I215L-I73HbsAg-E146L plasmid XhoI digestion, M is 15000bp Marker
  • Figure 117 is a photograph of TP4293TD37 cytopathic lesions caused by TP0 of Example 26
  • Figure 118 is a photograph of TP4293TD37 cytopathic lesions caused by TP1 of Example 26
  • Figure 119 is a photograph of TP4293TD37 cytopathies caused by TP2 of Example 26
  • Figure 120 is a photo of TP4293TD37 cell lesions caused by TP3 of Example 26
  • Figure 121 is a photograph of TP4293TD37 cytopathic lesions caused by TP4 of Example 26
  • Figure 122 is a schematic diagram showing the results of Western Blot detecting the expression of L8Lubiqutin and I215L proteins in the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-L8Lubiqutin-I215L-I73HbsAg-E146L in Example 29, wherein lane 1 is 293 blank cells, and lanes 2 and 3 are 293TD37 Samples of cells infected with pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L, M is Marker
  • Figure 123 is the vector map of pS5E1-L8Lubiqutin-IRES-I215L
  • Figure 124 is the vector map of pS5E4-I73Rhbsag-2A-E146L
  • Figure 125 is a vector map of pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF4L
  • Figure 126 is a schematic diagram of the results of CD8+ T cell response induced by pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L of Example 30
  • Figure 127 is a schematic diagram showing the results of CD4+ T cell response induced by pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L of Example 30
  • Figure 128 is a representative graph of the cellular immune response after intramuscular injection of pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L in Example 31
  • Figure 129 is a representative graph of the blank control immune response of Example 31
  • Figure 130 is the EP402R and pS5E1-IRES ligation product of Example 32 transformed into competent cell colony PCR verification electrophoresis detection results, wherein Nos. 1-6 are colonies, and M is a 2000bp Marker
  • Figure 131 is the pS5E1-EP402R-IRES plasmid digestion and electrophoresis detection verification of Example 32. Select colonies 1, 2, and 4 in Figure 130 for plasmid extraction and digestion verification, wherein the No. 1 swimming lane is the No. 1 plasmid digestion verification. , Lane 2 is the digestion verification of plasmid No. 2, lane 4 is the digestion verification of plasmid No. 4, M is Marker
  • Figure 132 is the result of electrophoresis detection of the digested products of pS5E1-EP402R-IRES vector and EP153R vector of Example 32, wherein lane 1 is pS5E1-EP402R-IRES, digested by NotI and XhoI, and lane 2 is EP153R fragment, digested by NotI and XhoI , M is 2000bp Marker
  • Figure 133 is the result of PCR verification electrophoresis of the transformed competent cell colony of pS5E1-EP402R-IRES vector and EP153R ligation product of Example 32, wherein Nos. 1-14 are colonies, and M is a 5000bp Marker
  • Figure 134 is the verification of pS5E1-F317L-IRES-A151R plasmid digestion and electrophoresis detection in Example 32, wherein lanes 1, 2, 3, 5, 11, and 13 are selected from 1, 2, 3, 5, and 11 in Figure 27. , No. 13 colony plasmids BamHI, EcoRV digestion identification, M is 15000bp Marker
  • Figure 135 is the electrophoresis detection result of the fusion PCR amplification of I177L-2A and K205 Rubiqutin fragments of Example 33, lane 1 is fragment K205R; lane 2 is fragment ubiqutin; lane 3 is fragment K205 Rubiqutin, M is 2000bp Marker; lane 4 is fragment 2A ; swimming lane 5 is fragment I177L; swimming lane 6 is fragment I177L-2A, M is 2000bp Marker
  • Figure 136 is the detection result of fragment pS5E4-EGFP vector digestion and electrophoresis of Example 33, wherein lane 1 is fragment pS5E4-EGFP, BamHI and XhoI double digestion gel recovery, M is 15000bp Marker
  • Figure 137 is the pS5E4-EGFP gel recovery vector of Example 33 and the I177L-2A fragment, K205 Rubiqutin seamless clone connection and transformed competent cell colony PCR verification electrophoresis detection results, wherein Nos. 1-3 are colonies, M is 15000bp Marker
  • Figure 138 is the electrophoresis detection result of picking the positive clones No. 1, 2, and 3 of Figure 137 to extract plasmids and verifying double-enzyme digestion with BamHI and XhoI in Example 33, wherein lanes 1, 2, and 3 are positive for No. 1, 2, and 3, respectively. Cloning BamHI, XhoI double digestion verification, M is 15000bp Marker
  • Figure 139 is the result of electrophoresis verification of pAd5LCL3 and pS5E1-EP402R-IRES-EP153R agarose gel of Example 34, wherein lane 1 is pAd5LCL3, and lane 2 is pS5E1-EP402R-IRES-EP153R
  • Figure 140 shows the electrophoresis detection results of the plasmid pAd5LCL3-EP402R-IRES-EP153R obtained by homologous recombination of the shuttle plasmid pS5E1-EP402R-IRES-EP153R and the adenovirus vector plasmid pAd5LCL3 of Example 34, wherein lanes 1-8 are pAd5LCL3-EP402R-IRES -EP153R clone, M is 15000bp Marker
  • Figure 141 shows that the positive plasmid No. 2 in Figure 140 was picked and transformed into competent cells in Example 34, and the plasmid was extracted and digested to verify the detection results, wherein the No. 1 swimming lane was pAd5LCL3-EP402R-IRES-EP153R plasmid XhoI digestion, No. 2 swimming lane For pAd5LCL3-EP402R-IRES-EP153R plasmid PacI digestion, lane 3 for pAd5LCL3-EP402R-IRES-EP153R plasmid BamHI digestion, M is 15000bp Marker
  • Figure 142 shows the result of electrophoresis detection of recombinant adenovirus vector pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin plasmid obtained by homologous recombination of shuttle plasmid pS5E4-I177L-2A-K205Rubiqutin and adenovirus vector plasmid pAd5LCL3-EP402R-IRES-EP153R in Example 34 , where lanes 1-7 are plasmids, and M is 15000bp Marker
  • Fig. 143 shows that the positive plasmid No. 1 of Fig. 142 was picked and transformed into competent cells in Example 34, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein lanes 1 and 2 were pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin plasmid XhoI digestion , lane 3 is pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin plasmid BamHI digestion, lane 4 is pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin plasmid PacI digestion, M is 15000Marker
  • Figure 144 is a photograph of TP4293TD37 cell lesions caused by TP0 of Example 35
  • Figure 145 is a photograph of TP4293TD37 cell lesions caused by TP1 of Example 35
  • Figure 146 is a photograph of TP4293TD37 cell lesions caused by TP2 of Example 35
  • Figure 147 is a photograph of TP4293TD37 cell lesions caused by TP3 of Example 35
  • Figure 148 is a photograph of TP4293TD37 cell lesions caused by TP4 of Example 35
  • Figure 149 is a schematic diagram of the results of Western Blot detection of EP153R protein in the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-EP402R-EP153R-K205R-I177Lubiqutin in Example 39
  • Figure 150 is a vector map of pS5E1-EP402R-IRES-EP153R
  • Figure 151 is the vector map of pS5E4-I177L-2A-K205Rubiqutin
  • Figure 152 is a vector map of pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin
  • Figure 153 is a schematic diagram showing the titer of IgG antibodies against African swine fever target protein EP402R in serum detected by the ELISA method of Example 40
  • Figure 154 is a schematic diagram showing the results of CD8+ T cell response induced by pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin in Example 40
  • Figure 155 is a schematic diagram of the results of CD4+ T cell response induced by pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin in Example 40
  • Figure 156 is a representative graph of the cellular immune response after intramuscular injection of pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin in Example 40
  • Figure 157 is a representative graph of the blank control immune response of Example 40
  • Figure 158 shows the results of enzyme digestion electrophoresis detection of F317L and pS5E1-IRES vector in Example 41, wherein lane 1 is pS5E1-IRES, double digestion with BamHI and EcorV, and lane 2 is fragment F317L, double digestion with BamHI and EcoRV, and M is 15000bp Marker
  • Figure 159 is the result of PCR electrophoresis detection of the transformed competent cell colony of F317L and pS5E1-IRES ligation product of Example 41, wherein Nos. 1-14 are colonies, and M is a 2000bp Marker
  • Figure 160 is the pS5E1-F317L-IRES plasmid digestion and electrophoresis detection verification of Example 41. Select colonies No. 9 and 10 in Figure 159 for plasmid extraction and digestion verification, wherein the No. 1 swimming lane is the No. 1 plasmid digestion verification, 2 Lane No. 9 is the digestion verification of plasmid No. 9, M is Marker
  • Figure 161 shows the results of electrophoresis detection of the fragment A151R and the pS5E1-F317L-IRES vector digested product of Example 41, wherein lane 1 is pS5E1-F317L-IRES, digested with NotI and XhoI; lanes 2 and 3 are the A151R fragment, NotI and XhoI Enzyme digestion, M is 15000bp Marker
  • Figure 162 is the result of PCR verification electrophoresis of the transformed competent cell colony with the pS5E1-F317L-IRES vector of Example 41 and the A151R ligation product, wherein Nos. 1-24 are colonies, and M is a 2000bp Marker
  • Figure 163 shows the verification of pS5E1-F317L-IRES-A151R plasmid digestion and electrophoresis in Example 41, wherein lanes 1, 2, 3, and 4 are the colony plasmids BamHI, EcoRV of No. 4, 15, 23, and 24 in Figure 162 Enzyme digestion identification, M is 2000bp Marker
  • Figure 164 is the PCR product electrophoresis detection result of the target fragments P34, 2A, pp62 of embodiment 42, wherein swimming lane 1 is fragment p34; 2 is fragment 2A; 3 is fragment pp62, M is 15000bp Marker
  • Figure 165 is the electrophoresis detection result of the fusion PCR amplification of the P34-2A fragment of Example 42, wherein the swimming lane 1 is the P34-2A fragment, and M is the 2000bp Marker
  • Figure 166 is the detection result of fragment pS5E4-EGFP vector digestion and electrophoresis of Example 42, wherein lane 1 is fragment pS5E4-EGFP, BamHI and XhoI double digestion gel recovery, M is 15000bp Marker
  • Figure 167 is the pS5E4-EGFP gel recovery vector of Example 42 and the P34-2A fragment, pp62 seamless clone connection and transformed competent cell colony PCR verification electrophoresis detection results, wherein No. 1-12 are colonies, M is 15000bp Marker
  • Figure 168 is the electrophoresis detection result of picking the positive clones No. 1, 2, 9, and 11 of Figure 167 to extract plasmids in Example 42 for verification by BamHI and XhoI double digestion, wherein lanes 1, 2, 3, and 4 are respectively 1 and 2 , 9, 11 positive clones BamHI, XhoI double digestion verification, M is 15000bp Marker
  • Figure 169 is the result of electrophoresis verification of pAd5LCL3 and pS5E1-F317L-IRES-A151R agarose gel of Example 43, wherein lane 1 is pAd5LCL3, lane 2 is pS5E1-F317L-IRES-A151R, and M is 15000bp Marker
  • Figure 170 shows the electrophoresis detection results of the plasmid pAd5LCL3-F317L-IRES-A151R obtained by homologous recombination of the shuttle plasmid pS5E1-F317L-IRES-A151R and the adenovirus vector plasmid pAd5LCL3 of Example 43, wherein lanes 1-7 are pAd5LCL3-F317L-IRES -A151R, M is 15000bp Marker
  • Figure 171 shows that the positive plasmid No. 3 in Figure 170 was picked and transformed into competent cells in Example 43, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results.
  • Lanes 1 and 2 were pAd5LCL3-P72-IRES-B602L plasmid XhoI digestion, M is 15000bp Marker
  • Figure 172 is the result of electrophoresis verification of the shuttle plasmid pS5E4-P34-2A-pp62 and the adenovirus vector plasmid pAd5LCL3-F317L-IRES-A151R agarose gel in Example 43, wherein the lane 1 is pS5E4-P34-2A-pp62, the lane 2 is pAd5LCL3-F317L-IRES-A151R, M is 15000bp Marker
  • Figure 173 is the electrophoresis detection result of the recombinant adenovirus vector pAd5LCL3-F317L-A151R-P34-pp62 plasmid obtained by homologous recombination of the shuttle plasmid pS5E4-P34-2A-pp62 of Example 43 and the adenovirus vector plasmid pAd5LCL3-F317L-IRES-A151R , in which lanes 1-4 are plasmids, and M is 15000bp Marker
  • Figure 174 shows that the positive plasmid No. 2 in Figure 173 was picked and transformed into competent cells in Example 43, and the plasmid was extracted and subjected to enzyme digestion to verify the detection results, wherein the No. 2 swimming lane was pAd5LCL3-F317L-A151R-P34-pp62 plasmid XhoI digestion, M is 15000bp Marker
  • Figure 175 is a photograph of TP4293TD37 cell lesions caused by TP0 of Example 44
  • Figure 176 is a photograph of TP4293TD37 cell lesions caused by TP1 of Example 44
  • Fig. 177 is a photograph of TP4293TD37 cell lesions caused by TP2 of Example 44
  • Figure 178 is a photograph of TP4293TD37 cell lesions caused by TP3 of Example 44
  • Figure 179 is a photograph of TP4293TD37 cell lesions caused by TP4 of Example 44
  • Figure 180 is a schematic diagram of the results of Western Blot detection of pp62 protein in the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-F317L-A151R-P34-pp62 in Example 48
  • Figure 181 is the vector map of pS5E1-F317L-IRES-A151R
  • Figure 182 is a vector map of pS5E4-P34-2A-pp62
  • Figure 183 is a vector map of pAd5LCL3-F317L-A151R-P34-pp62
  • Figure 184 is a schematic diagram showing the titer of IgG antibodies against African swine fever target protein pp62 in serum detected by the ELISA method of Example 49
  • Figure 185 is a schematic diagram showing the results of CD8+ T cell response induced by pAd5LCL3-F317L-A151R-P34-pp62 of Example 49
  • Figure 186 is a schematic diagram showing the results of CD4+ T cell response induced by pAd5LCL3-F317L-A151R-P34-pp62 of Example 49
  • Figure 187 is a representative graph of the cellular immune response after intramuscular injection of pAd5LCL3-F317L-A151R-P34-pp62 in Example 49
  • Figure 188 is a representative graph of the blank control immune response of Example 49
  • Amplification of wild-type human adenovirus type 5 ( VR-1516, gene sequence AC_000008.1) virus, collect and concentrate the virus solution, extract the adenovirus genome by HirtVirual DNA Extract method, use the cosmid method to construct the linear hAd5 genome into a circular supercos-Ad5 vector plasmid, use CRISPR/cas9 excises the E1 region of hAd5 adenovirus, and the designed gRNA is as follows:
  • hAd5-E1 upstream gRNA hAd5-E1 upstream gRNA
  • gRNA sites upstream and downstream of hAd5E1 region recover large fragment vector after cleavage, design primers, insert ITR and PIX sequences into upstream and downstream respectively by fusion PCR, and introduce SwaI restriction site, and then combine the fused fragment with The vector was seamlessly cloned to obtain an E1 knockout supercos-Ad5 ⁇ E1 adenovirus vector, and then the E3 region of the supercos-Ad5 ⁇ E1 plasmid was excised.
  • the gRNA was designed as follows:
  • hAd5-E3 upstream gRNA hAd5-E3 upstream gRNA
  • Design gRNA sites in the upstream and downstream of hAd5E3 region recover large fragment vector after cutting, design primers, perform fusion PCR on the excessively excised Fiber and pVIII sequences upstream and downstream of E3, and use seamless cloning to connect to obtain deletion E1 and E3 gene, and introduced the adenovirus vector plasmid pAd5 of SwaI restriction site.
  • Using the vector plasmid pAd5 obtained in Example 1 that has knocked out the E1 and E3 genes, and further knocking out the E4 gene, can increase the capacity of the adenovirus vector, reduce its immunogenicity, and use the PCR method to amplify part of the fiber and Introduce a single NdeI restriction site, and then use Gibson's seamless cloning method to connect the excess excised fragments to the vector to obtain a vector plasmid that lacks the E1, E3 and E4 genes, and introduces SwaI and I-sceI restriction sites.
  • Thermo Fisher's GeneArt TM CRISPR Search and Design tool (thermofisher.com/crisprdesign) software, input the first 400 bases of the fiber gene, the software automatically analyzes the 400-base sequence, providing 6 potential CRISPR target sequences .
  • GCTACTAAACAATTCCTTCC was selected as the targeting sequence, and the final gRNA was named Ad5-E4-up-gRNA.
  • the cleavage site and PAM site are shown in Figure 1 .
  • Thermo Fisher GeneArt TM CRISPR Search and Design tool (thermofisher.com/crisprdesign) software, input 300 bases downstream of E4, the software automatically analyzes, provides 6 potential CRISPR target sequences, select AGGTTCGCGTGCGGTTTTCT as the target sequence, The finally obtained gRNA was named Ad5-E4-down-gRNA, and the cleavage site and PAM site are shown in Figure 2.
  • the upstream and downstream primers were designed to amplify the DNA template of Ad5-E4-up-gRNA and the DNA template of Ad5-E4-down-gRNA by PCR respectively, and the amplification was performed using the GeneArt TM Precision gRNA Synthesis Kit.
  • Ad5-E4-up-gRNA-Forward TAATACGACTCACTATAGTACTAAACAATTCCT
  • Ad5-E4-up-gRNA-Reverse TTCTAGCTCTAAAACGGAAGGAATTGTTTAGTA
  • Ad5-E4-down-gRNA-Forward TAATACGACTCACTATAGGTTCGCGTGCGGTTT
  • Ad5-E4-down-gRNA-Reverse TTCTAGCTCTAAAACAGAAAACCGCACGCGAAC
  • the PCR reaction system for DNA template amplification of Ad5-E4-up-gRNA is: Phusion TM High-Fidelity PCR Master Mix (2X) 12.5ul, Tracr Fragment+T7 Primer Mix 1ul, 0.3 ⁇ M Ad5-E4-up-gRNA- Forward/Reverse primer mix working solution 1ul, make up to 25ul with water.
  • the PCR reaction system for DNA template amplification of Ad5-E4-down-gRNA is: Phusion TM High-Fidelity PCR Master Mix (2X) 12.5ul, Tracr Fragment+T7 Primer Mix 1ul, 0.3 ⁇ M Ad5-E4-down-gRNA- Forward/Reverse primer mix working solution 1ul, make up to 25ul with water.
  • the template DNA was transcribed in vitro using TranscriptAid TM Enzyme Mix to obtain Ad5-E4-up-gRNA and Ad5-E4-down-gRNA.
  • the reaction system for obtaining Ad5-E4-up-gRNA by in vitro transcription is: 8ul of NTP mix, 6ul of E1A-gRNA DNA template, 4ul of 5X TranscriptAidTM Reaction Buffer, and 2ul of TranscriptAidTM Enzyme Mix. After 4 hours of incubation at 37°C, 1 ul of DNase I was added and incubated at 37°C for 15 minutes.
  • the reaction system for obtaining Ad5-E4-down-gRNA by in vitro transcription is: 8ul of NTP mix, 6ul of E1B-gRNA DNA template, 4ul of 5X TranscriptAidTM Reaction Buffer, and 2ul of TranscriptAidTM Enzyme Mix. After 4 hours of incubation at 37°C, 1 ul of DNase I was added and incubated at 37°C for 15 minutes.
  • Wash Buffer1 and Wash Buffer2 are reagents in TranscriptAid TM Enzyme Mix kit.
  • the RNA sequences of Ad5-E4-up-gRNA and Ad5-E4-down-gRNA obtained by transcription are as follows:
  • Ad5-E4-up-gRNA G UACUAAACAAUUCCUUCC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
  • Ad5-E4-down-gRNA G GUUCGCGUGCGGUUUUCU GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAAAGUGGCACCGAGUCGGGUGCUUUU
  • the reaction system is 3 ⁇ g Cas9 protein, 6 ⁇ g Ad5-E4-up-gRNA, Ad5-E4-down- gRNA 6 ⁇ g, pAd5-REBP vector plasmid 3 ⁇ g, NEB buffer 3.1 5ul, supplemented with water to 50ul.
  • Lane 1 shows the results of Ad5-E4-up-gRNA, Ad5-E4-down-gRNA and cas9 "double-enzyme digestion" pAd5 vector plasmid, and a fragment with the target size of 2500bp-5000bp appeared, which shows that the digestion result is correct.
  • the vector was purified using the Axygen gel recovery kit.
  • Amplification primers :
  • Fiber-RH-F GAGTGCTACTAAACAATTCCTTCCTGGACCCAGAATATTGG
  • Fiber-ISceI-ITR-R TGGTGTTATTACCCTGTTATCCCTA GCAATTGAAAAATAAAAACACGTTG
  • the amplified sequence is:
  • the amplification system was as follows: 1 ul of 10 ⁇ M Fiber-RH-F primer; 1 ul of 10 ⁇ M Fiber-ISceI-ITR-R primer; 0.5 ul of template pAd5 (100 ng/ul); 25 ul of Q5 high-fidelity enzyme;
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 10sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • the electropherogram of the amplification result is shown in Figure 4. Lane 1 is the amplification result of the fiber partial fragment, and M is 2000maker. It can be seen that the amplification result is correct.
  • the Axygen gel recovery kit was used to purify the fragments.
  • Amplification primers :
  • ISceI-ITR-F TAGGGATAACAGGGTAAT AACACCACTCGACACGGCAC
  • ITR-RH-R GGCGTAGGTTCGCGTGCGGTTTTTCTGGGTGTTTTTTGTGGACTT
  • the amplified sequence is:
  • the amplification system is: 1 ul of 10 ⁇ M ISceI-ITR-F primer; 1 ul of 10 ⁇ M ITR-RH-R primer; 0.5 ul of template pAd5 (100ng/ul); 25 ul of Q5 high-fidelity enzyme;
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 10sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • the amplification results are shown in Figure 4.
  • Lane 2 is the amplification results of the ITR partial fragments, and M is 2000maker. It can be seen that the amplification results are correct.
  • the fragments were purified using the Axygen gel recovery kit.
  • the amplification system is: 1 ul of 10 ⁇ M Fiber-RH-F primer, 1 ul of 10 ⁇ M Fiber-ISceI-ITR-R primer, 0.5 ul of template pAd5 (100 ng/ul), 25 ul of Q5 high-fidelity enzyme, and water to 50 ul.
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 20sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • the amplification results are shown in Figure 5.
  • Lane 1 is the fusion fragment of Fiber-ITR, and M is 2000maker. It can be seen that the fusion result is correct. Fragments were purified using the Axygen gel recovery kit.
  • the ligation system is as follows: 100 ng of the gel-recovered product vector plasmid fragment, 50 ng of the gel-recovered product fiber-ITR fragment, 10 ul of Gibson premix, and water to 20 ul . Incubate for 40 minutes at 50°C.
  • Colony PCR was performed on the transformants using PCR amplification.
  • E4-cexu-F AGTGACGATTTGAGGAAGTTG
  • E4-cexu-R TCAATTGCAGAAAATTTCAAGTC
  • the reaction system was as follows: 1 ul of 10 ⁇ M E4-cexu-F primer, 1 ul of 10 ⁇ M E4-cexu-R primer, 10 ul of Q5 high-fidelity enzyme, replenished with water to 20 ul, and picked monoclonal colonies into the reaction system.
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 20sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • Agarose gel electrophoresis was performed to verify, as shown in Figure 6, except for Nos. 2, 8, 11, and 17, most of the colonies showed positive bands.
  • Thermo Fisher's GeneArt TM CRISPR Search and Design tool (thermofisher.com/crisprdesign) software, input the first 400 bases of 100k genes, the software automatically analyzes the 400 base sequences, providing 6 potential CRISPR target sequences .
  • the software Considering the length of the E2a knockout sequence and the requirements for constructing a live vector, ATAGGTGGCGTTCGTAGGCA was selected as the targeting sequence, and the final gRNA was named 100k-gRNA.
  • the cleavage site and PAM site are shown in Figure 8.
  • Thermo Fisher GeneArt TM CRISPR Search and Design tool (thermofisher.com/crisprdesign) software, input 300 bases downstream of E4, the software automatically analyzes, provides 6 potential CRISPR target sequences, select TACCCCGGTAATAAGGTTCA as the target sequence, The finally obtained gRNA was named protease-gRNA, and the cleavage site and PAM site are shown in Figure 9.
  • protease-gRNA-Foward TAATACGACTCACTATAG CCCCGGTAATAAGGT
  • the PCR reaction system for DNA template amplification of 100k-gRNA is: Phusion TM High-Fidelity PCR Master Mix (2X) 12.5ul, Tracr Fragment+T7 Primer Mix 1ul, 0.3 ⁇ M 100k-gRNA-Forward/Reverse primer mixed working solution 1ul , replenish water to 25ul.
  • the PCR reaction system for DNA template amplification of protease-gRNA is: Phusion TM High-Fidelity PCR Master Mix (2X) 12.5ul, Tracr Fragment+T7 Primer Mix 1ul, 0.3 ⁇ M protease-gRNA-Forward/Reverse primer mixed working solution 1ul , replenish water to 25ul.
  • the template DNA was transcribed in vitro using TranscriptAid TM Enzyme Mix to obtain 100k-gRNA and protease-gRNA.
  • the reaction system for obtaining 100k-gRNA by in vitro transcription is: 8ul of NTP mix, 6ul of 100k-gRNA DNA template, 4ul of 5X TranscriptAidTM Reaction Buffer, and 2ul of TranscriptAidTM Enzyme Mix. After 4 hours of incubation at 37°C, 1 ul of DNase I was added and incubated at 37°C for 15 minutes.
  • the reaction system for obtaining protease-gRNA by in vitro transcription is: NTP mix 8ul, protease-gRNA DNA template 6ul, 5X TranscriptAid TM Reaction Buffer 4ul, TranscriptAid TM Enzyme Mix 2ul. After 4 hours of incubation at 37°C, 1 ul of DNase I was added and incubated at 37°C for 15 minutes.
  • Wash Buffer1 and Wash Buffer2 are reagents in TranscriptAid TM Enzyme Mix kit.
  • the RNA sequences of 100k-gRNA and protease-gRNA obtained by transcription are as follows:
  • protease-gRNA G CCCCGGUAAUAAGGUUCA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
  • the reaction system is Cas9 protein 3 ⁇ g; 100k-gRNA 6 ⁇ g; protease-gRNA 6 ⁇ g; Example 2. 3 ⁇ g of the obtained vector plasmid; NEB buffer 3.1 5ul; add water to 50ul.
  • Lane 1 is the result of 100k-gRNA, protease-gRNA and cas9 "double-enzyme digestion" vector plasmid, and a fragment with a target size of 1000-2500bp appeared, which shows that the restriction enzyme digestion result is correct.
  • the vector was purified using the Axygen gel recovery kit.
  • the amplification system was as follows: 1 ul of 10 ⁇ M 100k-F primer; 1 ul of 10 ⁇ M 100k-ORF6/7-R primer; 0.5 ul of template pAd5 ⁇ E4 (100 ng/ul); 25 ul of Q5 high-fidelity enzyme;
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 20sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • ORF6/7-F ACTTGTATGTGTTGGGAATTGTA
  • the amplification system was: 1 ul of ORF6/7-F primer; 1 ul of 10 ⁇ M ORF6/7-R primer; 0.5 ul of template ORF6/7 expression cassette gene (100 ng/ul); 25 ul of Q5 high-fidelity enzyme;
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 10sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • Protease-R ATGGATCACAACCCCACCATGAACCTTATTACCGGGGTACCCA
  • the amplification system was as follows: 1 ul of 10 ⁇ M ORF6/7-Protease-F primer; 1 ul of 10 ⁇ M Protease-R primer; 0.5 ul of template pAd5 ⁇ E4 (100 ng/ul); 25 ul of Q5 high-fidelity enzyme;
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 10sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • the amplification system is: 10 ⁇ M 100k-F primer 1ul; 10 ⁇ M Protease-R primer 1ul; template 100k gel recovery product (50ng/ul) 1ul template E4 ORF6/7 expression sealant recovery product (50ng/ul) 1ul template E4 ORF6/ 7. 1ul of the recovery product (50ng/ul) of expression frame glue; 25ul of Q5 high-fidelity enzyme; replenish water to 50ul.
  • the PCR program was as follows: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 50sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C.
  • the amplification results are shown in Figure 12, where lane 1 is the fragment 100k, the E4 ORF6/7 expression box, and the protease fusion PCR product. It can be seen that the amplification results are correct. Fragments were purified using the Axygen gel recovery kit.
  • the ligation system is as follows: The gel recovery product is 100ng of the vector fragment after E2a knockout, Gel recovery product 100k, E4 ORF6/7 expression cassette, protease fusion PCR fragment 50ng, Gibson premix 10ul, water to 20ul. Incubate for 40 minutes at 50°C.
  • Colony PCR was performed on the transformants using PCR amplification.
  • DBP-upsteam-F GTTGGGCTCGCATGTGCCG
  • DBP-downsteam-R ACTCCCATGGATCACAACCC
  • the reaction system was: 1 ul of 10 ⁇ M DBP-upsteam-F primer, 1 ul of 10 ⁇ M DBP-downsteam-R primer, 10 ul of Q5 high-fidelity enzyme, replenished with water to 20 ul, and picked monoclonal colonies into the reaction system.
  • the PCR program was: initial denaturation at 98°C, 10sec, 1 cycle; denaturation at 98°C, 5sec; annealing at 60°C, 30sec; extension at 72°C, 20sec, 35 cycles; extension at 72°C, 5min, 1 cycle; hold at 4°C. Perform agarose gel electrophoresis to verify, as shown in Figure 13, positive bands appear at 9, 18, 21, and 24.
  • the backbone of the shuttle plasmid pS5E1 adopts basic elements such as puc origin and amp (2796bp) (the pS5E1 backbone is synthesized by Beijing Bomed Gene Technology Co., Ltd.), the partial sequence of ITR in the left arm of Ad5 (355bp), and the partial sequence of PIX and PIVa2 in the right arm (2100bp). ), and CMV-MCS (Seq ID NO.12) (944bp) SV40 early polyA (Seq ID NO.13) (160bp).
  • puc-Ad5-right arm-F TAATGCAGCTGGCTTATCGAAACGTGGAATGCGAGACCGTCT
  • Ad5-right arm-CMV-R ACACACAAGCAGGGAGCAGATACAAGGGTGGGAAAGAATATATAAG
  • CMV-SV40-R TAAACAAGTTGGGGTGGGCGAAGTGATCAGCGGGTTTAAACGGG
  • SV40-R AGAGGTCGACGGTATACAGAC
  • SV40-Ad5-left arm-F TGTCTGTATACCGTCGACCTCTCCGAAAAACACCTGGGCGAGTCTCC
  • Ad5-left arm-puc-R ACACTATAGAATACACGGAATTCTTAATTAAATCATCAATAATATACCTTATTTTG
  • pCDNA3.1(+) as template (purchased from Thermo Fisher Scientific) and CMV-F and CMV-SV40-R as primers, amplify the CMV promoter MCS fragment of pS5E1 shuttle plasmid; amplification system: pCDNA3.1(+) plasmid 50ng, 10uM CMV-F primer 1ul, 10uM CMV-SV40-R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul; PCR program: 98°C, 10s; 98°C, 5s, 60 °C, 30s, 72°C, 1min, 35 cycles; 72°C, 5min.
  • lane 1 is the CMV-MCS fragment
  • lane 2 is the SV40 earlypolyA fragment
  • M 2000Marker
  • pAd5LCL3 plasmid As template, SV40-Ad5-left arm-F and Ad5-left arm-puc-R as primers, amplify the left arm of pS5E1 shuttle plasmid, amplification system: pAd5LCL3 plasmid 50ng, 10uM SV40-Ad5- Left arm-F primer 1ul, 10uM Ad5-left arm-puc-R primer 1ul, Q5 high-fidelity enzyme 20ul, replenish water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 20s, 35 cycles; 72°C, 5min.
  • pAd5LCL3 plasmid as template, puc-Ad5-right arm-F and Ad5-right arm-CMV-R as primers, amplify the right arm of pS5E1 shuttle plasmid, amplification system: pAd5LCL3 plasmid 50ng, 10uM puc-Ad5- 1ul of right arm-F primer, 1ul of 10uM Ad5-right arm-CMV-R primer, 20ul of Q5 high-fidelity enzyme, and water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 15s, 35 cycles; 72°C, 5min.
  • lane 1 is the CMV-MCS-SV40 earlypolyA fusion fragment
  • lane 2 is PUC
  • lane 3 is the right arm of Ad5
  • lane 4 is the left arm of Ad5.
  • the fragments were purified using the Axygen gel recovery kit, and then the four fragments of pS5E1 backbone, Ad5 left arm, Ad5 right arm, and CMV-MCS-SV40 earlypolyA were connected using Bomed's seamless cloning kit.
  • the connection system was 2 ⁇ Smealess Cloning Mix 10ul, pS5E1 backbone fragment 50ng, Ad5 left arm 50ng, Ad5 right arm 50ng, CMV-MCS-SV40 polyA 50ng, add water to 20ul, and incubate at 50°C for 40 minutes to obtain the ligation product plasmid pS5E1.
  • the ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Colonies were selected for agarose gel verification, and the results were shown in Figure 17, with positive bands appearing.
  • plasmid pS5E1 PacI single digestion M is 15000bp Marker, the digestion result is correct, the human adenovirus type 5 vector E1 region shuttle plasmid pS5E1 was successfully constructed, and its vector map is shown in Figure 51.
  • IRES-EcoRV-F ccg GATATC TGTCGTCATCATCCTTATAGTCC
  • IRES-NotI-R aaatat GCGGCCGC GGTTGTGGCCATTATCATCGTG
  • Amplification system Q5 enzyme 25ul, 10uM primer IRES-EcoRV-F 1ul, 10uM primer IRES-NotI-R 1ul, template IRES template 2ul, add water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60 °C, 30s, 72°C, 20s, 35 cycles; 72°C, 5min.
  • the electrophoresis detection of the amplification results is shown in Figure 19, where lanes 1 and 2 are the PCR amplification products of the IRES fragments, and M is the 15000bp ladder. It can be seen that the amplification results are correct.
  • Enzyme digestion reaction system vector pS5E1, IRES fragment ⁇ 2ug, EcoRV and NotI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul; Reaction conditions: 37°C, 30min; 65°C, 20min inactivation; gel recovery and purification.
  • the electrophoresis detection of the digested product is shown in Figure 20, wherein lane 1 is fragment IRES EcoRV, digested by NotI, and lane 2 is pS5E1 EcoRV, digested by NotI, and M is 15000bp ladder.
  • the pS5E1 vector is ligated with the IRES fragment
  • Amplification system Q5 enzyme 10ul, 10uM primer IRES-EcoRV-F 1ul, 10uM primer IRES-NotI-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72 °C, 20s, 35 cycles; 72°C, 5min. Perform electrophoresis verification, as shown in Figure 21, where Nos. 1-9 are colonies, and M is Marker, as can be seen from Figure 21, positive bands appear in Nos. 2 and 6.
  • P72-BamHI-F cgcGGATCCgccaccATGGCCAGCGGCGGAGCTTT
  • Amplification system Q5 enzyme 25ul, 10uM primer P72-BamHI-F 1ul, 10uM primer P72-his-EcoRV-R 1ul, template P72 1ul, replenish water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 40s, 35 cycles; 72°C, 5min.
  • Enzyme digestion reaction system vector pS5E1-IRES, P72 fragment ⁇ 2ug, EcoRV and BamHI each 1ul; 10 ⁇ cutsmart buffer 5ul; replenish water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification. The electrophoresis detection of the digested products is shown in Figure 23, where lane 1 is the fragment pS5E1-IRES, NotI digested, and lane 2 is P72, NotI digested, and M is 15000bp Marker.
  • the target fragment P72 is connected to pS5E1-IRES
  • Amplification system Q5 enzyme 10ul, 10uM primer P72-BamHI-F 1ul, 10uM primer P72-his-EcoRV-R 1ul, replenish water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s , 72°C, 20s, 35 cycles; 72°C, 5min.
  • Carry out electrophoresis verification as shown in Figure 24, in which Nos. 1-10 are colonies, and M is Marker. It can be seen from Figure 24 that No. 2 and No. 5 appear positive bands.
  • Plasmid digestion verification (BamHI & EcoRV), select 2 and 5 for plasmid extraction, and enzyme digestion verification. The results are shown in Figure 25, in which plasmid No. 5 was a positive plasmid.
  • B602L-XhoI-R cggCTCGAGTCAGTGGTGGTGGTGATGGTGGGCGTAATCGGGCACGTCGT
  • Amplification system Q5 enzyme 25ul, 10uM primer B602L-NotI-F 1ul, 10uM primer B602L-XhoI-R 1ul, template P72 1ul, replenish water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C , 30s, 72°C, 40s, 35 cycles; 72°C, 5min.
  • Enzyme digestion reaction system vector pS5E1-P72-IRES, B602L fragment 2ug, NotI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification. The electrophoresis detection of the digested product is shown in Figure 26, wherein lane 1 is pS5E1-P72-IRES, digested with NotI and XhoI, and lane 2 is the B602L fragment, digested with NotI and XhoI, and M is 15000bp Marker.
  • the pS5E1-P72-IRES vector is ligated with the B602L fragment
  • Ligation system pS5E1-P72-IRES 100ng; B602L fragment 50ng; T4 DNA ligase 1ul; 10 ⁇ ligase buffer 1ul; water to 10ul. Reaction conditions: room temperature, 30 min. The ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Amplification system Q5 enzyme 10ul, 10uM primer B602L-NotI-F 1ul, 10uM primer B602L-XhoI-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72 °C, 20s, 35 cycles; 72°C, 5min; electrophoresis verification, as shown in Figure 27, where Nos. 1-7 are colonies, M is Marker, as can be seen from Figure 27, a positive band appears.
  • the backbone of the shuttle plasmid pS5E4 adopts basic elements such as puc origin and amp, the left arm ITR sequence of the Ad5E4 region (370bp), the right arm part of the fiber gene sequence (1746bp), and the EF1 ⁇ -EGFP-HBV polyA gene.
  • EF1 ⁇ -EGFP-HBV polyA gene was synthesized by Biomed.
  • Ad5E4-left arm-EF1a-R caatcccccccttttcttttaaaaAACACCACTCGACACGGCAC
  • EF1 ⁇ -Ad5E4-right arm-F GGAAAGAACCAGCTGGGGCTCTAGCAATTGAAAAATAAACACGTTGA
  • Ad5E4-right arm-puc-R TAATACGACTCACTATAGGGAGACCCAAAATGTAACCACTGTGAG
  • the PCR program was: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 40sec, 35 cycles; 72°C, 5min.
  • pAd5LCL3 As template and puc-Ad5E4-left arm-F and Ad5E4-left arm-EF1a-R as primers, amplify the left arm fragment of pS5E1 shuttle plasmid.
  • Amplification system pAd5LCL3 plasmid 50ng, 10uM puc-Ad5E4-left arm-F primer 1ul, 10uM Ad5E4-left arm-EF1a-R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 10sec, 35 cycles; 72°C, 5min.
  • pAd5LCL3 As template and EF1 ⁇ -Ad5E4-right arm-F and Ad5E4-right arm-puc-R as primers, amplify the right arm fragment of pS5E4-EGFP shuttle plasmid; amplification system: pAd5LCL3 plasmid 50ng, 10uM EF1 ⁇ - Ad5E4-right arm-F primer 1ul, 10uM Ad5E4-right arm-puc-R primer 1ul, Q5 high-fidelity enzyme 20ul; replenish water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 40sec, 35 cycles; 72°C, 5min.
  • PCR amplification of pS5E4-EGFP shuttle plasmid backbone amplification system: pS5E1 backbone plasmid 50ng, 10uM puc-F primer 1ul, 10uM puc-R primer 1ul , Q5 high-fidelity enzyme 20ul; replenish water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 1min20sec, 35 cycles; 72°C, 5min.
  • lane 1 is the left arm of the pS5E4-EGFP shuttle plasmid
  • lane 2 is the right arm of the pS5E4-EGFP shuttle plasmid
  • lane 3 is EF1 ⁇ -EGFP-HBV
  • lane 4 is pS5E4-EGFP Shuttle plasmid backbone
  • M is 2000Marker. It can be seen from Figure 29 that the amplification result is correct.
  • the left arm of pS5E4-EGFP shuttle plasmid, the right arm of pS5E4-EGFP shuttle plasmid, EF1 ⁇ -EGFP-HBV, and pS5E4-EGFP shuttle plasmid backbone were connected using the seamless cloning kit from Biomed.
  • connection system was 2 ⁇ Smealess Cloning Mix 10ul, pS5E4-EGFP shuttle plasmid left arm fragment 50ng, pS5E4-EGFP shuttle plasmid right arm fragment 50ng, EF1 ⁇ -EGFP-HBV fragment 50ng, pS5E4-EGFP shuttle plasmid backbone fragment 50ng, add water to 20ul, incubate at 50°C for 40 minutes; the ligation product was transformed into DH5 ⁇ competent cells, spread on plates containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • P30-BamHI-F cgcGGATCCGCCACC ATGGACTTCATCCTGAACATCA
  • P30-2A-R CTCCGCTTCC GGCGTAGTCGGGCACGTCGTA
  • P2A-F ACGACGTGCCCGACTACGCC GGAAGCGGAGCTACTAACTTC
  • P2A-R CTGGAAGAACTCGCTGTCCAT AGGTCCAGGGTTCTCCTCCACGT
  • 2A-P54-F CCCTGGACCT ATGGACAGCGAGTTCTTCCAG
  • P54-XhoI-R ccg CTCGAG TTAGAGGGAGTTTTTCCAGGTC
  • Amplification system P30 gene synthetic fragment 50ng, 10uM P30-BamHI-F primer 1ul, 10uM P30 -2A-R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 20sec, 35 cycles; 72°C, 5min.
  • Amplification system P54 gene synthetic fragment 50ng, 10uM 2A-P54-F primer 1ul, 10uM P54 -XhoI-R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 20sec, 35 cycles; 72°C, 5min.
  • lane 1 is the amplified fragment of P30
  • lane 2 is the amplified fragment of P54
  • lane 3 is the amplified fragment of 2A
  • M1 and M2 are 2000bp ladders.
  • Amplification system P30 gel recovery fragment 50ng, P54 gel recovery fragment 50ng, P2A gel recovery fragment 50ng, 10uM P30-BamHI-F primer 1ul, 10uM P54-XhoI-R primer 1ul, Q5 high-fidelity enzyme 25ul; water to 50ul;
  • the PCR program was: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 50sec, 35 cycles; 72°C, 5min.
  • the fusion results are shown in Figure 33, where lane 1 is the P30-2A-P54 fragment, and M is Maker.
  • Enzyme digestion reaction system vector pS5E4-EGFP, P30-2A-P54 fragment 2ug, BamHI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Axygen kit gel recovery and purification. The results of enzyme digestion are shown in Figure 34, wherein lanes 1 and 2 are pS5E4-EGFP, BamHI, XhoI double-enzyme digestion gel recovery, 3, 4 are fragment P30-2A-P54, BamHI, XhoI double enzyme digestion gel recovery, M is 15000bp Marker.
  • Ligation system pS5E4 (100ng), P30-2A-P54 fragment (50ng), 1ul of T4 DNA ligase, 1ul of 10 ⁇ ligase buffer, and water to 10ul. Reaction conditions: room temperature, 30 min. The ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Reaction conditions 37°C, 1h; 65°C, 20min inactivation.
  • lane 1 is pAd5LCL3
  • lane 2 is pS5E1-P72-IRES-B602L.
  • Reaction system enzyme digestion reaction solution 37.5ul; dephosphorylation enzyme 1ul; dephosphorylation buffer 5ul; replenish water to 50ul. Reaction conditions: 37°C, 1h; 65°C, 5min inactivation.
  • the digestion result is as follows As shown in Figure 39, lane 1 is pAd5LCL3-P72-IRES-B602L plasmid XhoI digestion, lane 2 is pAd5LCL3-P72-IRES-B602L plasmid PacI digestion, M is 15000Marker, it can be seen from Figure 39, the result of digestion Correct, the adenovirus vector plasmid pAd5LCL3-P72-IRES-B602L was successfully constructed.
  • Reaction conditions 37°C, 1h; 65°C, 20min inactivation.
  • lane 1 is pAd5LCL3
  • lane 2 is pS5E1-P72-IRES-B602L.
  • Reaction system enzyme digestion reaction solution 37.5ul; dephosphorylation enzyme 1ul; dephosphorylation buffer 5ul; replenish water to 50ul. Reaction conditions: 37°C, 1h; 65°C, 5min inactivation.
  • 1 lane is pAd5LCL3-P72-B602L-P30-P54 plasmid XhoI digestion
  • the second lane is pAd5LCL3-P72-B602L-P30-P54 plasmid PacI digestion
  • M is 15000Marker, as can be seen from Figure 42 , the result of enzyme digestion was correct, and the adenovirus vector plasmid pAd5LCL3-P72-B602L-P30-P54 was successfully constructed, and its vector map was shown in Figure 55.
  • Example 7 Packaging of recombinant adenovirus
  • Linearization of plasmid pAd5LCL3-P72-B602L-P30-P54 The plasmid to be transfected was digested with PacI, incubated at 37°C for 40min, and then inactivated at 65°C for 20min.
  • Transfection Dilute the linearized 2 ⁇ g plasmid and PEI with 100ul serum-free medium respectively; add the plasmid dilution to the PEI dilution solution, pipette 5 times or vortex for 10 seconds to mix, and incubate at room temperature for 10 minutes to form Transfection complex. During the incubation, the cell culture medium was gently aspirated from the plate, 2 mL of fresh growth medium was added, and 10 minutes later, the transfection complex was added to the cells in the fresh medium.
  • Cell culture Culture the transfected 293TD37 cells in a 37°C, 5% CO 2 incubator for 72-96 hours; 72-96 hours after viral plasmid transfection, collect the cell suspension in a 6-well plate in a 1.5ml centrifuge tube The middle is TP0.
  • Continuous inoculation freeze and thaw the collected cell suspension at -80°C for 3 times, centrifuge at 2000g at 4°C for 10 minutes, take 500ul of supernatant to infect 293TD37 cells (293TD37 cells need to be prepared one day in advance), 37°C, 5% CO 2 Incubate for 60 minutes, supplement with 2 mL of FBS medium, culture at 37°C, 5% CO 2 for 72 hours, and collect the cell suspension, namely TP1; repeat the previous steps to collect the cell suspension, namely TP2. Continue to inoculate until TP4 cells become diseased.
  • Cytopathic When the 293TD37 cells were cultured from TP0 to TP4, the cells gradually became diseased, until the 293TD37 cells were completely diseased at TP4.
  • the cytopathic conditions caused by TP0 to TP4 are shown in Figures 43-47, respectively, and TP4 has been completely diseased.
  • the cells were adhered to grow into monolayer cells, the medium was discarded, and the recombinant adenovirus was serially diluted 10-3 to 10-6 times with serum-free DMEM maintenance solution, and each dilution was inoculated into 2 wells. 250uL per well, 1 hour after infection, discard the supernatant, supplement with complete medium, and then incubate at 37°C in a 5% carbon dioxide incubator.
  • virus titer mean x 1013 x 4 x 10 (-n) .
  • the FFU of pAd5LCL3-P72-B602L-P30-P54 virus was 2 ⁇ 10 8 FFU/mL, and the titer was high.
  • 293TD37 cells To prepare 293TD37 cells, take the well-grown cells in the T75 culture flask, discard the supernatant, wash the cells with PBS, digest with 0.25% trypsin, and add 10 mL of fresh DMEM medium containing 10% fetal bovine serum to terminate the digestion, then mix by pipetting. , 293TD37 cells were seeded into 6-well plates (5 ⁇ 10 5 cells/mL, 2 mL/well), incubated at room temperature for 1 hour to make them adhere to the wall, and the degree of adherence was examined by microscopy after incubation. Infection was performed with pAd5LCL3-P72-B602L-P30-P54 virus particles at a titer of 5 MOI/well.
  • the detection method is as follows:
  • Virus infection prepare a 12-well plate of A549 cells, each well of cells is 2.5 ⁇ 10 5 /well, discard the medium, wash once with PBS, inoculate adenovirus with 1 ⁇ 10 9 VP/well/0.5ml, and infect A549 cells, wild-type adenovirus type 5 as positive control, were incubated at 37°C, 5% CO 2 for 1 h, the virus liquid was discarded, supplemented with 5% complete medium, and cultured at 37° C., 5% CO 2 for 48 h.
  • RCA (average positive cell field) ⁇ (374 field/well) ⁇ (dilution factor))/Total VPs in 0.5ml viral sample
  • Judgment standard is that the level of RCA is less than 1RCA/3 ⁇ 10 10 vp. According to statistics, the level of RCA of pAd5LCL3-P72-B602L-P30-P54 is less than 1RCA/3 ⁇ 10 10 vp, indicating that the replication-deficient pAd5LCL3-P72-B602L-P30-P54 virus prepared by the present invention can be stable in 293TD37 cells Packaging, will not convert to wild type or has a low chance of converting to wild type.
  • Example 12 Immunological evaluation of African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-P72-B602L-P30-P54 in mouse model
  • mice Twenty SPF mice (6-8 weeks old) were randomly divided into 4 groups with 5 mice in each group. Mice were immunized with pAd5LCL3-P72-B602L-P30-P54 according to the groupings shown in Table 1.
  • the injection method is: intramuscular injection in the posterior inner thigh; injection dose: 100ul.
  • Table 1 Grouping of mice in vaccine immunization detection
  • mice after intramuscular injection of pAd5LCL3-P72-B602L-P30-P54, mice can produce higher concentrations of IgG antibodies against P72 and P30 proteins.
  • the average titer of the high-dose group was above 10 5
  • the average titer of the middle-dose group was also 70,000, which was significantly different from the control group. titer of antibodies.
  • mice 10 SPF mice (6-8 weeks old) were randomly divided into 2 groups with 5 mice in each group. Mice were immunized with pAd5LCL3-P72-B602L-P30-P54 according to the groupings shown in Table 2.
  • the injection method is: intramuscular injection in the posterior inner thigh; injection dose: 100ul.
  • mice were sacrificed 14 days after immunization, spleen lymphocytes were isolated, PK15 cells transfected with shuttle plasmids pS5E1-P72-IRES-B602L and pS5E4-P30-2A-P54 were used to stimulate and culture for 6 hours, and protein secretion blockers were added to block them. cytokine secretion. After 6 hours, Fc receptors were blocked, dead cells and cell surface molecular markers were stained, and after cells were fixed and perforated, intracellular cytokines were stained. Cell surface markers include CD4 and CD8, and intracellular cytokines include IFN ⁇ and IL2. Flow cytometry (CyExpert) was used to analyze the expression levels of IFN ⁇ and IL2 in CD4+T cells and CD8+T cells stimulated with target proteins.
  • FIG. 58 The immune responses of CD8+ T cells and CD4+ T cells induced by pAd5LCL3-P72-B602L-P30-P54 are shown in Figure 58 and Figure 59, and the representative results are shown in Figure 60 and Figure 61, of which Figure 60 is the intramuscular injection of pAd5LCL3
  • Figure 60 is the intramuscular injection of pAd5LCL3
  • FIG. 61 is the representative graph of the immune response of the blank control.
  • the pAd5LCL3-P72-B602L-P30-P54 recombinant adenovirus has good immunogenicity and can induce high levels of serum IgG antibodies in mice.
  • the high-dose 1*10 ⁇ 8FFU and the middle-dose 1*10 ⁇ 7FFU induced high titers. Since the expression of P72 and B602L antigens, P30 and P54 antigens are regulated by the same expression elements, respectively, P72 and P30 serum IgG antibodies can represent the high immunogenicity of these four antigens.
  • the results of cellular immune response showed that intramuscular injection of 1*10 ⁇ 7FFU adenovirus vector vaccine could induce specific cellular immune responses in the immunized mice.
  • Example 13 Immunological evaluation of African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-P72-B602L-P30-P54 on target animals (ternary pigs)
  • African swine fever multi-antigen recombinant adenovirus pAd5LCL3-P72-B602L-P30-P54 vaccine Animal immunization: Use 1*10 ⁇ 9FFU pAd5LCL3-P72-B602L-P30-P54 vaccine to immunize ternary pigs. Four weeks later, pig blood samples were collected, serum was separated, and the immunized serum samples were tested using IDVET African swine fever detection kit. The specific immunization methods are shown in Table 3:
  • Table 3 Grouping of ternary pigs for vaccine immunization
  • S/P% (OD SAMPLE -OD NC )/(OD PC -OD NC )*100
  • the average net OD of the positive control is greater than 0.350; OD PC >0.350
  • the experimental results show that the recombinant adenovirus pAd5LCL3-P72-B602L-P30-P54 vaccine can induce sufficient immune response in the ternary pig immunization test.
  • CTL Cytotoxic T cell
  • African swine fever multi-antigen recombinant adenovirus pAd5LCL3-P72-B602L-P30-P54 vaccine Animal immunization: use 1 ⁇ 10 8 FFU of pAd5LCL3-P72-B602L-P30-P54 vaccine, immunize ternary pigs, and collect pig blood samples after four weeks .
  • Porcine peripheral blood lymphocyte isolation use the pig peripheral blood lymphocyte isolation kit from Tianjin Haoyang Huake Biotechnology Co., Ltd. to separate lymphocytes from the collected pig blood samples, and use a counter to count effector cells.
  • Cytotoxic T cell (CTL) killing assay Lactate dehydrogenase cytotoxicity detection kit (purchased from Biyuntian) was used to detect cytotoxic T cell (CTL) killing assay. Specific steps: 1. Prepare PK15 cells one night in advance (the cells were purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences), and infect with African swine fever pAd5LCL3-P72-B602L-P30-P54 vaccine and adenovirus vector control vaccine (25MOI , 18h in advance).
  • the infected PK15 cells were digested with trypsin, resuspended in serum-free medium and diluted to 1 ⁇ 10 5 /ml as target cells. Add target cells to a 96-well bottom cell culture plate, adding 100ul to each well. 3 effector cells were naturally released to control wells without target cells, only 100ul culture medium was added.
  • Killing activity (%) [(OD experimental group-OD total natural release)/(OD maximum release group-OD total natural release)] ⁇ 100%
  • the African swine fever vaccine pAd5LCL3-P72-B602L-P30-P54 of this example can significantly enhance Strong specific immune response to African swine fever virus.
  • Example 14 Construction of the E1 region shuttle plasmid pS5E1-C129Rubiqutin-IRES-MGF5L6L of African swine fever adenovirus type 5 vector
  • IRES-EcoRV-F ccg GATATC TGTCGTCATCATCCTTATAGTCC
  • IRES-NotI-R aaatat GCGGCCGC GGTTGTGGCCATTATCATCGTG
  • Amplification system Q5 enzyme 25ul, 10uM primer IRES-EcoRV-F 1ul, 10uM primer IRES-NotI-R 1ul, template IRES template 2ul, add water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60 °C, 30s, 72°C, 20s, 35 cycles; 72°C, 5min.
  • the electrophoresis detection of the amplification results is shown in Figure 62, in which lanes 1 and 2 are the PCR amplification products of the IRES fragments, and M is the 15000bp Marker. It can be seen that the amplification results are correct.
  • Enzyme digestion reaction system vector pS5E1, IRES fragment ⁇ 2ug, EcoRV and NotI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul; Reaction conditions: 37°C, 30min; 65°C, 20min inactivation; gel recovery and purification.
  • the electrophoresis detection of the digested products is shown in Figure 63, wherein lane 1 is the fragment IRES EcoRV, NotI digested, and lane 2 is pS5E1 EcoRV, NotI digested, and M is 15000bp Marker.
  • the pS5E1 vector is ligated with the IRES fragment
  • Amplification system Q5 enzyme 10ul, 10uM primer IRES-EcoRV-F 1ul, 10uM primer IRES-NotI-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72 °C, 20s, 35 cycles; 72°C, 5min.
  • Perform electrophoresis verification as shown in Figure 64, where Nos. 1-9 are colonies, and M is Marker, as can be seen from Figure 64, positive bands appear in Nos. 2 and 6.
  • MGF5L6L-NotI-F aaggaaaaaaaGCGGCCGCgccaccATGCTGGTGATCTTCCTGGG
  • MGF5L6L-XhoI-R catgCTCGAG TCAGGCGTAGTCAGGCACAT
  • Amplification system Q5 enzyme 25ul, 10uM primer MGF5L6L-NotI-F 1ul, 10uM primer MGF5L6L-XhoI-R 1ul, template MGF5L6L 1ul, replenish water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C , 30s, 72°C, 40s, 35 cycles; 72°C, 5min.
  • Enzyme digestion reaction system vector pS5E1-IRES, MGF5L6L fragment ⁇ 2ug, NotI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification. The electrophoresis detection of the digestion products is shown in Figure 66, wherein lane 1 is pS5E1-IRES, NotI and XhoI double digestion, and lane 2 is fragment MGF5L6L, NotI and XhoI double digestion, M is 15000bp Marker.
  • the target fragment MGF5L6L is connected to pS5E1-IRES
  • Amplification system Q5 enzyme 10ul, 10uM primer MGF5L6L-NotI-F 1ul, 10uM primer MGF5L6L-XhoI-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72 °C, 30s, 35 cycles; 72°C, 5min. Perform electrophoresis verification, as shown in Figure 67, where Nos. 1-12 are colonies, and M is a 2000bp Marker.
  • CP129R-BamHI-F cgcGGATCCgccaccATGGAGCACCCCAGCACAAA
  • CP129R-ubiqutin-R GGGTTTTCACGAAAATCTGCATGGCGTAATCGGGCACGTCGTAA
  • ubiqutin-F ATGCAGATTTTCGTGAAAACCC
  • ubiqutin-EcoRV-R ccgGATATC TTACTTGTCTTCTGGTTTGTTGA
  • Amplification system Q5 enzyme 25ul, primer CP129R-BamHI-F 1ul, primer CP129R-ubiqutin-R 1ul, template C129R 2ul, replenish water to 50ul; Reaction conditions: 98°C for 30s; 98°C for 10s, 68°C for 30s, 72°C for 15s, 35 cycles; 5 min at 72°C.
  • Amplification system Q5 enzyme 25ul, primer ubiqutin-F 1ul, primer ubiqutin-EcoRV-R 1ul, template ubiqutin 2ul, make up to 50ul with water; Reaction conditions: 98°C for 30s; 98°C for 10s, 68°C for 30s, 72°C for 15s, 35 cycle; 72 °C 5min.
  • Amplification system Q5 enzyme 25ul, upstream primer CP129R-BamHI-F, downstream primer ubiqutin-EcoRV-R, template fragment CP129R and fragment ubiqutin each 50ng, replenish water to 50ul; Reaction conditions: 98°C; 98°C5s, 68°C30s , 72°C for 30s, 35 cycles; 72°C for 7min.
  • Enzyme digestion reaction system vector pS5E1-IRES-MGF5L6L, CP129 Rubiqutin fragment 2ug, EcoRV and BamHI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification. The electrophoresis detection of the digestion products is shown in Figure 69, wherein lane 1 is pS5E1-IRES-MGF5L6L, digested by EcoRV and BamHI; lane 2 is CP129 Rubiqutin fragment, digested by EcoRV and BamHI, M is 15000bp Marker, 2000bp Marker.
  • Ligation system pS5E1-IRES-MGF5L6L 100ng; CP129 Rubiqutin fragment 50ng; T4 DNA ligase 1ul; 10 ⁇ ligase buffer 1ul; water to 10ul. Reaction conditions: room temperature, 30 min. The ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Amplification system Q5 enzyme 10ul, 10uM primer CP129R-BamHI-F 1ul, 10uM primer ubiqutin-EcoRV-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72 °C, 30s, 35 cycles; 72°C, 5min; electrophoresis verification, as shown in Figure 70, where Nos. 1-5 are colonies, and M is 2000bp Marker. As can be seen from Figure 70, positive bands appeared in Nos. 1 and 2.
  • the backbone of the shuttle plasmid pS5E4 adopts basic elements such as puc origin and amp, the left arm ITR sequence of the Ad5E4 region (370bp), the right arm part of the fiber gene sequence (1746bp), and the EF1 ⁇ -EGFP-HBV polyA gene.
  • EF1 ⁇ -EGFP-HBV polyA gene was synthesized by Biomed.
  • Ad5E4-left arm-EF1 ⁇ -R caatcccccccttttcttttaaaaAACACCACTCGACACGGCAC
  • EF1 ⁇ -Ad5E4-right arm-F GGAAAGAACCAGCTGGGGCTCTAGCAATTGAAAAATAAACACGTTGA
  • Ad5E4-right arm-puc-R TAATACGACTCACTATAGGGAGACCCAAAATGTAACCACTGTGAG
  • the PCR program was: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 40sec, 35 cycles; 72°C, 5min.
  • pAd5LCL3 As template and puc-Ad5E4-left arm-F and Ad5E4-left arm-EF1 ⁇ -R as primers, amplify the left arm fragment of pS5E1 shuttle plasmid.
  • Amplification system pAd5LCL3 plasmid 50ng, 10uM puc-Ad5E4-left arm-F primer 1ul, 10uM Ad5E4-left arm-EF1 ⁇ -R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 10sec, 35 cycles; 72°C, 5min.
  • pAd5LCL3 As template and EF1 ⁇ -Ad5E4-right arm-F and Ad5E4-right arm-puc-R as primers, amplify the right arm fragment of pS5E4-EGFP shuttle plasmid; amplification system: pAd5LCL3 plasmid 50ng, 10uM EF1 ⁇ - Ad5E4-right arm-F primer 1ul, 10uM Ad5E4-right arm-puc-R primer 1ul, Q5 high-fidelity enzyme 20ul; replenish water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 40sec, 35 cycles; 72°C, 5min.
  • PCR amplification of pS5E4-EGFP shuttle plasmid backbone amplification system: pS5E1 backbone plasmid 50ng, 10uM puc-F primer 1ul, 10uM puc-R primer 1ul , Q5 high-fidelity enzyme 20ul; replenish water to 40ul.
  • the PCR program was: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C, 1min20sec, 35 cycles; 72°C, 5min.
  • lane 1 is the left arm of the pS5E4-EGFP shuttle plasmid
  • lane 2 is the right arm of the pS5E4-EGFP shuttle plasmid
  • lane 3 is EF1 ⁇ -EGFP-HBV
  • lane 4 is pS5E4-EGFP Shuttle plasmid backbone
  • M is 2000Marker.
  • the left arm of pS5E4-EGFP shuttle plasmid, the right arm of pS5E4-EGFP shuttle plasmid, EF1 ⁇ -EGFP-HBV, and pS5E4-EGFP shuttle plasmid backbone were connected using the seamless cloning kit from Biomed.
  • connection system was 2 ⁇ Smealess Cloning Mix 10 ⁇ l, pS5E4-EGFP shuttle plasmid left arm fragment 50ng, pS5E4-EGFP shuttle plasmid right arm fragment 50ng, EF1 ⁇ -EGFP-HBV fragment 50ng, pS5E4-EGFP shuttle plasmid backbone fragment 50ng, add water to 20 ⁇ l, incubate at 50°C for 40 minutes; the ligation product was transformed into DH5 ⁇ competent cells, spread on plates containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • PS5E4-CP312R-HamHI-F ccaagctgtgaccggcgcctacGGATCCGCCACCATGACAACCCACAT
  • CP312R-2A-R GAAGTTAGTAGCTCCGCTTCCGGCGTAATCAGGCACGTCGTA
  • CP312R-2A-F TACGACGTGCCTGATTACGCCGGAAGCGGAGCTACTAACTTC
  • 2A-MGF110-4L-R GCCCAGAAACACCACCAGCATAGGTCCAGGGTTCTCCTCCA
  • MGF110-4L-F ATGCTGGTGGTGTTTCTGGG
  • MGF110-4L-XhoI-R CGGGTTTAAACGGGCCCTCTAGACTCGAGTCACAGGTCCTTCT
  • HBV(jd)-R TAAGGGTCAATGTCCATGCC
  • MGF110-4L fragment with MGF110-4L gene synthetic fragment as template and MGF110-4L-F and MGF110-4L-XhoI-R as primers; amplification system: MGF110-4L gene synthetic fragment 50ng, 10uM MGF110- 4L-F primer 1ul, 10uM MGF110-4L-XhoI-R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 20sec , 35 cycles; 72°C, 5min.
  • lane 1 is the amplified fragment of CP312R
  • lane 2 is the amplified fragment of 2A
  • lane 3 is the amplified fragment of MGF110-4L
  • M is the 2000bp Marker.
  • Amplification system CP312R gel recovery fragment 50ng, 2A gel recovery fragment 50ng, MGF110-4L gel recovery fragment 50ng, 10uM PS5E4-CP312R-HamHI-F primer 1ul, 10uM MGF110-4L-XhoI-R primer 1ul, Q5 high-fidelity enzyme 25ul; replenish water to 50ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 50sec, 35 cycles; 72°C, 5min.
  • the fusion results are shown in Figure 76, where lane 1 is the CP312R-2A-MGF110-4L fragment, and M is the 2000bp Marker.
  • Enzyme digestion reaction system vector pS5E4-EGFP 2ug, BamHI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation.
  • Axygen kit gel recovery and purification The results of gel recovery are shown in Figure 77, wherein lane 1 is the fragment CP312R-2A-MGF110-4L gel recovery, lane 2 is the vector pS5E4-EGFP, BamHI, XhoI double digestion gel recovery, M is 15000bp Marker.
  • Ligation system pS5E4-EGFP gel recovery product (100ng), CP312R-2A-MGF110-4L fragment (50ng), 2 ⁇ Smealess Cloning Mix 5ul, replenish water to 10ul. Reaction conditions: 50°C, 40min.
  • the ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Reaction conditions 37°C, 1h; 65°C, 20min inactivation.
  • lane 1 is pS5E1-C129Rubiqutin-IRES-MGF5L6L
  • lane 2 is pAd5LCL3.
  • Reaction system 37.5 ⁇ l of enzyme digestion reaction solution; 1 ⁇ l of dephosphorylase; 5 ⁇ l of dephosphorylation buffer; water to 50 ⁇ l. Reaction conditions: 37°C, 1h; 65°C, 5min inactivation.
  • Reaction conditions 37°C, 1h; 65°C, 20min inactivation.
  • lane 1 is pS5E4-CP312R-2A-MGF110-4L
  • lane 2 is pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L.
  • Reaction system 37.5 ⁇ l of enzyme digestion reaction solution; 1 ⁇ l of dephosphorylase; 5 ⁇ l of dephosphorylation buffer; water to 50 ⁇ l. Reaction conditions: 37°C, 1h; 65°C, 5min inactivation.
  • Example 17 Packaging of recombinant adenovirus
  • Transfection Dilute the linearized 2 ⁇ g plasmid and PEI with 100 ⁇ l serum-free medium respectively; add the plasmid dilution to the PEI dilution solution, pipette 5 times or vortex for 10 seconds to mix, and incubate at room temperature for 10 minutes to form Transfection complex. During the incubation, the cell culture medium was gently aspirated from the plate, 2 mL of fresh growth medium was added, and 10 minutes later, the transfection complex was added to the cells in the fresh medium.
  • Cell culture Culture the transfected 293TD37 cells in a 37°C, 5% CO 2 incubator for 72-96 hours; 72-96 hours after viral plasmid transfection, collect the cell suspension in a 6-well plate in a 1.5ml centrifuge tube The middle is TP0.
  • Continuous inoculation freeze and thaw the collected cell suspension at -80°C for 3 times, centrifuge at 2000g for 10 minutes at 4°C, take 500 ⁇ l of supernatant to infect 293TD37 cells (293TD37 cells need to be prepared one day in advance), 37°C, 5% CO 2 Incubate for 60 minutes, supplement with 2 mL of FBS medium, culture at 37°C, 5% CO 2 for 72 hours, and collect the cell suspension, namely TP1; repeat the previous steps to collect the cell suspension, namely TP2. Continue to inoculate until the cells become diseased.
  • Cytopathic When the 293TD37 cells were cultured from TP0 to TP4, the cells gradually became diseased until the 293TD37 cells were completely diseased.
  • the cytopathic conditions caused by TP0 to TP4 are shown in Figures 86-90, respectively, and TP4 has been completely diseased.
  • the cells were adhered to grow into monolayer cells, the medium was discarded, and the recombinant adenovirus was serially diluted 10-3 to 10-6 times with serum-free DMEM maintenance solution, and each dilution was inoculated into 2 wells. 250uL per well, 1 hour after infection, discard the supernatant, supplement with complete medium, and then incubate at 37°C in a 5% carbon dioxide incubator.
  • virus titer mean x 1013 x 4 x 10 (-n) .
  • the FFU of pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L virus was 1.9 ⁇ 10 8 FFU/mL, and the titer was high.
  • Example 19 Detection of stability of African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L
  • 293TD37 cells To prepare 293TD37 cells, take the well-grown cells in the T75 culture flask, discard the supernatant, wash the cells with PBS, digest with 0.25% trypsin, and add 10 mL of fresh DMEM medium containing 10% fetal bovine serum to terminate the digestion, then mix by pipetting. , 293TD37 cells were seeded into 6-well plates (5 ⁇ 10 5 cells/mL, 2 mL/well), incubated at room temperature for 1 hour to make them adhere to the wall, and the degree of adherence was examined by microscopy after incubation.
  • Infection was performed with pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L virus particles at a titer of infection of 5 MOI/well. 48 hours after 293TD37 cells developed lesions, the cells were collected, freeze-thawed 3 times, and then centrifuged at 2000 g to collect the supernatant.
  • the collected 5th, 10th, 15th, 20th, 25th, and 30th passages of the virus liquid were tested, and it was found that the genome of the virus was still intact, indicating that the replication-deficient pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L virus was able to express in 293TD37 cells.
  • the detection method is as follows:
  • Virus infection prepare a 6-well plate of A549 cells, each well of cells is 2.5 ⁇ 10 5 /well, discard the medium, wash once with PBS, inoculate adenovirus with 1 ⁇ 10 9 vp/well of virus, infect A549 cells, Wild-type adenovirus type 5 was used as a control. After 1 h at 37°C, 5% CO 2 , the virus solution was discarded, and 5% complete medium was supplemented. The cells were cultured at 37° C. and 5% CO 2 for 48 h.
  • RCA (average positive cell field) ⁇ (374 field/well) ⁇ (dilution factor))/Total VPs in 0.5ml viral sample
  • Judgment standard is that the level of RCA is less than 1RCA/3 ⁇ 10 10 vp. According to statistics, the level of RCA of pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L is less than 1RCA/3 ⁇ 10 10 vp, indicating that the replication-deficient pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L virus prepared by the present invention is in 293TD37 It is stably packaged in cells without conversion to wild type or with a low probability of conversion to wild type.
  • 293TD37 cells were prepared one day in advance, placed in a 12-well cell culture plate, and 293TD37 cells were infected with the African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L virus. 1ml of cells were washed with PBS, and samples were prepared for Western Blot detection; HA antibody was used to detect the target protein, and the HA antibody was purchased from Abcam.
  • C129Rubiquitin, MGF5L6L and CP312R have HA tags, and the size of C129Rubiquitin fusion protein is 34kda, the size of MGF5L6L protein is 25kda, and the size of CP312R protein is 35kda.
  • the experimental results are shown in Figure 91.
  • the vaccine can clearly see the MGF5L6L protein, which shows that the protein expression level of pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L is relatively high.
  • Example 22 Immunological evaluation of African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L in mouse model
  • mice 10 SPF mice (6-8 weeks old) were randomly divided into 2 groups with 5 mice in each group. Mice were immunized with pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L according to the groupings shown in Table 5.
  • the injection method is: intramuscular injection in the posterior inner thigh; injection dose: 100ul.
  • mice were sacrificed 14 days after immunization, spleen lymphocytes were isolated, and PK15 cells transfected with shuttle plasmids pS5E1-C129Rubiqutin-MGF5L6L and pS5E4-CP312R-MGF110-4L were stimulated and cultured for 6 hours, and protein secretion blockers were added to block cytokines. secretion. After 6 hours, Fc receptors were blocked, dead cells and cell surface molecular markers were stained, and after cells were fixed and perforated, intracellular cytokines were stained. Cell surface markers include CD4 and CD8, and intracellular cytokines include IFN ⁇ and IL2. Flow cytometry (CyExpert) was used to analyze the expression levels of IFN ⁇ and IL2 in CD4+T cells and CD8+T cells stimulated with target proteins.
  • FIG. 96 and 97 The CD8+ T cell and CD4+ T cell immune responses induced by pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L are shown in Figures 96 and 97, and the representative results are shown in Figures 98 to 99, of which Figure 98 is intramuscular injection
  • Figure 98 is intramuscular injection
  • FIG. 99 is the representative graph of the immune response of the blank control.
  • mice 14 days after immunization of mice, after spleen cells were stimulated with the target protein, the levels of IFN ⁇ , TNF ⁇ and IL2 expressed in CD8+ T cells were significantly higher than those in the Ad5 vector control group (Control) (P ⁇ 0.05). After CD4+ T cells were stimulated, the levels of IFN ⁇ , TNF ⁇ and IL2 expressed were significantly higher than those in the Ad5 vector control group (Control) (P ⁇ 0.05).
  • the results of cellular immune response showed that intramuscular injection of 1*10 ⁇ 7FFU adenovirus vector vaccine could induce specific cellular immune responses in the immunized mice.
  • I215L-NotI-F aaggaaaaaaaGCGGCCGCgccaccATGGTGAGCAGGTTTCTGATC
  • I215L-XhoI-R catgCTCGAG TCAGGCGTAATCGGGCACAT
  • Amplification system Q5 enzyme 25ul, 10uM primer I215L-NotI-F 1ul, 10uM primer I215L-XhoI-R 1ul, template I215L 1ul, replenish water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C , 30s, 72°C, 40s, 35 cycles; 72°C, 5min.
  • Enzyme digestion reaction system vector pS5E1-IRES, I215L fragment ⁇ 2ug, NotI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification.
  • the electrophoresis detection of the digestion product is shown in Figure 100, wherein the lane vector is pS5E1-IRES NotI and XhoI double digestion, the swimming lane I215L is the fragment I215L NotI and XhoI double digestion, and M is 15000bp, 2000bp Marker.
  • the target fragment I215L is connected to pS5E1-IRES
  • Amplification system Q5 enzyme 10ul, 10uM universal primer CMV-F 1ul, 10uM primer I215L-XhoI-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C , 30s, 35 cycles; 72°C, 5min.
  • Perform electrophoresis verification as shown in Figure 101, where Nos. 1-11 are colonies, and M is a 2000bp Marker. It can be seen from Figure 101 that 1, 2, 5, 6, 7, 8, and 11 are positive colonies.
  • L8L-ubiqutin-R AAGGGTTTTCACGAAAATCTGCATGGCGTAGTCGGGCACGTCGT
  • ubiqutin-F ATGCAGATTTTCGTGAAAACCC
  • ubiqutin-EcoRV-R ccgGATATC TTACTTGTCTTCTGGTTTGTTGA
  • Amplification system Q5 enzyme 25ul, primer L8L-BamHI-F 1ul, primer L8L-ubiqutin-R 1ul, template L8L 2ul, make up to 50ul with water; Reaction conditions: 98°C for 30s; 98°C for 10s, 68°C for 30s, 72°C for 15s , 35 cycles; 72 °C 5min.
  • Amplification system Q5 enzyme 25ul, primer ubiqutin-F 1ul, primer ubiqutin-EcoRV-R 1ul, template ubiqutin 2ul, make up to 50ul with water; reaction conditions: 98°C for 30s; 98°C for 10s, 68°C for 30s, 72°C for 15s, 35 cycle; 72 °C 5min.
  • Amplification system Q5 enzyme 25ul, upstream primer L8L-BamHI-F, downstream primer ubiqutin-EcoRV-R template fragment L8L, fragment ubiqutin each 50ng, replenish water to 50ul; Reaction conditions: 98°C; 98°C for 5s, 68°C for 30s, 72°C for 30s, 35 cycles; 72°C for 7min.
  • Enzyme digestion reaction system vector pS5E1-IRES-I215L, L8Lubiqutin fragment ⁇ 2ug, EcoRV and BamHI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification.
  • lane 1 is the pS5E1-IRES-I215L plasmid
  • lane 2 is the pS5E1-IRES-I215L plasmid EcoRV and BamHI digestion
  • lane 3 is the L8Lubiqutin fragment EcoRV and BamHI digestion
  • M is 15000bp Marker.
  • Ligation system pS5E1-IRES-I215L 100ng; L8 Lubiqutin fragment 50ng; T4 DNA ligase 1ul; 10 ⁇ ligase buffer 1ul; water to 10ul. Reaction conditions: room temperature, 30 min. The ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Amplification system Q5 enzyme 10ul, 10uM universal primer CMV-F 1ul, 10uM primer ubiqutin-EcoRV-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C , 40s, 35 cycles; 72°C, 5min; electrophoresis verification, as shown in Figure 105, where Nos. 1-24 are colonies, and M is a 2000bp Marker.
  • the shuttle plasmid pS5E4-EGFP in the E4 region of the human adenovirus type 5 vector was successfully constructed.
  • pS5E4-I73R-BamHI-F ccaagctgtgaccggcgcctacGGATCCgccaccATGGAGACACAGAAG
  • I73R-hbsag-R AGCCGCTGGTGGGTGTTCTCCATGGCGTAGTCAGGCACATCGTA
  • hbsag-F ATGGAGAACACCACCAGCGGC
  • hbsag-2A-R TGAAGTTAGTAGCTCCGCTTCCGATGTACACCCAGAGGCAGAA
  • 2A-E146L-R ACAAAGTCTGTTGTTCCGCCCATAGGTCCAGGGTTCTCCTCCA
  • E146L-pS5E4-XhoI-R CGGGTTTAAACGGGCCCTCTAGACTCGAGTTAGATGATTCTCTGC
  • 2A gene synthesis fragment as template, 2A-F and 2A-E146L-R as primers, amplify 2A fragment; amplification system: 2A gene synthesis fragment 50ng, 10uM 2A-F primer 1ul, 10uM 2A-E146L-R Primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 20sec, 35 cycles; 72°C, 5min.
  • E146L fragment with E146L gene synthesis fragment as template and E146L-F and E146L-pS5E4-XhoI-R as primers; amplification system: E146L gene synthesis fragment 50ng, 10uM E146L-F primer 1ul, 10uM E146L-pS5E4 -XhoI-R primer 1ul, Q5 high-fidelity enzyme 20ul; water to 40ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 30sec, 35 cycles; 72°C, 5min.
  • the target fragment was purified using Axygen gel recovery kit.
  • Amplification system I73R gel recovery fragment 50ng, hbsag gel recovery fragment 50ng, 10uM pS5E4-I73R-HamHI-F primer 1ul, 10uM hbsag-2A-R primer 1ul, Q5 high-fidelity enzyme 25ul; replenish water to 50ul; PCR program: 98°C, 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 20sec, 35 cycles; 72°C, 5min.
  • Amplification system 2A gel recovery fragment 50ng, E146L gel recovery fragment 50ng, 10uM 2A-F primer 1ul, 10uM E146L-pS5E4-XhoI-R primer 1ul, Q5 high-fidelity enzyme 25ul; replenish water to 50ul; PCR program: 98°C , 10sec; 98°C, 5sec, 60°C, 30sec, 72°C, 30sec, 35 cycles; 72°C, 5min.
  • the fusion results are shown in Figure 107, where lane 1 is the I73Rhbsag fragment, lane 2 is the 2A-E146L fragment, and M is the 2000bp Marker.
  • Enzyme digestion reaction system vector pS5E4-EGFP 2ug, BamHI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Axygen kit gel recovery and purification.
  • Ligation system pS5E4-EGFP gel recovery product (100ng), I73Rhbsag fragment (50ng), 2A-E146L fragment (50ng), 2 ⁇ Smealess Cloning Mix 5ul, replenish water to 10ul. Reaction conditions: 50°C, 40min.
  • the ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Example 25 Recombination of shuttle plasmids pS5E1-L8Lubiqutin-IRES-I215L, pS5E4-I73Rhbsag-2A-E146L and pAd5LCL3 to construct pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L plasmid
  • Reaction conditions 37°C, 1h; 65°C, 20min inactivation.
  • lane 1 is pAd5LCL3
  • lane 2 is pS5E1-L8Lubiqutin-IRES-I215L.
  • Reaction system enzyme digestion reaction solution 37.5ul; dephosphorylation enzyme 1ul; dephosphorylation buffer 5ul; replenish water to 50ul. Reaction conditions: 37°C, 1h; 65°C, 5min inactivation.
  • Reaction conditions 37°C, 1h; 65°C, 20min inactivation.
  • lane 1 is pS5E4-I73Rhbsag-2A-E146L
  • lane 2 is pAd5LCL3-L8Lubiqutin-IRES-I215L.
  • Reaction system enzyme digestion reaction solution 37.5ul; dephosphorylation enzyme 1ul; dephosphorylation buffer 5ul; replenish water to 50ul. Reaction conditions: 37°C, 1h; 65°C, 5min inactivation.
  • Example 26 Packaging of recombinant adenovirus
  • Plasmid pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L Linearization The plasmid to be transfected was digested with PacI, incubated at 37°C for 40min, and then inactivated at 65°C for 20min.
  • Transfection Dilute the linearized 2 ⁇ g plasmid and PEI with 100ul serum-free medium respectively; add the plasmid dilution to the PEI dilution solution, pipette 5 times or vortex for 10 seconds to mix, and incubate at room temperature for 10 minutes to form Transfection complex. During the incubation, the cell culture medium was gently aspirated from the plate, 2 mL of fresh growth medium was added, and 10 minutes later, the transfection complex was added to the cells in the fresh medium.
  • Cell culture Culture the transfected 293TD37 cells in a 37°C, 5% CO 2 incubator for 72-96 hours; 72-96 hours after viral plasmid transfection, collect the cell suspension in a 6-well plate in a 1.5ml centrifuge tube The middle is TP0.
  • Continuous inoculation freeze and thaw the collected cell suspension at -80°C for 3 times, centrifuge at 2000g at 4°C for 10 minutes, take 500ul of supernatant to infect 293TD37 cells (293TD37 cells need to be prepared one day in advance), 37°C, 5% CO 2 Incubate for 60 minutes, supplement with 2 mL of FBS medium, culture at 37°C, 5% CO 2 for 72 hours, and collect the cell suspension, namely TP1; repeat the previous steps to collect the cell suspension, namely TP2. Continue to inoculate until the cells become diseased.
  • Cytopathic When the 293TD37 cells were cultured from TP0 to TP4, the cells gradually became diseased, until the 293TD37 cells were completely diseased at TP4.
  • the cytopathic conditions caused by TP0 to TP4 are shown in Figures 117-121, respectively.
  • 293TD37 cells take the well-grown cells in the T75 culture flask, discard the supernatant, wash the cells with PBS, digest with 0.25% trypsin, and add 10 mL of fresh DMEM medium containing 10% fetal bovine serum to terminate the digestion, then mix by pipetting. , inoculated into 6-well plates (5 ⁇ 105/mL, 2ml per well), and cultured at 37°C in a 5% CO 2 carbon dioxide incubator. After 24 hours, the cells were adhered to grow into monolayer cells, the medium was discarded, and the recombinant adenovirus was serially diluted 10-3 to 10-6 times with serum-free DMEM maintenance solution, and each dilution was inoculated into 2 wells.
  • virus titer mean x 1013 x 4 x 10 (-n) .
  • the FFU of pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L virus was 2.2 ⁇ 10 8 FFU/mL, and the titer was high.
  • Example 28 Detection of stability of African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L
  • 293TD37 cells To prepare 293TD37 cells, take the well-grown cells in the T75 culture flask, discard the supernatant, wash the cells with PBS, digest with 0.25% trypsin, and add 10 mL of fresh DMEM medium containing 10% fetal bovine serum to terminate the digestion, then mix by pipetting. , 293TD37 cells were seeded into 6-well plates (5 ⁇ 10 5 cells/mL, 2 mL/well), incubated at room temperature for 1 hour to make them adhere to the wall, and the degree of adherence was examined by microscopy after incubation.
  • Infection was performed with pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L virus particles at a titer of infection of 5 MOI/well. 48 hours after 293TD37 cells developed lesions, the cells were collected, freeze-thawed 3 times, and then centrifuged at 2000 g to collect the supernatant.
  • the detection method is as follows:
  • Virus infection prepare a 6-well plate of A549 cells, each well of cells is 2.5 ⁇ 10 5 /well, discard the medium, wash once with PBS, inoculate adenovirus with 1 ⁇ 10 9 vp/well of virus, infect A549 cells, Wild-type adenovirus type 5 was used as a control. After 1 h at 37°C, 5% CO 2 , the virus solution was discarded, and 5% complete medium was supplemented. The cells were cultured at 37° C. and 5% CO 2 for 48 h.
  • RCA (average positive cell field) ⁇ (374 field/well) ⁇ (dilution factor))/Total VPs in 0.5ml viral sample
  • Judgment standard is the level of RCA ⁇ 1RCA/3 ⁇ 10 10 vp. According to statistics, the level of RCA of pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L is less than 1RCA/3 ⁇ 10 10 vp, indicating that the replication-deficient pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L virus prepared by the present invention can stably package in 293TD37 cells , will not convert to wild type or have a low chance of converting to wild type.
  • lane 1 is 293 blank cells
  • lanes 2 and 3 are samples of 293TD37 cells infected with pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L; it can be clearly seen that L8Lubiquitin fusion protein and I215L protein are expressed normally, so It can be seen that the pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L vaccine can express the target protein normally in 293 cells.
  • Example 31 Immunological evaluation of African swine fever multi-antigen recombinant adenovirus vaccine pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L mouse model
  • mice 10 SPF mice (6-8 weeks old) were randomly divided into 2 groups with 5 mice in each group. Mice were immunized with pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L according to the groupings shown in Table 6.
  • the injection method is: intramuscular injection in the posterior inner thigh; injection dose: 100ul.
  • mice were sacrificed 14 days after immunization, spleen lymphocytes were isolated, and PK15 cells transfected with shuttle plasmids pS5E1-L8Lubiqutin-I215L and pS5E4-I73Rhbsag-E146L were used to stimulate and culture for 6 hours, and protein secretion blockers were added to block cytokine secretion. After 6 hours, Fc receptors were blocked, dead cells and cell surface molecular markers were stained, and after cells were fixed and perforated, intracellular cytokines were stained. Cell surface markers include CD4 and CD8, and intracellular cytokines include IFN ⁇ and IL2. Flow cytometry (CyExpert) was used to analyze the expression levels of IFN ⁇ and IL2 in CD4+T cells and CD8+T cells stimulated with target proteins.
  • CD8+ T cell and CD4+ T cell immune responses induced by pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L are shown in Figure 126 and Figure 127, and representative results are shown in Figure 128 to Figure 129, of which Figure 128 is an intramuscular injection of pAd5LCL3
  • Figure 128 is an intramuscular injection of pAd5LCL3
  • the representative graph of the cellular immune response after -L8Lubiqutin-I215L-I73Rhbsag-E146L, and FIG. 129 is the representative graph of the immune response of the blank control.
  • mice 14 days after immunization of mice, after spleen cells were stimulated by the target protein, the levels of IFN ⁇ , TNF ⁇ and IL2 expressed in CD8+ T cells were significantly higher than those in the Ad5 vector control group (Control) (P ⁇ 0.05). After CD4+T cells were stimulated, the levels of IFN ⁇ , TNF ⁇ and IL2 expressed were significantly higher than those in the Ad5 vector control group (Control) (P ⁇ 0.05).
  • the results of cellular immune response showed that intramuscular injection of 1*10 ⁇ 7FFU adenovirus vector vaccine could induce specific cellular immune responses in the immunized mice.
  • the shuttle plasmid pS5E1 in the E1 region of the human adenovirus type 5 vector was successfully constructed.
  • EP402R-BamHI-F cgc GGATCC gccaccATGATCATCATCGTGATCTTCC
  • EP402R-EcoRV ccg GATATC ttaAGCGTAGTCTGGGACGTCGT
  • Amplification system Q5 enzyme 25ul, 10uM primer EP402R-BamHI-F 1ul, 10uM primer EP402R-EcoRV 1ul, template EP402R 1ul, add water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s , 72°C, 45s, 35 cycles; 72°C, 5min.
  • Enzyme digestion reaction system vector pS5E1-IRES, EP402R fragment ⁇ 2ug, EcoRV and BamHI each 1ul; 10 ⁇ cutsmart buffer 5ul; replenish water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification. The electrophoresis detection of the digested products is shown in Figure 23, wherein lane 1 is fragment EP402R, which is double digested by BamHI and EcoRV, and lane 2 is pS5E1-IRES, which is double digested by BamHI and EcoRV, and M is 15000bp Marker.
  • the target fragment EP402R is connected to pS5E1-IRES
  • Amplification system Q5 enzyme 10ul, 10uM primer EP402R-BamHI-F 1ul, 10uM primer EP402R-EcoRV-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72 °C, 1min, 35 cycles; 72°C, 5min. Perform electrophoresis verification, as shown in Figure 130, where Nos. 1-6 are colonies, and M is a 2000bp Marker.
  • EP153R-NotI-F ATAAGAATGCGGCCGCgccaccATGTTCAGCAACAAGAAGTACAT
  • EP153R-XhoI-R AAAACTCGAGTCACTTGCTACAGATGTACAG
  • Amplification system Q5 enzyme 25ul, 10uM primer EP153R-NotI-F 1ul, 10uM primer EP153R-XhoI-R 1ul, template EP153R 1ul, replenish water to 50ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C , 30s, 72°C, 20s, 35 cycles; 72°C, 5min.
  • Enzyme digestion reaction system vector pS5E1-EP402R-IRES, EP153R fragment ⁇ 2ug, NotI and XhoI each 1ul; 10 ⁇ cutsmart buffer 5ul; water to 50ul. Reaction conditions: 37°C, 30min; 65°C, 20min inactivation. Gel recovery and purification. The electrophoresis detection of the digested product is shown in Figure 132, wherein lane 1 is pS5E1-EP402R-IRES, digested with NotI and XhoI, and lane 2 is the EP153R fragment, digested with NotI and XhoI, and M is a 2000bp Marker.
  • Ligation system pS5E1-EP402R-IRES 100ng; EP153R fragment 50ng; T4 DNA ligase 1ul; 10 ⁇ ligase buffer 1ul; water to 10ul. Reaction conditions: room temperature, 30 min. The ligation product was transformed into DH5 ⁇ competent cells, spread on a plate containing ampicillin resistance, and incubated at 37°C for 12-16 hours.
  • Amplification system Q5 enzyme 10ul, 10uM universal primer CMV-F 1ul, 10uM primer EP153R-XhoI-R 1ul, add water to 20ul; PCR program: 98°C, 10s; 98°C, 5s, 60°C, 30s, 72°C , 2min30s, 35 cycles; 72°C, 5min; electrophoresis verification, as shown in Figure 133, where Nos. 1-14 are colonies, and M is a 5000bp Marker.
  • Example 33 Construction of the shuttle plasmid pS5E4-I177L-2A-K205Rubiqutin in the E4 region of the African swine fever adenovirus type 5 vector
  • the shuttle plasmid pS5E4-EGFP in the E4 region of the human adenovirus type 5 vector was successfully constructed.
  • EF1 ⁇ -BamHI-I177L-F ccaagctgtgaccggcgcctacGGATCCGCCACCATGTGGAAGGTGAA
  • I177L-2A-F CTACGACGTGCCCGATTACGCCGGAAGCGGAGCTACTAACTTC
  • 2A-K205R-R AACTGCTCTCTGGGCTCCACCATAGGTCCAGGGTTCTCCTCCA
  • K205R-F ATGGTGGAGCCCAGAGAGCA
  • K205R-ubiqutin-R GGGTTTTCACGAAAATCTGCATGGCGTAATCGGGCACATCGT
  • ubiqutin-F ATGCAGATTTTCGTGAAAACCC

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Abstract

一种非洲猪瘟病毒疫苗，所述疫苗共包括5组，每组分别是通过构建非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体，并经293TD37细胞包装而获得。其中，每组的非洲猪瘟病毒的四种抗原基因分别为1、P72、B602L、P30和P54；2、C129Rubiqutin、MGF5L6L、CP312R和MGF110-4L；3、L8Lubiqutin、I215L、I73Rhbsag和E146L；4、EP402R、EP153R、I177L和K205Rubiqutin；5、F317L、A151R、P34和pp62。非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体的构建，主要通过CRISPR/cas9敲除腺病毒载体的E1、E3、E2a和E4基因，再将E4的ORF6/7表达框构建在E2a区，并构建了E1、E4区域的穿梭质粒，分别用于表达合适的四种抗原基因，从而获得全新的腺病毒载体。该载体相对于第一代腺病毒载体，增加了约3kb的载体容量，再通过293TD37细胞系进行包装，获得滴度较高的重组腺病毒，用于制作非洲猪瘟的重组腺病毒疫苗。

Description

一种非洲猪瘟的重组腺病毒疫苗及其构建方法
本申请主张中国在先申请,申请号:2020106427453，申请日2020年7月6日的优先权；主张中国在先申请,申请号:2020106427538，申请日2020年7月6日的优先权；主张中国在先申请,申请号:2020106427557，申请日2020年7月6日的优先权；主张中国在先申请,申请号:2020106427449，申请日2020年7月6日的优先权；本申请主张中国在先申请,申请号:2020106427542，申请日2020年7月6日的优先权；其所有的内容作为本发明的一部分。
技术领域
本发明涉及基因工程技术领域和免疫学领域，具体而言，涉及一种非洲猪瘟病毒的重组腺病毒疫苗及其构建方法。
背景技术
非洲猪瘟(ASF)是一种具有高度传染性的猪病毒病。在家猪中可导致接近100％的高死亡率。ASF是由ASF病毒(ASFVirus,ASFV)引起，ASFV是一种较大的双链DNA病毒,主要在巨噬细胞的细胞质中复制，具有20面体结构，直径为175～215nm,基因组全长170～190kb，含有151个开放阅读框，可编码150～200种蛋白，具有囊膜的双股线性DNA病毒。构成ASFV病毒粒子的结构蛋白有P30、P72、P49、P54、P220、P62、pB602L、CD2v蛋白等，基于一个或两个亚单位疫苗迄今未能诱导免疫强大到足以对疫苗接种者具有显著保护作用。
我国已于2018年发现ASF疫情，带来了巨大的直接和间接经济损失。因此，迫切需要开发针对ASFV的疫苗。有报道，ASFV疫苗的先前的研究主要集中灭活疫苗和减毒疫苗。然而，灭活疫苗不能诱导有效的保护性应答；减毒疫苗的生物安全是其使用的主要限制因素，减毒毒株在我国是不允许研究的。然而在现阶段不能进行活病毒实验的情况下，需要提供疫苗以引发针对尽可能多的抗原的免疫应答。
因此需要开发新型的ASFV疫苗。潜在的候选疫苗是活载体疫苗。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。有利的是，需要用尽可能少的活载体输送尽可能多的病原体蛋白质。
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。腺病毒5型作为表达非洲猪瘟抗原蛋白的载体具有很多优势：①腺病毒表达载体是复制缺陷型的，只能在其独特的互补细胞系中生产制备，同时腺病毒无需整合进宿主细胞基因组中，目的基因在宿主细胞基因组外游离状态下表达，整合突变致癌可能性小，基因毒性低，制备疫苗安全性好；②重组的腺病毒载体可获得较高的滴度，利于大规模生产，工厂化效率高，生产成本低；③目前对腺病毒5型的结构、特性、功能的研究较为深入，腺病毒载体容易复制，操作简单，利于研究；④普通的一代腺病毒载体基因组敲除了6K的基因，能插入外源基因7.5k，具有较大的容量；⑤腺病毒相对稳定，经得起纯化、浓缩、保存。
现有技术报道了一些活载体疫苗。例如将ASFV p32、p54、p72和pp62基因分别重组入人腺病毒Ad5载体中进行“鸡尾酒”式免疫,获得了良好的抗原特异性CTL反应；之后他们又将ASFV A151R、B119L、B602L、EP402RΔPRR、B438L和K205R-A104R共7个ASFV抗原基因,重组入复制缺陷型腺病毒载体,通过“鸡尾酒”式混合免疫后能够诱导强烈体液免疫反应和细胞免疫应答。但“鸡尾酒” 式免疫，每个ASFV抗原基因都必须重组入一个复制缺陷型腺病毒载体，因此需要的载体数量非常多，有在免疫过程中受到针对腺病毒载体的免疫反应的风险。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。CN109652449A公开了EP153R与EP402R两种抗原基因共表达的重组腺病毒载体，CN109735567A公开了EP153R与P54两种抗原基因共表达的重组腺病毒载体。
但为了进一步增强对ASF的特异性免疫反应，还需进一步提高腺病毒载体的抗原基因容量，需用尽可能少的活载体输送尽可能多的病原体蛋白质，以引发针对尽可能多的抗原的免疫应答。
CN110269932A公开了将ASFV的A104R、A151R、B119L、B602L、CD2v、K205R、P49等5-7种抗原基因基于腺病毒载体融合在一起用于制备活载体疫苗。但多个抗原基因融合存在降低免疫原性和可能导致免疫失效的风险，因此要提高疫苗活性，还是必须要在每一个腺病毒载体上表达完全独立的抗原基因。
ASFV的P30蛋白是一种很重要的结构蛋白，由CP204L基因编码。研究发现P30能够诱导宿主细胞产生抑制细胞内化的中和抗体，从而使得疾病发作延迟甚至保护细胞抵抗病毒感染，因此P30在阻断病毒与细胞相互作用中有着重要的作用。P30作为病毒的早期蛋白，感染细胞后主要分布于细胞质中，在感染后4小时即可在胞浆内检测到；P30也是最具有抗原性的ASFV蛋白之一，有很强的免疫原性，在感染动物体内能诱导机体产生病毒中和抗体，因此通常被用做诊断抗原。ASFV的P54蛋白，由E183L基因编码，其抗体具有一定的病毒中和能力，此外，P30蛋白和P54蛋白与易感细胞上的两个不同的受体或结合位点相互作用，能缓和病程。P72蛋白是ASFV的主要检测抗原之一，大小约75ku。稳定性好，变异小。以P72蛋白为抗原，已经研发出一系列检测产品。由B602L基因编码的pB602L蛋白，可刺激基体产生较高水平的抗体。但目前现有技术中还没有四种抗原基因共表达的重组腺病毒载体，也没有关于ASFV的P72、B602L、P30、P54四种抗原基因共表达的重组腺病毒载体并运用于活载体疫苗的开发。
发明内容
为解决上述问题，本发明提供了一种非洲猪瘟病毒疫苗，所述疫苗是通过构建非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体，并经293TD37细胞包装而获得。其中，非洲猪瘟病毒的四种抗原基因共设计了五组，任意一组的四种抗原基因都可用于制备非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体，即非洲猪瘟病毒疫苗。本发明可以极大地提高腺病毒载体疫苗的容量，使用在一个腺病毒载体上同时表达非洲猪瘟四个独立抗原的方式，增强对非洲猪瘟病毒的特异性免疫反应。
非洲猪瘟病毒的抗原基因共计有160多种，发明人经过大量筛选实验，从中选出了20种免疫效果更强的抗原基因，分别为：P72、B602L、P30、P54、CP129R、MGF5L6L、CP312R、MGF110-4L、L8L、I215L、I73R、E146L、EP402R、EP153R、I177L、K205R、F317L、A151R、P34、pp62；这20种抗原基因根据基因片段大小和蛋白结构，被分成五组，每组的4种抗原基因能在本发明提供的重组腺病毒载体pAd5LCL3中得以共表达，即能在同一载体中完全独立表达四种抗原基因。这五组抗原基因疫苗(包括了5个重组腺病毒载体pAd5LCL3)组成了完整的非洲猪瘟病毒疫苗，取得了非常好的免疫效果。这五组抗原基因疫苗中，每一组的四种抗原基因，都可以很好地搭配组装在同一重组腺病毒载体中，从而得以完全独立表达这四种抗原基因。
一方面，本发明提供了一种可同时表达多种抗原基因的重组腺病毒载体pAd5LCL3，所述的重组腺病毒载体pAd5LCL3缺失E1、E3、E4和E2a基因，具有可分别同时表达一个或多个外源的抗原基因的E1区和E4区；所述抗原基因可以是任意来源的大小合适的抗原基因。
进一步地，所述重组腺病毒载体pAd5LCL3的E1区和E4区可表达四个不同或相同来源的抗原基因。
进一步地，所述重组腺病毒载体pAd5LCL3的E4区域缺失了ORF1～ORF7的序列。
进一步地，所述重组腺病毒载体pAd5LCL3的E2a区域(又称DNA Binding Protein，DBP)缺失。
进一步地，所述重组腺病毒载体pAd5LCL3的E4区域的E4promoter、ORF6、ORF7、polyA序列放在了E2a位置。
进一步地，所述重组腺病毒载体pAd5LCL3的E1区预置了SwaⅠ酶切位点。
进一步地，所述重组腺病毒载体pAd5LCL3的E4区预置了I-sceI酶切位点。
研究发现腺病毒复制相关的基因为E1、E2、E3、E4，缺失这些基因，并不影响腺病毒结构蛋白的表达，但会使得腺病毒无法进行复制和包装；因此构建这些复制相关的细胞系，能使敲除复制基因的复制缺陷型的腺病毒载体能够在其特有的细胞系中进行复制包装。同时，研究发现，只要表达腺病毒的E4基因中ORF6或是ORF3均能够代替整个E4基因，使敲除了E4的腺病毒能够复制包装。通过进一步对E4、E2a基因进行序列的研究分析，E4基因能够在E2a处表达。因此，本发明对E4基因进行序列分析，找出了E4的promoter、ORF6/7、polyA这几个基本元素，将其整合成一个完整的表达框，将其构建在敲除了E2a基因的序列位置，使ORF6、ORF7基因正常表达，最终获得了敲除E1、E3、E4、E2a，并将E4表达框置于E2a位置的复制缺陷型腺病毒5型载体pAd5LCL3，能在含有DBP序列的293TD37细胞中进行复制包装。
研究发现，E4基因中含有ORF1,2,3,4,5,6,7共7个表达框，其中ORF6/7是不能缺失的，一旦缺失了，将会明显影响腺病毒的包装和抗原基因的表达，因此需要将ORF6/7补回，同时为了获得更大的载体空间，需要将ORF6/7在E2a处表达，从而制得容量更大、表达效果更好的腺病毒载体。
进一步地，所述可同时表达四种抗原基因的重组腺病毒载体pAd5LCL3必须通过由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的293TD37细胞才能实现重组腺病毒包装，293TD37细胞的细胞株保藏编号为：CCTCC NO:C201996，保藏于中国典型培养物保藏中心。
普通的293细胞中含有腺病毒5型的E1基因，敲除E1、E3的腺病毒能在该细胞系中复制，但敲除了E4、E2a基因的腺病毒则不能在293细胞中复制。
293TD37细胞株由本发明小组发明，并已申请发明专利CN201911033247.2，于2019年5月8日保藏与中国典型培养物保藏中心，保藏编号为CCTCC NO:C201996，分类命名是人胚肾转化细胞AY293-TD-37，该细胞株包含腺病毒的E2a-DBP基因和E4-ORF6/7基因，能够用于包装E2a-DBP基因和E4基因缺乏的二代腺病毒而形成完整的具有感染性的二代腺病毒颗粒，该二代腺病毒与第一代腺病毒相比，出现RCA的几率大大降低，为制备活载体疫苗奠定了基础，并且由于E2a-DBP和E4基因的同时缺失使得包装容量相较与E2a突变或E4缺失的二代腺病毒再次增加，进一步提高了腺病毒载体外源基因的插入量，对增强腺病毒载体的应用水平具有重要意义。
本发明提供了重组腺病毒载体pAd5LCL3的构建方法，主要利用CRISPR/cas9敲除腺病毒环状载体质粒的E1、E3、E4和E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置。
所述可同时表达四种抗原基因的重组腺病毒载体pAd5LCL3的构建方法，包含以下步骤：
1)利用CRISPR/cas9敲除腺病毒环状载体质粒的E1基因，引入SwaⅠ酶切位点，融合后的片段与载体无缝克隆，利用CRISPR/cas9敲除E3基因，再使用无缝克隆方式连接，获得缺失E1和E3基因的腺病毒载体质粒pAd5。
2)再利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5的E4基因，使用PCR扩增并引入I-sceI酶切位点，再使用无缝克隆方法，获得缺失E1、E3和E4基因的腺病毒载体质粒pAd5△E4。
在已经敲除了E1、E3基因的基础上，敲除E4基因，能提高腺病毒载体的容量，降低其免疫原性，同时可以在E4区域插入外源基因，外源基因可以在E4位置大量表达，而不影响腺病载体的包装。在E1、E4基因处表达外源基因，能避免多个外源基因在同一区域表达的相互干扰，更利于表达，同时减少了不必要的E4相关基因，降低了腺病毒的免疫原性，使腺病毒能较为长期得存在于宿主细胞中，使外源基因更长期表达。
E4区基因在免疫原性中起着关键作用，大量E4区域基因的表达会使宿主产生较为强烈的免疫反应，诱导抗体的产生，不利于是腺病毒载体在宿主中长期表达目的蛋白，因此敲在E4区域敲除不必要的基因可以降低腺病毒载体的免疫原性，使载体能够在较长时间内进行表达。
为了使E4基因能敲除完全，便于大载体质粒的连接，使用CRISPR/cas9方法将E4区域的上游Fiber基因、以及E4的基因，进行敲除，使用PCR的方法扩增部分fiber以及引入I-sceI单酶切位点，再使用Gibson的无缝克隆方法，将多余切除的片段连接至载体上，重新获得E4敲除引进了I-sceI单酶切位点的载体质粒。使用I-sceI将载体质粒线性化，构建E4区域的穿梭质粒，使外源基因重组到E4区域，并在E4区域大量表达。
3)利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5△E4的E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置，再使用无缝克隆方法，获得缺失E1、E3、E4和E2a基因的腺病毒载体质粒pAd5LCL3。
E4区域的ORF1～ORF5的序列进行敲除，保留E4promoter、ORF6、ORF7、polyA序列，并将其插入E2a位置，因此E4位置可以进行表达外源基因。同时还敲除了E2a区的DBP序列。腺病毒E2a基因是一个DNA binding protein，与腺病毒的复制相关，敲除这个基因并不影响腺病毒的结构蛋白以及腺病毒的包装。DBP缺失可以阻止或极大减低回复突变。E2a和E4部分序列的敲除，增加了约3kb的载体容量。
现有的腺病毒载体构建普遍采用穿梭质粒，需要寻找单一的酶切位点。本发明创造性地采用CRISPR/cas9来构建重组腺病毒载体，通过比对，选择合适的E1、E3、E4、E2a敲除位点，根据E1、E3、E4、E2a序列的位置，敲除的基因碱基的数量，选择CRISPR位点，并设计了最佳的gRNA，从而完成重组腺病毒载体的构建。
再一方面，本发明提供了一种重组腺病毒疫苗，所述疫苗在pAd5LCL3的E1区和E4区包含目的基因。
研究证明，表E3区对于外源蛋白的表达水平不高，而在E1和E4区进行抗原基因的表达，表达水平更高，因此可以将四个抗原分别表达在E1区和E4区。
E3基因由于跟复制相关，需要敲除，让其复制缺陷；E3的作用是与腺病毒的免疫逃逸相关；敲除E3区可以增加腺病毒载体的容量；并使腺病毒载体能够正常包装。
进一步地，所述目的基因为病毒、细菌、肿瘤的基因或基因片段。
进一步地，所述目的基因为非洲猪瘟病毒基因。
再一方面，本发明提供了一种非洲猪瘟病毒疫苗，其特征在于，所述疫苗为通过构建一种非洲猪瘟病毒的四种抗原基因共表达的重组腺病毒载体，再经293TD37细胞包装而获得。
进一步地，所述非洲猪瘟病毒的四种抗原基因共表达的重组腺病毒载体需通过由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的293TD37细胞进行重组腺病毒包装，293TD37细胞的细胞株保藏编号为：CCTCC NO:C201996，保藏于中国典型培养物保藏中心。
进一步地，所述四种抗原基因分别为以下五组抗原基因中的任意一组：第一组：P72、B602L、P30和P54；第二组：C129Rubiqutin、MGF5L6L、CP312R和MGF110-4L；第三组：L8Lubiqutin、I215L、I73Rhbsag和E146L；第四组：EP402R、EP153R、I177L和K205Rubiqutin；第五组：F317L、A151R、P34和pp62。23、如权利要求12所述的疫苗，其特征在于，所述第一组中，P72和B602L表达在E1区，P30和P54表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-P72-B602L-P30-P54；所述第二组中，CP129Rubiqutin是在CP129R上添加分子佐剂ubiqutin获得，CP129Rubiqutin和MGF5L6L表达在E1区，CP312R和MGF110-4L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；所述第三组中，L8Lubiqutin是在L8L上添加分子佐剂ubiqutin获得，I73Rhbsag是在173R上添加分子佐剂hbsag获得，L8Lubiqutin和I215L表达在E1区，I73Rhbsag和E146L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；所述第四组中，K205Rubiqutin是在K205R上添加分子佐剂ubiqutin获得，EP402R和EP153R表达在E1区，I177L和K205Rubiqutin表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；所述第五组中，F317L和A151R表达在E1区，P34和pp62表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-F317L-A151R-P34-pp62。
发明人经过大量筛选实验，从160多种非洲猪瘟病毒的抗原基因中，选出了20种免疫效果更强的抗原基因，这20种抗原基因根据基因片段大小和蛋白结构，被分成五组，每组的4种抗原基因能在本发明提供的重组腺病毒载体pAd5LCL3中得以共表达，即能在同一载体中完全独立表达四种抗原基因。
每组的非洲猪瘟病毒的四种抗原基因分别为1、P72、B602L、P30和P54；2、C129Rubiqutin、MGF5L6L、CP312R和MGF110-4L；3、L8Lubiqutin、I215L、I73Rhbsag和E146L；4、EP402R、EP153R、I177L和K205Rubiqutin；5、F317L、A151R、P34和pp62。
进一步地，所述P72、B602L、P30、P54和pAd5LCL3-P72-B602L-P30-P54分别具有如序列表中Seq ID NO.1、Seq ID NO.2、Seq ID NO.3、Seq ID NO.4、Seq ID NO.6所示的核苷酸序列；所述CP129R、ubiqutin、MGF5L6L、CP312R、MGF110-4L、pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L分别具有如序列表中Seq ID NO.14、Seq ID NO.15、Seq ID NO.16、Seq ID NO.17、Seq ID NO.18、Seq ID NO.19所示的核苷酸序列；所述L8L、ubiqutin、I215L、I73R、hbsag、E146L、pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L分别具有如序列表中Seq ID NO.20、Seq ID NO.15、Seq ID NO.22、Seq ID NO.23、Seq ID NO.24、Seq ID NO.25、Seq ID NO.26所示的核苷酸序列；所述EP402R、EP153R、I177L、K205R、ubiqutin、pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin分别具有如序列表中Seq ID NO.27、Seq ID NO.28、Seq ID NO.29、Seq ID NO.30、Seq ID NO.31、Seq ID NO.32所示的核苷酸序列；所述F317L、A151R、P34、pp62、pAd5LCL3-F317L-A151R-P34-pp62分别具有如序列表中Seq ID NO.33、Seq ID NO.34、Seq ID NO.36、Seq ID NO.36、Seq ID NO.37所示的核苷酸序列。
再一方面，本发明提供了一种如上所述的一种非洲猪瘟病毒疫苗的构建方法，主要包含以下步骤：
1)利用CRISPR/cas9敲除腺病毒环状载体质粒的E1基因，引入SwaⅠ酶切位点，融合后的片段与载体无缝克隆，再利用CRISPR/cas9敲除E3基因，再使用无缝克隆方式连接，获得缺失E1和E3基因的腺病毒载体质粒pAd5；
2)再利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5的E4基因，使用PCR扩增并引入I-sceI酶切位点，再使用无缝克隆方法，获得缺失E1、E3和E4基因的腺病毒载体质粒pAd5△E4；
3)利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5△E4的E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置，再使用无缝克隆方法，获得缺失E1、E3、E4和E2a基因的腺病毒载体质粒pAd5LCL3；
4)构建腺病毒E1区域穿梭质粒，pS5E1通过DNA连接酶分别与第一组的P72、IRES、B602L、或第二组的CP129Rubiqutin、IRES、MGF5L6L、或第三组的L8Lubiqutin、IRES、I215L、或第四组的EP402R、IRES、EP153R、或第五组的F317L、IRES、A151R基因片段连接，构建非洲猪瘟腺病毒5型载体E1区域穿梭质粒，分别为第一组：pS5E1-P72-IRES-B602L；第二组：pS5E1-CP129Rubiqutin-IRES-MGF5L6L；第三组pS5E1-L8Lubiqutin-IRES-I215L；第四组：pS5E1-EP402R-IRES-EP153R；第五组：pS5E1-F317L-IRES-A151R。
5)构建腺病毒E4区域穿梭质粒，分别与第一组的P30、2A、P54；或第二组的CP312R、2A、MGF5L6L；或第三组的I73Rhbsag、2A、E146L；或第四组的I177L、2A、K205Rubiqutin；或第五组的P34、2A、pp62基因通过融合PCR技术得到基因片段，分别为P30-2A-P54、CP312R-2A-MGF5L6L、I73Rhbsag-2A-E146L、I177L-2A-K205Rubiqutin、P34-2A-pp62，通过对穿梭质粒pS5E4-EGFP酶切，敲除EGFP，并通过DNA连接酶与基因片段连接，构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒，分别为第一组：pS5E4-P30-2A-P54；第二组：pS5E4-CP312R-2A-MGF5L6L；第三组：pS5E4-I73Rhbsag-2A-E146L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pS5E4-P34-2A-pp62。
6)将分别将E1区域穿梭质粒pS5E1-P72-IRES-B602L、或pS5E1-CP129Rubiqutin-IRES-MGF5L6L、或pS5E1-L8Lubiqutin-IRES-I215L、或pS5E1-EP402R-IRES-EP153R、或pS5E1-F317L-IRES-A151R与腺病毒载体质粒pAd5LCL3同源重组，得到腺病毒载体质粒第一组：pAd5LCL3-P72-IRES-B602L；第二组pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L；第三组：pAd5LCL3-L8Lubiqutin-IRES-I215L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pAd5LCL3-F317L-IRES-A151R。
7)分别将E4区域穿梭质粒第一组：pS5E4-P30-2A-P54；第二组：pS5E4-CP312R-2A-MGF5L6L；第三组：pS5E4-I73Rhbsag-2A-E146L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pS5E4-P34-2A-pp62与腺病毒载体质粒第一组：pAd5LCL3-P72-IRES-B602L；第二组pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L；第三组：pAd5LCL3-L8Lubiqutin-IRES-I215L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pAd5LCL3-F317L-IRES-A151R同源重组，获得四种抗原基因共表达的重组腺病毒疫苗第一组：pAd5LCL3-P72-B602L-P30-P54；第二组：pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；第三组：pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；第四组：pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；第五组：pAd5LCL3-F317L-A151R-P34-pp62。
进一步地，步骤1)所述的腺病毒环状载体质粒来源于在A549细胞中扩增野生型人腺病毒5型病毒，收集并浓缩病毒液，采用HirtViral DNA Extract方法提取腺病毒5型基因组，使用cosmid方法将线性的腺病毒5型基因组构建成环状的腺病毒环状载体质粒。
进一步地，步骤3)所述的ORF6/7表达框基因具有如序列表中Seq ID NO.7所示的核苷酸序列；步骤4)所述的IRES具有如序列表中Seq ID NO.8所示的核苷酸序列；步骤5)所述的2A具有如序列表中Seq ID NO.9所示的核苷酸序列。
进一步地，步骤4)所述的穿梭质粒pS5E1骨架采用puc origin、amp基本元素，Ad5左臂ITR部分序列，右臂PIX、PIVa2部分序列，以及CMV-MCS SV40 early polyA；步骤5)所述的E4区域穿梭质粒pS5E4-EGFP的骨架采用puc origin、amp基本元素，Ad5E4区域左臂ITR序列，右臂部分fiber基因序列，以及EF1α-EGFP-HBV polyA基因；其中puc origin、amp基本元素具有如序列表可见中Seq ID NO.10所示的核苷酸序列，EF1α-EGFP-HBV polyA基因具有如序列表可见中Seq ID NO.11所示的核苷酸序列。
穿梭质粒pS5E1的骨架由北京博迈德基因技术有限公司合成，合成采用puc origin、amp等基本元素(2796bp)，Ad5左臂ITR部分序列(400bp)，右臂PIX、PIVa2部分序列(2100bp)，以及CMV-MCS(944bp)SV40 early polyA(160bp)。经PCR扩增和基因片段纯化后，进行无缝克隆连接，连接产物转化至感受态细胞，涂布氨苄抗性平板，培养后挑选阳性克隆进行酶切验证，获得腺病毒E1区域穿梭质粒pS5E1。
穿梭质粒pS5E4的骨架采用puc origin、amp等基本元素，Ad5E4区域左臂ITR序列(370bp)，右臂部分fiber基因序列(1746bp)，以及EF1α-EGFP-HBV polyA基因。经PCR扩增和基因片段纯化后，进行无缝克隆连接，连接产物转化至感受态细胞，涂布氨苄抗性平板，培养后挑选阳性克隆进行酶切验证，获得腺病毒E4区域穿梭质粒pS5E4-EGFP。
进一步地，步骤6)所述E1区域穿梭质粒穿梭质粒与腺病毒载体质粒pAd5LCL3同源重组，是通过PacI和SwaI对穿梭质粒和腺病毒载体质粒pAd5LCL3进行酶切，酶切产物去磷酸化，OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段，转化产物涂布平板，挑取菌落，进行XhoI酶切验证。
进一步地，步骤7)所述E4区域穿梭质粒与腺病毒载体质粒同源重组，是通过PacI和I-sceI对E4区域穿梭质粒和腺病毒载体质粒进行酶切，酶切产物去磷酸化，OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段，转化产物涂布平板，挑取菌落，进行XhoI酶切验证。
再一方面，本发明提供了一种重组腺病毒载体的包装方法，主要步骤为：分别将所述的重组腺病毒疫苗第一组：pAd5LCL3-P72-B602L-P30-P54；第二组：pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；第三组：pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；第四组：pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；第五组：pAd5LCL3-F317L-A151R-P34-pp62；用PacI酶切，线性化后的质粒用于转染；转染由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的293TD37细胞，收集细胞悬液。
其中的293TD37细胞株于2019年5月8日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:C201996，分类命名是人胚肾转化细胞AY293-TD37，该细胞株包含腺病毒的E2a和E4-ORF6/7基因，是通过将HEK293细胞经过基因工程改造所获得，能用于包装缺失E2a和E4基因的二代重组腺病毒而形成具有感染性的二代腺病毒颗粒。
进一步地，所述重组腺病毒载体的包装方法，主要由以下步骤制得：
1)将分别将所述的重组腺病毒疫苗第一组：pAd5LCL3-P72-B602L-P30-P54；第二组：pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；第三组：pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；第四组： pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；第五组：pAd5LCL3-F317L-A151R-P34-pp62；用PacI酶切，线性化后的质粒用于转染；使用PEI转染试剂转染293TD37细胞；
2)转染后的293TD37细胞于37℃，5％CO 2培养箱中培养72-96小时后收集细胞悬液，即为TP0代腺病毒；
3)TP0代腺病毒感染293TD37细胞于37℃，5％CO 2培养箱中培养72小时，收集细胞悬液即TP1代腺病毒；
4)重复3)，收集细胞悬液即TP2代腺病毒；
5)持续接毒直至细胞发生病变。
再一方面，本发明提供了293TD37细胞用于包装非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体的用途，所述四种抗原基因分别为第一组：P72、B602L、P30和P54；第二组：C129Rubiqutin、MGF5L6L、CP312R和MGF110-4L；第三组：L8Lubiqutin、I215L、I73Rhbsag和E146L；第四组：EP402R、EP153R、I177L和K205Rubiqutin；第五组：F317L、A151R、P34和pp62；其中第一组中，P72和B602L表达在E1区，P30和P54表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-P72-B602L-P30-P54；所述第二组中，CP129Rubiqutin是在CP129R上添加分子佐剂ubiqutin获得，CP129Rubiqutin和MGF5L6L表达在E1区，CP312R和MGF110-4L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；所述第三组中，L8Lubiqutin是在L8L上添加分子佐剂ubiqutin获得，I73Rhbsag是在173R上添加分子佐剂hbsag获得，L8Lubiqutin和I215L表达在E1区，I73Rhbsag和E146L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；所述第四组中，K205Rubiqutin是在K205R上添加分子佐剂ubiqutin获得，EP402R和EP153R表达在E1区，I177L和K205Rubiqutin表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；所述第五组中，F317L和A151R表达在E1区，P34和pp62表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-F317L-A151R-P34-pp62；
其中，所述293TD37细胞是由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的，细胞株保藏编号为：CCTCC NO:C201996，保藏于中国典型培养物保藏中心。
本发明提供了一种非洲猪瘟病毒疫苗，所述疫苗是通过构建非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体，并经293TD37细胞包装而获得。其中，非洲猪瘟病毒的四种抗原基因共设计了五组，任意一组的四种抗原基因都可用于制备非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体，即非洲猪瘟病毒疫苗。非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体的构建，主要通过CRISPR/cas9敲除腺病毒载体的E1、E3、E2a和E4基因，构建了E1、E4区域的穿梭质粒，分别用于表达四种抗原基因，从而获得全新的腺病毒载体。本发明的有益效果主要体现在：
①提供了全新的腺病毒5型载体CRISPR/cas9的构建方法，自主设计最佳敲除位点和gRNA，避免了过去载体构建用穿梭质粒敲除，需要找单一的酶切位点。
②由于E4区基因在免疫原性中起着关键作用，大量E4区域基因的表达会使宿主产生较为强烈的免疫反应，诱导抗体的产生，不利于腺病毒载体在宿主中长期表达目的蛋白，本发明在E4区域敲除不必要的基因可以降低腺病毒载体的免疫原性，使载体能够在较长时间内进行表达。
②本发明将E4区域的ORF1～ORF5的序列进行敲除，保留E4promoter、ORF6、ORF7、polyA序列，并将其插入E2a位置，因此E4位置可以进行表达外源基因。
③本发明还进一步敲除了DBP(E2a)序列，DBP缺失可以阻止或极大减低回复突变。④E2a和E4部分序列的敲除，相对于一代载体，增加了约3kb的载体容量。
⑤腺病毒载体的E2a、E4敲除，并将E4promoter-ORF6/7-polyA放于E2a区域，由此可以用E2a(DBP序列)互补的细胞系进行拯救，同时，外源基因可以在E1、E4区域同时表达而不相互干扰，目前该腺病毒疫苗在我们公司构建的互补细胞系—293TD37细胞系中的拯救，该细胞系能够持久的表达DBP蛋白。
⑥本发明构建了E1、E4区域的穿梭质粒，用于E1、E4区域外源基因表达。
⑦本发明经293TD37细胞系包装制备得到的重组腺病毒病毒的滴度较高。
基于以上原则，本发明可以极大地提高腺病毒载体的疫苗的容量，使用在一个腺病毒载体上同时表达非洲猪瘟四个独立抗原的方式，增强对非洲猪瘟病毒的特异性免疫反应，可以使家猪获得更好的免疫保护。
附图说明
图1为实施例2的Ad5-E4-up-gRNA裂解位点和PAM位点示意图
图2为实施例2的Ad5-E4-down-gRNA裂解位点和PAM位点示意图
图3为实施例2的Ad5-E4-up-gRNA、Ad5-E4-down-gRNA以及cas9“双酶切”载体质粒电泳检测结果，其中泳道1为Ad5-E4-up-gRNA、Ad5-E4-down-gRNA以及cas9“双酶切”，M为maker
图4为实施例2含有部分敲除的fiber、ITR片段扩增电泳检测结果，其中泳道1为fiber部分片段扩增结果，泳道2为ITR部分片段扩增结果，M为maker
图5为实施例2的Fiber-ITR融合片段电泳检测结果，其中泳道1为Fiber-ITR融合片段，M为maker
图6为实施例2的菌落PCR验证电泳检测结果，其中泳道1-24为菌落，M为maker
图7为实施例2对图6的阳性克隆菌落质粒进行BamHI、XhoI酶切验证的电泳检测结果，其中1-5为BamHI酶切，6-10为XhoI酶切，1、10为pAd5对照(为确实E4基因)，M为maker
图8为实施例3的100k-gRNA裂解位点和PAM位点示意图
图9为实施例3的protease-gRNA裂解位点和PAM位点示意图
图10为实施例3的100k-gRNA、protease-gRNA以及cas9“双酶切”载体质粒电泳检测结果，泳道1为100k-gRNA、protease-gRNA以及cas9“双酶切”载体质粒结果，M为maker
图11为实施例3的100k、E4 ORF6/7表达框、protease PCR扩增电泳检测结果，其中泳道1为E4ORF6/7表达框，泳道2为100k，M为Marker
图12为实施例3的100k、E4 ORF6/7表达框、Protease片段的融合PCR电泳检测结果，其中泳道1为片段100k、E4 ORF6/7表达框、protease融合PCR产物，M为Marker
图13为实施例3的菌落PCR验证电泳检测结果，其中泳道1-24为菌落，M为maker
图14为实施例3对图13的菌落挑取9、18、21、24号阳性克隆菌落进行XhoI酶切验证的电泳检测结果，其中泳道1为9号阳性克隆XhoI酶切，泳道2为18号阳性克隆XhoI酶切，泳道3为21号阳性克隆XhoI酶切，泳道4为24号阳性克隆XhoI酶切，泳道5为对照质粒pAd5LCL3的XhoI酶切，M为maker
图15为实施例4的CMV-MCS和SV40 earlypolyA片段扩增电泳检测结果，其中泳道1为CMV-MCS片段，泳道2为SV40 earlypolyA片段，M为2000Marker
图16为实施例4的CMV-MCS-SV40 earlypolyA、PUC、Ad5右臂、Ad5左臂扩增电泳检测结果， 其中泳道1为CMV-MCS-SV40 earlypolyA融合片段，泳道2为PUC，泳道3为Ad5右臂，泳道4为Ad5左臂，M为2000Marker
图17为实施例4的pS5E1骨架、Ad5左臂、Ad5右臂、CMV-MCS-SV40 earlypolyA四个片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中泳道1-6为菌落，M为Marker
图18为实施例4的选取图17中的1-6号菌落酶切验证的电泳检测结果，其中左1-6为质粒pS5E1 NcoI单酶切，右1-6为质粒pS5E1 PacI单酶切，M为15000bp Marker
图19为实施例4的IRES片段PCR扩增电泳检测结果，其中泳道1、2为IRES片段PCR扩增产物，M为15000bp Marker
图20为实施例4的片段IRES与pS5E1载体酶切电泳检测结果，其中泳道1为片段IRES EcoRV、NotI酶切，泳道2为pS5E1 EcoRV、NotI酶切，M为15000bp Marker
图21为实施例4的pS5E1载体与IRES片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-9号为菌落，M为Marker
图22为实施例4的pS5E1-IRES质粒NotI、EcoRV酶切电泳检测验证，选取图21的2、6进行质粒提取，酶切验证，其中1号泳道为2号质粒NotI、EcoRV酶切鉴定，2号泳道为6号质粒NotI、EcoRV酶切鉴定
图23为实施例4的P72与pS5E1-IRES载体酶切电泳检测结果，其中泳道1为片段pS5E1-IRES、NotI酶切，泳道2为P72、NotI酶切，M为15000bp Marker
图24为实施例4的P72与pS5E1-IRES连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-10号为菌落，M为Marker
图25为实施例4的pS5E1-P72-IRES质粒酶切电泳检测验证，选取图24中的2、5号菌落进行质粒提取，酶切验证，其中2号泳道为2号质粒酶切验证，5号泳道为5号质粒酶切验证，M为Marker
图26为实施例4的片段B602L与pS5E1-P72-IRES载体酶切产物电泳检测结果，其中泳道1为pS5E1-P72-IRES，NotI、XhoI酶切，泳道2为B602L片段，NotI、XhoI酶切，M为15000bp Marker
图27为实施例4的B602L与pS5E1-P72-IRES连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-7号为菌落，M为Marker
图28为实施例4的pS5E1-P72-IRES-B602L质粒酶切电泳检测验证，其中1、2、4、6号泳道为选取图27中的1、2、4、6号菌落质粒NotI、XhoI酶切鉴定，M为15000bp Marker
图29为实施例5的pS5E4-EGFP穿梭质粒左臂、pS5E4-EGFP穿梭质粒右臂、EF1a-EGFP-HBV、pS5E4-EGFP穿梭质粒骨架扩增电泳检测结果，其中泳道1为pS5E4-EGFP穿梭质粒左臂，泳道2为pS5E4-EGFP穿梭质粒右臂，泳道3为EF1α-EGFP-HBV，泳道4为pS5E4-EGFP穿梭质粒骨架，M为2000Marker
图30为实施例5的pS5E4-EGFP穿梭质粒左臂、pS5E4-EGFP穿梭质粒右臂、EF1α-EGFP-HBV、pS5E4-EGFP穿梭质粒骨架四个片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中泳道1-20为菌落，M为Marker
图31为实施例5的选取图30中的3、4、5、6号菌落酶切验证的电泳检测结果，其中1-4为3、4、5、6号阳性克隆PacI单酶切，5-8为3、4、5、6号阳性克隆HindIII单酶切，M1、M3为15000bp Marker，M2为2000bp Marker
图32为实施例5的片段P30、P54、2A的PCR扩增电泳检测结果，其中泳道1为P30扩增片段， 泳道2为P54扩增片段，泳道3为2A扩增片段，M1、M2为2000bp Marker。
图33为实施例5的片段P30-2A-P54融合PCR扩增电泳检测结果，其中泳道1为P30-2A-P54片段，M为Maker
图34为实施例5的片段P30-2A-P54与pS5E4-EGFP载体酶切电泳检测结果，其中泳道1、2为pS5E4-EGFP，BamHI、XhoI双酶切胶回收，泳道3、4：片段P30-2A-P54 BamHI、XhoI双酶切胶回收，M为15000bp Marker
图35为实施例5的pS5E4与P30-2A-P54片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-20号为菌落，M为2000bp Marker
图36为实施例5挑取图35的2、19号阳性克隆提取质粒进行BamHI、XhoI双酶切验证的电泳检测结果，其中泳道2为2号阳性克隆BamHI、XhoI双酶切验证，泳道19为19号阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker
图37为实施例6的pAd5LCL3与pS5E1-P72-IRES-B602L琼脂糖凝胶验证电泳检测结果，其中泳道1为pAd5LCL3，泳道2为pS5E1-P72-IRES-B602L
图38为实施例6的穿梭质粒pS5E1-P72-IRES-B602L和腺病毒载体质粒pAd5LCL3同源重组获得pAd5LCL3-P72-IRES-B602L质粒的电泳检测结果，其中泳道1-7为pAd5LCL3-P72-IRES-B602L克隆，M为15000bp Marker
图39为实施例6挑取图38的1号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1号泳道为pAd5LCL3-P72-IRES-B602L质粒XhoI酶切，2号泳道为pAd5LCL3-P72-IRES-B602L质粒PacI酶切，M为15000bp Marker
图40为实施例6对穿梭质粒pS5E4-P30-2A-P54和腺病毒载体质粒pAd5LCL3-P72-IRES-B602L琼脂糖凝胶验证电泳检测结果，其中泳道1为pS5E4-P30-2A-P54，泳道2为pAd5LCL3-P72-IRES-B602L，M为15000bp Marker
图41为实施例6的穿梭质粒pS5E4-P30-2A-P54和腺病毒载体质粒pAd5LCL3-P72-IRES-B602L同源重组获得重组腺病毒载体pAd5LCL3-P72-B602L-P30-P54质粒的电泳检测结果，其中泳道1-8为菌落，M为15000bp Marker
图42为实施例6的挑取图41的4号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1号泳道为pAd5LCL3-P72-B602L-P30-P54质粒XhoI酶切，2号泳道为pAd5LCL3-P72-B602L-P30-P54质粒PacI酶切，M为15000bp Marker
图43为实施例7的293TD37转染pAd5LCL3-P72-B602L-P30-P54质粒TP0后72小时的细胞照片
图44为实施例7的293TD37感染TP0后TP1的细胞照片
图45为实施例7的293TD37感染TP1后TP2细胞照片
图46为实施例7的293TD37感染TP2后TP3细胞照片
图47为实施例7的TP4引起的TP4293TD37细胞病变照片
图48为实施例11用Western Blot检测非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54中P30蛋白结果示意图
图49为实施例12对比非洲猪瘟多抗原重组腺病毒pAd5LCL3-P72-B602L-P30-P54、非相关抗原pAd5-FMDO腺病毒组和生理盐水组疫苗诱导的细胞毒性T细胞(CTL)杀伤实验结果图
图50为pAd5LCL3的载体图谱
图51为pS5E1的载体图谱
图52为pS5E1-P72-IRES-B602L的载体图谱
图53为pS5E4-EGFP的载体图谱
图54为pS5E4-P30-2A-P54的载体图谱
图55为pAd5LCL3-P72-B602L-P30-P54的载体图谱
图56为实施例11用Western Blot检测非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54中P54、P72表达结果示意图，其中M，预染Makker；泳道1，P54抗体血清；泳道2，P72抗体血清；泳道3：293TD37细胞对照
图57为实施例12用间接ELISA法检测血清中针对非洲猪瘟目的蛋白P72和P30的IgG抗体滴度结果示意图(ns,P≥0.05；*,P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001)，其中左图为蛋白P72的IgG抗体滴度，右图为P30的IgG抗体滴度
图58为实施例12非洲猪瘟重组腺病毒疫苗Ad5LCL3-P72-B602L-P30-P54诱导的CD8+T细胞反应示意图
图59为实施例12非洲猪瘟重组腺病毒疫苗Ad5LCL3-P72-B602L-P30-P54诱导的CD4+T细胞反应示意图
图60为实施例12非洲猪瘟重组腺病毒疫苗Ad5LCL3-P72-B602L-P30-P54诱导的细胞免疫反应代表图
图61为实施例12空白对照免疫反应代表图
图62为实施例14的IRES片段PCR扩增电泳检测结果，其中泳道1、2为IRES片段PCR扩增产物，M为15000bp Marker
图63为实施例14的片段IRES与pS5E1载体酶切电泳检测结果，其中泳道1为片段IRES EcoRV、NotI酶切，泳道2为pS5E1 EcoRV、NotI酶切，M为15000bp Marker
图64为实施例14的pS5E1载体与IRES片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-9号为菌落，M为Marker
图65为实施例14的pS5E1-IRES质粒NotI、EcoRV酶切电泳检测验证，选取图64的2、6进行质粒提取，酶切验证，其中1号泳道为2号质粒NotI、EcoRV酶切鉴定，2号泳道为6号质粒NotI、EcoRV酶切鉴定
图66为实施例14的MGF5L6L与pS5E1-IRES载体酶切电泳检测结果，其中泳道1为pS5E1-IRES，NotI和XhoI双酶切，泳道2为片段MGF5L6L，NotI和XhoI双酶切，M为15000bp Marker
图67为实施例14的MGF5L6L与pS5E1-IRES连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-12号为菌落，M为2000bp Marker
图68为实施例14的pS5E1-IRES-MGF5L6L质粒酶切电泳检测验证，选取图67中的2、9、11号菌落进行质粒提取，酶切验证，其中2号泳道为2号质粒酶切验证，9号泳道为9号质粒酶切验证，11号泳道为11号质粒酶切验证，M为Marker
图69为实施例14的片段CP129Rubiqutin与pS5E1-IRES-MGF5L6L载体酶切产物电泳检测结果，其中泳道1为pS5E1-IRES-MGF5L6L质粒，EcoRV和BamHI酶切，泳道2为C129Rubiqutin片段，EcoRV和BamHI酶切，M为15000bp Marker、2000bp Marker
图70为实施例14的pS5E1-IRES-MGF5L6L与CP129Rubiqutin连接产物转化感受态细胞菌落PCR 验证电泳检测结果，其中1-5号为菌落，M为2000bp Marker
图71为实施例14的pS5E1-CP129Rubiqutin-IRES-MGF5L6L质粒酶切电泳检测验证，其中1、2号泳道为选取图70中的1、2号菌落质粒BamHI、EcoRV酶切鉴定，M为2000bp Marker
图72为实施例15的pS5E4-EGFP穿梭质粒左臂、pS5E4-EGFP穿梭质粒右臂、EF1α-EGFP-HBV、pS5E4-EGFP穿梭质粒骨架扩增电泳检测结果，其中泳道1为pS5E4-EGFP穿梭质粒左臂，泳道2为pS5E4-EGFP穿梭质粒右臂，泳道3为EF1α-EGFP-HBV，泳道4为pS5E4-EGFP穿梭质粒骨架，M为2000Marker
图73为实施例15的pS5E4-EGFP穿梭质粒左臂、pS5E4-EGFP穿梭质粒右臂、EF1α-EGFP-HBV、pS5E4-EGFP穿梭质粒骨架四个片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中泳道1-20为菌落，M为Marker
图74为实施例15的选取图30中的3、4、5、6号菌落酶切验证的电泳检测结果，其中1-4为3、4、5、6号阳性克隆PacI单酶切，5-8为3、4、5、6号阳性克隆HindIII单酶切，M1、M3为15000bp Marker，M2为2000bp Marker
图75为实施例15的片段CP312R、MGF110-4L、2A的PCR扩增电泳检测结果，其中泳道1为CP312R扩增片段，泳道2为2A扩增片段，泳道3为MGF110-4L扩增片段，M为2000bp Marker
图76为实施例15的片段CP312R-2A-MGF110-4L融合PCR扩增电泳检测结果，其中泳道1为CP312R-2A-MGF110-4L片段，M为2000bp Marker
图77为实施例15的片段CP312R-2A-MGF110-4L与pS5E4-EGFP载体酶切电泳检测结果，其中泳道1为片段CP312R-2A-MGF110-4L胶回收，泳道2为片段pS5E4-EGFP，BamHI和XhoI双酶切胶回收，M为15000bp Marker
图78为实施例15的pS5E4与CP312R-2A-MGF110-4L片段连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-12号为菌落，M为15000bp Marker
图79为实施例15挑取图78的1、2、3、4号阳性克隆提取质粒进行BamHI、XhoI双酶切验证的电泳检测结果，其中泳道1、2、3、4分别为1、2、3、4号阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker
图80为实施例16的pAd5LCL3与pS5E1-CP129Rubiqutin-IRES-MGF5L6L琼脂糖凝胶验证电泳检测结果，其中泳道1为pS5E1-CP129Rubiqutin-IRES-MGF5L6L，泳道2为pAd5LCL3
图81为实施例16的穿梭质粒pS5E1-C129Rubiqutin-IRES-MGF5L6L和腺病毒载体质粒pAd5LCL3同源重组获得pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L质粒的电泳检测结果，其中泳道1-5为pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L克隆，M为15000bp Marker
图82为实施例16挑取图81的4号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1、2号泳道为pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L质粒XhoI酶切，M为15000bp Marker
图83为实施例16对穿梭质粒pS5E4-CP312R-2A-MGF110-4L和腺病毒载体质粒pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L琼脂糖凝胶验证电泳检测结果，其中泳道1为pS5E4-CP312R-2A-MGF110-4L，泳道2为pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L，M为15000bp Marker
图84为实施例16的穿梭质粒pS5E4-CP312R-2A-MGF110-4L和腺病毒载体质粒pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L同源重组获得重组腺病毒载体 pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒的电泳检测结果，其中泳道1-6为质粒，M为15000bp Marker
图85为实施例16的挑取图84的3号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1号泳道为pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒XhoI酶切，2号泳道为pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒PacI酶切，M为15000bp Marker
图86为实施例17的TP0引起的293TD37细胞照片
图87为实施例17的TP1引起的293TD37细胞照片
图88为实施例17的TP2引起的293TD37细胞照片
图89为实施例17的TP3引起的293TD37细胞照片
图90为实施例17的TP4引起的293TD37细胞病变照片
图91为实施例21用Western Blot检测非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L中CP312R蛋白结果示意图
图92为pS5E1-C129Rubiqutin-IRES-MGF5L6L的载体图谱
图93为pS5E4-EGFP的载体图谱
图94为pS5E4-CP312R-2A-MGF110-4L的载体图谱
图95为pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L的载体图谱
图96为实施例22的pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L诱导的CD8+T细胞反应结果示意图
图97为实施例22的pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L诱导的CD4+T细胞反应结果示意图
图98为实施例22的肌肉注射pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L后细胞免疫反应代表图
图99为实施例22的空白对照免疫反应代表图
图100为实施例23的I215L与pS5E1-IRES载体酶切电泳检测结果，其中泳道载体为pS5E1-IRES，NotI和XhoI双酶切，泳道I215L为片段I215L，NotI和XhoI双酶切，M为15000bp、2000bp Marker
图101为实施例23的I215L与pS5E1-IRES连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-11号为菌落，M为2000bp Marker
图102为实施例23的pS5E1-IRES-I215L质粒酶切电泳检测验证，选取图101中的5、6、7、8号菌落进行质粒提取，酶切验证，其中2号泳道为2号质粒酶切验证，9号泳道为9号质粒酶切验证，11号泳道为11号质粒酶切验证，M为Marker
图103为实施例23的L8L和ubiqutin融合后的L8Lubiqutin片段，并经胶回收纯化试剂盒纯化后的电泳检测结果，其中泳道1为L8Lubiqutin片段，M为Marker
图104为实施例23的片段L8Lubiqutin与pS5E1-IRES-I215L载体酶切产物电泳检测结果，其中泳道1为pS5E1-IRES-I215L质粒；泳道2为pS5E1-IRES-I215L质粒EcoRV和BamHI酶切；泳道3为L8Lubiqutin片段EcoRV和BamHI酶切，M为15000bp Marker
图105为实施例23的pS5E1-IRES-I215L载体与L8Lubiqutin连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-24号为菌落，M为2000bp Marker
图106为实施例23的pS5E1-L8Lubiqutin-IRES-I215L质粒酶切电泳检测验证，其中4、6、9、14、 17、18号泳道为选取图105中的4、6、9、14、17、18号菌落质粒BamHI、EcoRV酶切鉴定，M为15000bp Marker
图107为实施例24的融合PCR扩增I73Rhbsag和2A-E146L片段的电泳检测结果，其中泳道1为I73Rhbsag片段，泳道2为2A-E146L片段，M为2000bp Marker
图108为实施例24的片段pS5E4-EGFP载体酶切电泳检测结果，其中泳道1为片段pS5E4-EGFP，BamHI和XhoI双酶切胶回收，M为15000bp Marker
图109为实施例24的pS5E4-EGFP胶回收载体与I73Rhbsag片段、2A-E146L无缝克隆连接并转化感受态细胞菌落PCR验证电泳检测结果，其中1-12号为菌落，M为15000bp Marker
图110为实施例24挑取图109的1、2、3号阳性克隆提取质粒进行BamHI、XhoI双酶切验证的电泳检测结果，其中泳道1、2、3分别为1、2、3号阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker
图111为实施例25的pAd5LCL3与pS5E1-L8Lubiqutin-IRES-I215L琼脂糖凝胶验证电泳检测结果，其中泳道1为pAd5LCL3，泳道2为pS5E1-L8Lubiqutin-IRES-I215L
图112为实施例25的穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L和腺病毒载体质粒pAd5LCL3同源重组获得pAd5LCL3-L8Lubiqutin-IRES-I215L质粒的电泳检测结果，其中泳道1-7为pAd5LCL3-L8Lubiqutin-IRES-I215L克隆，M为15000bp Marker
图113为实施例25挑取图112的6号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1号泳道为pAd5LCL3-P72-IRES-B602L质粒XhoI酶切，M为15000bp Marker
图114为实施例25对穿梭质粒pS5E4-I73Rhbsag-2A-E146L和腺病毒载体质粒pAd5LCL3-L8Lubiqutin-IRES-I215L琼脂糖凝胶验证电泳检测结果，其中泳道1为pS5E4-I73Rhbsag-2A-E146L，泳道2为pAd5LCL3-L8Lubiqutin-IRES-I215L，M为15000bp Marker
图115为实施例25的穿梭质粒pS5E4-I73Rhbsag-2A-E146L和腺病毒载体质粒pAd5LCL3-L8Lubiqutin-IRES-I215L同源重组获得重组腺病毒载体pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L质粒的电泳检测结果，其中泳道1-8为质粒，M为15000bp Marker
图116为实施例25的挑取图115的2号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中2号泳道为pAd5LCL3-L8Lubiqutin-I215L-I73HbsAg-E146L质粒XhoI酶切，M为15000bp Marker
图117为实施例26的TP0引起的TP4293TD37细胞病变照片
图118为实施例26的TP1引起的TP4293TD37细胞病变照片
图119为实施例26的TP2引起的TP4293TD37细胞病变照片
图120为实施例26的TP3引起的TP4293TD37细胞病变照片
图121为实施例26的TP4引起的TP4293TD37细胞病变照片
图122为实施例29用Western Blot检测非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-L8Lubiqutin-I215L-I73HbsAg-E146L表达L8Lubiqutin和I215L蛋白结果示意图，其中泳道1为293空白细胞，泳道2、3为293TD37细胞感染pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L的样品，M为Marker
图123为pS5E1-L8Lubiqutin-IRES-I215L的载体图谱
图124为pS5E4-I73Rhbsag-2A-E146L的载体图谱
图125为pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF4L的载体图谱
图126为实施例30的pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L诱导的CD8+T细胞反应结果示意图
图127为实施例30的pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L诱导的CD4+T细胞反应结果示意图
图128为实施例31的肌肉注射pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L后细胞免疫反应代表图
图129为实施例31的空白对照免疫反应代表图
图130为实施例32的EP402R与pS5E1-IRES连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-6号为菌落，M为2000bp Marker
图131为实施例32的pS5E1-EP402R-IRES质粒酶切电泳检测验证，选取图130中的1、2、4号菌落进行质粒提取，酶切验证，其中1号泳道为1号质粒酶切验证，2号泳道为2号质粒酶切验证，4号泳道为4号质粒酶切验证，M为Marker
图132为实施例32的pS5E1-EP402R-IRES载体与EP153R载体酶切产物电泳检测结果，其中泳道1为pS5E1-EP402R-IRES，NotI、XhoI酶切，泳道2为EP153R片段，NotI、XhoI酶切，M为2000bp Marker
图133为实施例32的pS5E1-EP402R-IRES载体与EP153R连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-14号为菌落，M为5000bp Marker
图134为实施例32的pS5E1-F317L-IRES-A151R质粒酶切电泳检测验证，其中1、2、3、5、11、13号泳道为选取图27中的1、2、3、5、11、13号菌落质粒BamHI、EcoRV酶切鉴定，M为15000bp Marker
图135为实施例33的融合PCR扩增I177L-2A和K205Rubiqutin片段的电泳检测结果，泳道1为片段K205R；泳道2为片段ubiqutin；泳道3为片段K205Rubiqutin，M为2000bp Marker；泳道4为片段2A；泳道5为片段I177L；泳道6为片段I177L-2A，M为2000bp Marker
图136为实施例33的片段pS5E4-EGFP载体酶切电泳检测结果，其中泳道1为片段pS5E4-EGFP，BamHI和XhoI双酶切胶回收，M为15000bp Marker
图137为实施例33的pS5E4-EGFP胶回收载体与I177L-2A片段、K205Rubiqutin无缝克隆连接并转化感受态细胞菌落PCR验证电泳检测结果，其中1-3号为菌落，M为15000bp Marker
图138为实施例33挑取图137的1、2、3号阳性克隆提取质粒进行BamHI、XhoI双酶切验证的电泳检测结果，其中泳道1、2、3分别为1、2、3号阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker
图139为实施例34的pAd5LCL3与pS5E1-EP402R-IRES-EP153R琼脂糖凝胶验证电泳检测结果，其中泳道1为pAd5LCL3，泳道2为pS5E1-EP402R-IRES-EP153R
图140为实施例34的穿梭质粒pS5E1-EP402R-IRES-EP153R和腺病毒载体质粒pAd5LCL3同源重组获得pAd5LCL3-EP402R-IRES-EP153R质粒的电泳检测结果，其中泳道1-8为pAd5LCL3-EP402R-IRES-EP153R克隆，M为15000bp Marker
图141为实施例34挑取图140的2号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1号泳道为pAd5LCL3-EP402R-IRES-EP153R质粒XhoI酶切，2号泳道为pAd5LCL3-EP402R-IRES-EP153R质粒PacI酶切，3号泳道为pAd5LCL3-EP402R-IRES-EP153R质粒BamHI酶切，M为15000bp Marker
图142为实施例34的穿梭质粒pS5E4-I177L-2A-K205Rubiqutin和腺病毒载体质粒 pAd5LCL3-EP402R-IRES-EP153R同源重组获得重组腺病毒载体pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒的电泳检测结果，其中泳道1-7为质粒，M为15000bp Marker
图143为实施例34的挑取图142的1号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中泳道1、2为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒XhoI酶切，泳道3为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒BamHI酶切，泳道4为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒PacI酶切，M为15000Marker
图144为实施例35的TP0引起的TP4293TD37细胞病变照片
图145为实施例35的TP1引起的TP4293TD37细胞病变照片
图146为实施例35的TP2引起的TP4293TD37细胞病变照片
图147为实施例35的TP3引起的TP4293TD37细胞病变照片
图148为实施例35的TP4引起的TP4293TD37细胞病变照片
图149为实施例39用Western Blot检测非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-EP402R-EP153R-K205R-I177Lubiqutin中EP153R蛋白结果示意图
图150为pS5E1-EP402R-IRES-EP153R的载体图谱
图151为pS5E4-I177L-2A-K205Rubiqutin的载体图谱
图152为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin的载体图谱
图153为实施例40的ELISA法检测血清中针对非洲猪瘟目的蛋白EP402R的IgG抗体滴度结果示意图
图154为实施例40的pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin诱导的CD8+T细胞反应结果示意图
图155为实施例40的pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin诱导的CD4+T细胞反应结果示意图
图156为实施例40的肌肉注射pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin后细胞免疫反应代表图
图157为实施例40的空白对照免疫反应代表图
图158为实施例41的F317L与pS5E1-IRES载体酶切电泳检测结果，其中泳道1为pS5E1-IRES，BamHI和EcorV双酶切，泳道2为片段F317L，BamHI和EcoRV双酶切，M为15000bp Marker
图159为实施例41的F317L与pS5E1-IRES连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-14号为菌落，M为2000bp Marker
图160为实施例41的pS5E1-F317L-IRES质粒酶切电泳检测验证，选取图159中的9、10号菌落进行质粒提取，酶切验证，其中1号泳道为1号质粒酶切验证，2号泳道为9号质粒酶切验证，M为Marker
图161为实施例41的片段A151R与pS5E1-F317L-IRES载体酶切产物电泳检测结果，其中泳道1为pS5E1-F317L-IRES，NotI和XhoI酶切；泳道2、3为A151R片段，NotI和XhoI酶切，M为15000bp Marker
图162为实施例41的pS5E1-F317L-IRES载体与A151R连接产物转化感受态细胞菌落PCR验证电泳检测结果，其中1-24号为菌落，M为2000bp Marker
图163为实施例41的pS5E1-F317L-IRES-A151R质粒酶切电泳检测验证，其中1、2、3、4号泳道为选取图162中的4、15、23、24号菌落质粒BamHI、EcoRV酶切鉴定，M为2000bp Marker
图164为实施例42的目的片段P34、2A、pp62的PCR产物电泳检测结果，其中泳道1为片段p34；2为片段2A；3为片段pp62，M为15000bp Marker
图165为实施例42的融合PCR扩增P34-2A片段的电泳检测结果，其中泳道1为P34-2A片段，M为2000bp Marker
图166为实施例42的片段pS5E4-EGFP载体酶切电泳检测结果，其中泳道1为片段pS5E4-EGFP，BamHI和XhoI双酶切胶回收，M为15000bp Marker
图167为实施例42的pS5E4-EGFP胶回收载体与P34-2A片段、pp62无缝克隆连接并转化感受态细胞菌落PCR验证电泳检测结果，其中1-12号为菌落，M为15000bp Marker
图168为实施例42挑取图167的1、2、9、11号阳性克隆提取质粒进行BamHI、XhoI双酶切验证的电泳检测结果，其中泳道1、2、3、4分别为1、2、9、11号阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker
图169为实施例43的pAd5LCL3与pS5E1-F317L-IRES-A151R琼脂糖凝胶验证电泳检测结果，其中泳道1为pAd5LCL3，泳道2为pS5E1-F317L-IRES-A151R，M为15000bp Marker
图170为实施例43的穿梭质粒pS5E1-F317L-IRES-A151R和腺病毒载体质粒pAd5LCL3同源重组获得pAd5LCL3-F317L-IRES-A151R质粒的电泳检测结果，其中泳道1-7为pAd5LCL3-F317L-IRES-A151R，M为15000bp Marker
图171为实施例43挑取图170的3号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中1、2号泳道为pAd5LCL3-P72-IRES-B602L质粒XhoI酶切，M为15000bp Marker
图172为实施例43对穿梭质粒pS5E4-P34-2A-pp62和腺病毒载体质粒pAd5LCL3-F317L-IRES-A151R琼脂糖凝胶验证电泳检测结果，其中泳道1为pS5E4-P34-2A-pp62，泳道2为pAd5LCL3-F317L-IRES-A151R，M为15000bp Marker
图173为实施例43的穿梭质粒pS5E4-P34-2A-pp62和腺病毒载体质粒pAd5LCL3-F317L-IRES-A151R同源重组获得重组腺病毒载体pAd5LCL3-F317L-A151R-P34-pp62质粒的电泳检测结果，其中泳道1-4为质粒，M为15000bp Marker
图174为实施例43的挑取图173的2号阳性质粒转化至感受态细胞，提取质粒进行酶切验证检测结果，其中2号泳道为pAd5LCL3-F317L-A151R-P34-pp62质粒XhoI酶切，M为15000bp Marker
图175为实施例44的TP0引起的TP4293TD37细胞病变照片
图176为实施例44的TP1引起的TP4293TD37细胞病变照片
图177为实施例44的TP2引起的TP4293TD37细胞病变照片
图178为实施例44的TP3引起的TP4293TD37细胞病变照片
图179为实施例44的TP4引起的TP4293TD37细胞病变照片
图180为实施例48用Western Blot检测非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-F317L-A151R-P34-pp62中pp62蛋白结果示意图
图181为pS5E1-F317L-IRES-A151R的载体图谱
图182为pS5E4-P34-2A-pp62的载体图谱
图183为pAd5LCL3-F317L-A151R-P34-pp62的载体图谱
图184为实施例49的ELISA法检测血清中针对非洲猪瘟目的蛋白pp62的IgG抗体滴度结果示意图
图185为实施例49的pAd5LCL3-F317L-A151R-P34-pp62诱导的CD8+T细胞反应结果示意图
图186为实施例49的pAd5LCL3-F317L-A151R-P34-pp62诱导的CD4+T细胞反应结果示意图
图187为实施例49的肌肉注射pAd5LCL3-F317L-A151R-P34-pp62后细胞免疫反应代表图
图188为实施例49的空白对照免疫反应代表图
具体实施方式
下面结合附图对本发明的优选实施例作进一步详细描述，需要指出的是，以下所述实施例旨在便于对本发明的理解，而对其不起任何限定作用。
实施例1缺失E1和E3基因的腺病毒载体质粒pAd5的构建
在A549细胞
Figure PCTCN2021104793-appb-000001
中扩增野生型人腺病毒5型(
Figure PCTCN2021104793-appb-000002
VR-1516，基因序列AC_000008.1)病毒，收集并进行浓缩病毒液，采用HirtVirual DNA Extract方法提取腺病毒基因组，使用cosmid的方法将线性的hAd5基因组构建成环状的supercos-Ad5载体质粒，利用CRISPR/cas9对hAd5腺病毒E1区域进行切除，设计gRNA如下：
hAd5-E1上游gRNA:
Figure PCTCN2021104793-appb-000003
hAd5-E1下游gRNA:
Figure PCTCN2021104793-appb-000004
在hAd5E1区域的上下游设计gRNA位点，切割后回收大片段载体，设计引物，通过融合PCR将ITR，PIX序列分别插入上、下游，并引入SwaI酶切位点，然后将融合后的片段与载体进行无缝克隆，获得E1敲除的supercos-Ad5△E1腺病毒载体，而后对supercos-Ad5△E1质粒进行E3区域的切除，设计gRNA如下：
hAd5-E3上游gRNA:
Figure PCTCN2021104793-appb-000005
hAd5-E3下游gRNA:
Figure PCTCN2021104793-appb-000006
在hAd5E3区域的上下游设计gRNA位点，切割后回收大片段载体，后设计引物，对E3上下游过多切除的Fiber，以及pVIII序列进行融合PCR，使用无缝克隆的方式连接，获得缺失E1和E3基因，并引入SwaI酶切位点的腺病毒载体质粒pAd5。
实施例2缺失E1、E3和E4基因的腺病毒载体质粒pAd5△E4的构建
采用实施例1获得的已经敲除了E1、E3基因的载体质粒pAd5，进一步敲除敲除E4基因，能提高腺病毒载体的容量，降低其免疫原性，并使用PCR的方法扩增部分fiber以及引入NdeI单酶切位点，再使用Gibson的无缝克隆方法，将多余切除的片段连接至载体上，获得缺失E1、E3和E4基因，并引入SwaI和I-sceI酶切位点的载体质粒pAd5△E4。
1、目的基因E4CRISPR靶序列的选择
1)E4基因上游fiber基因CRISPR靶序列的选择
使用赛默飞GeneArt TMCRISPR Search and Design tool(thermofisher.com/crisprdesign)软件，输入fiber基因的前400个碱基，软件自动分析该400个碱基的序列，提供了6个潜在的CRISPR靶序列。考虑到E4基因敲除序列的长度，以及构建活载体的要求，选择GCTACTAAACAATTCCTTCC作为靶向序列，最终获得的gRNA命名为Ad5-E4-up-gRNA，裂解位点和PAM位点如图1所示。
2)E4下游非编码序列CRISPR靶序列的选择
使用赛默飞GeneArt TMCRISPR Search and Design tool(thermofisher.com/crisprdesign)软件，输入E4下游的300个碱基，软件自动分析，提供了6个潜在的CRISPR靶序列，选择AGGTTCGCGTGCGGTTTTCT作为靶向序列，最终获得的gRNA命名为Ad5-E4-down-gRNA，裂解位点和PAM位点如图2所示。
2、Ad5-E4-up-gRNA和Ad5-E4-down-gRNA的DNA扩增
1)Ad5-E4-up-gRNA的DNA模板设计
5’-TAATACGACTCACTATAG TACTAAACAATTCCTTCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’
2)Ad5-E4-down-gRNA的DNA模板设计
5’-TAATACGACTCACTATAG GTTCGCGTGCGGTTTTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’
3、设计扩增Ad5-E4-up-gRNA和Ad5-E4-down-gRNA的DNA模板的上下游引物
设计上下游引物分别进行PCR扩增Ad5-E4-up-gRNA的DNA模板和Ad5-E4-down-gRNA的DNA模板，使用GeneArt TM Precision gRNA Synthesis Kit试剂盒进行扩增。
引物设计：
Ad5-E4-up-gRNA-Forward：TAATACGACTCACTATAGTACTAAACAATTCCT
Ad5-E4-up-gRNA-Reverse：TTCTAGCTCTAAAACGGAAGGAATTGTTTAGTA
Ad5-E4-down-gRNA-Forward：TAATACGACTCACTATAGGTTCGCGTGCGGTTT
Ad5-E4-down-gRNA-Reverse：TTCTAGCTCTAAAACAGAAAACCGCACGCGAAC
4、扩增Ad5-E4-up-gRNA和Ad5-E4-down-gRNA的DNA模板
1)准备0.3μM的Ad5-E4-up-gRNA-Forward/Reverse引物混合工作液
10μM的Ad5-E4-up-gRNA-Forward引物3ul，10μM的Ad5-E4-up-gRNA-Reverse引物3ul，补充水至100ul。
2)准备0.3μM的Ad5-E4-down-gRNA-Forward/Reverse引物混合工作液
10μM的Ad5-E4-down-gRNA-Forward引物3ul，10μM的Ad5-E4-down-gRNA-Reverse引物3ul，补充水至100ul。
3)PCR反应体系
Ad5-E4-up-gRNA的DNA模板扩增的PCR反应体系为：Phusion TM High-Fidelity PCR Master Mix(2X)12.5ul，Tracr Fragment+T7 Primer Mix 1ul，0.3μM Ad5-E4-up-gRNA-Forward/Reverse引物混合工作液1ul，补水至25ul。
Ad5-E4-down-gRNA的DNA模板扩增的PCR反应体系为：Phusion TM High-Fidelity PCR Master Mix(2X)12.5ul，Tracr Fragment+T7 Primer Mix 1ul，0.3μM Ad5-E4-down-gRNA-Forward/Reverse引物混合 工作液1ul，补水至25ul。
4)PCR程序
起始变性98℃，10sec，1循环；变性98℃，5sec；退火55℃，15sec，32循环；延伸72℃，1min，1循环；保持4℃。
5、体外转录获得Ad5-E4-up-gRNA和Ad5-E4-down-gRNA
使用TranscriptAid TM Enzyme Mix对模板DNA进行体外转录，获得Ad5-E4-up-gRNA和Ad5-E4-down-gRNA。
体外转录获得Ad5-E4-up-gRNA的反应体系为：NTP mix 8ul，E1A-gRNA DNA模板6ul，5X TranscriptAid TM Reaction Buffer 4ul，TranscriptAid TM Enzyme Mix 2ul。于37℃孵育4小时后加入1ul的DNase I，于37℃孵育15分钟。
体外转录获得Ad5-E4-down-gRNA的反应体系为：NTP mix 8ul，E1B-gRNA DNA模板6ul，5X TranscriptAid TM Reaction Buffer 4ul，TranscriptAid TM Enzyme Mix 2ul。于37℃孵育4小时后加入1ul的DNase I，于37℃孵育15分钟。
体外转录获得Ad5-E4-up-gRNA、Ad5-E4-down-gRNA
6、体外转录产物的纯化
1)将转录后的反应体系用无核酸酶水补充至200ul；
2)加入100ul的Binding buffer，充分混匀；
3)加入300ul的乙醇(>96％)，充分混匀；
4)将混合液转移到the Gene JET TM RNA Purification Micro Column中，14000×g离心30-60秒，弃下液；
5)加入700ul的Wash Buffer1(加入13mL乙醇)，14000×g离心30-60秒，弃下液；
6)加入700ul的Wash Buffer2(加入30mL乙醇)，14000×g离心30-60秒，弃下液，重复上述步骤一次；
7)14000×g空离60秒，将所有的洗脱液完全去除，将空管放置于1.5mL的收集管中；
8)在柱子中心加入10ul的无核酸酶水，14000×g离心60秒收集gRNA。
其中，Wash Buffer1和Wash Buffer2均为TranscriptAid TM Enzyme Mix试剂盒中的试剂，转录获得的Ad5-E4-up-gRNA和Ad5-E4-down-gRNA的RNA序列如下所示：
Ad5-E4-up-gRNA:G UACUAAACAAUUCCUUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
Ad5-E4-down-gRNA:G GUUCGCGUGCGGUUUUCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
7、CRISPR/Cas9“酶切”
使用Ad5-E4-up-gRNA、Ad5-E4-down-gRNA和cas9双酶切实施例1获得载体质粒，反应体系为Cas9蛋白3μg，Ad5-E4-up-gRNA 6μg，Ad5-E4-down-gRNA 6μg，pAd5-REBP载体质粒3μg，NEB buffer 3.1 5ul，补充水至50ul。
酶切反应于37℃孵育过夜。取3ul样品进行琼脂糖凝胶验证，实验结果电泳图如图3所示。泳道1为Ad5-E4-up-gRNA、Ad5-E4-down-gRNA以及cas9“双酶切”pAd5载体质粒结果，出现了目的大小在2500bp-5000bp的片段，可见酶切结果正确。使用Axygen胶回收试剂盒将载体进行纯化。
8、获得含有部分敲除的fiber、ITR片段并引入I-SceI酶切位点，敲除使用含有敲除部分fiber的引物，扩增fiber片段并引入I-SceI酶切位点
1)片段fiber的扩增
扩增引物：
Fiber-RH-F：GAGTGCTACTAAACAATTCCTTCCTGGACCCAGAATATTGG
Fiber-ISceI-ITR-R：TGGTGTTATTACCCTGTTATCCCTA GCAATTGAAAAATAAACACGTTG
扩增序列为：
Figure PCTCN2021104793-appb-000007
扩增体系为：10μM Fiber-RH-F引物1ul；10μM Fiber-ISceI-ITR-R引物1ul；模板pAd5(100ng/ul)0.5ul；Q5高保真酶25ul；补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，10sec，35循环；延伸72℃，5min，1循环；保持4℃。扩增结果电泳图如图4所示，泳道1为fiber部分片段扩增结果，M为2000maker，可见扩增结果正确，使用Axygen胶回收试剂盒将片段进行纯化。
2)ITR片段的扩增
扩增引物：
ISceI-ITR-F： TAGGGATAACAGGGTAATAACACCACTCGACACGGCAC
ITR-RH-R：GGCGTAGGTTCGCGTGCGGTTTTCTGGGTGTTTTTTGTGGACTT
扩增序列为：
Figure PCTCN2021104793-appb-000008
扩增体系为：10μM ISceI-ITR-F引物1ul；10μM ITR-RH-R引物1ul；模板pAd5(100ng/ul)0.5ul；Q5高保真酶25ul；补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，10sec，35循环；延伸72℃，5min，1循环；保持4℃。扩增结果如图4所示，泳道2为ITR部分片段扩增结果，M为2000maker，可见扩增结果正确，使用Axygen胶回收试剂盒将片段进行纯化。
3)融合PCR获得Fiber-ITR的融合片段
扩增体系为：10μM Fiber-RH-F引物1ul，10μM Fiber-ISceI-ITR-R引物1ul，模板pAd5(100ng/ul)0.5ul，Q5高保真酶25ul，补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，20sec，35循环；延伸72℃，5min，1循环；保持4℃。扩增结果如图5所示，泳道1为Fiber-ITR的融合片段，M为2000maker，可见融合结果正确。使用Axygen胶回收试剂盒将片段进行纯化。
9、载体连接
使用NEB的Gibson使Fiber-ITR片段与敲除E4后的载体质粒连接，连接体系如下所示：胶回收产物载体质粒片段100ng，胶回收产物fiber-ITR片段50ng，Gibson预混液10ul，补水至20ul。于50℃，孵育40分钟。
10、转化
取出卡那抗性培养基平板，将制备好的NEB 10β感受态细胞放在冰上融化后，加入10ul连接产物，用移液器轻轻吸打均匀，在冰上放置30分钟；将离心管放置42℃水浴，热击90秒，利用卡那青霉素抗性筛选转化子。
11、菌落PCR进行转化子筛选
使用PCR扩增方式对转化子进行菌落PCR验证。
设计菌落PCR的下游引物
E4-cexu-F：AGTGACGATTTGAGGAAGTTG
E4-cexu-R：TCAATTGCAGAAAATTTCAAGTC
反应体系为：10μM E4-cexu-F引物1ul，10μM E4-cexu-R引物1ul，Q5高保真酶10ul，补水至20ul，挑取单克隆菌落于反应体系中。PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，20sec，35循环；延伸72℃，5min，1循环；保持4℃。进行琼脂糖凝胶电泳验证，如图6所示，除了2、8、11、17号，大部分菌落出现了阳性条带。
12、质粒酶切验证
挑取4个阳性克隆菌落，提取质粒，进行BamHI、XhoI酶切验证，酶切结果如图7所示，从图7可以看出，2-5号质粒BamHI、XhoI酶切结果均正确，同时测序结果正确，即获得了缺失E1、E3和E4基因的腺病毒载体质粒pAd5△E4。
实施例3缺失E1、E3、E4和E2a基因的腺病毒载体质粒pAd5LCL3的构建
1、目的基因E2a CRISPR靶序列的选择
1)E2a基因上游100k基因CRISPR靶序列的选择
使用赛默飞GeneArt TMCRISPR Search and Design tool(thermofisher.com/crisprdesign)软件，输入100k基因的前400个碱基，软件自动分析该400个碱基的序列，提供了6个潜在的CRISPR靶序列。考虑到E2a基因敲除序列的长度，以及构建活载体的要求，选择ATAGGTGGCGTTCGTAGGCA作为靶向序列，最终获得的gRNA命名为100k-gRNA，裂解位点和PAM位点如图8所示。
2)E2a下游非编码序列CRISPR靶序列的选择
使用赛默飞GeneArt TMCRISPR Search and Design tool(thermofisher.com/crisprdesign)软件，输入E4下游的300个碱基，软件自动分析，提供了6个潜在的CRISPR靶序列，选择TACCCCGGTAATAAGGTTCA作为靶向序列，最终获得的gRNA命名为protease-gRNA，裂解位点和PAM位点图9所示。
2、100k-gRNA和protease-gRNA的DNA扩增
1)100k-gRNA的DNA模板设计
5’-TAATACGACTCACTATAG AGGTGGCGTTCGTAGGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’
2)protease-gRNA的DNA模板设计
5’-TAATACGACTCACTATAG CCCCGGTAATAAGGTTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3’
3、设计扩增100k-gRNA和protease-gRNA的DNA模板的上下游引物
设计上下游引物分别进行PCR扩增100k-gRNA的DNA模板和protease-gRNA的DNA模板，使用GeneArt TM Precision gRNA Synthesis Kit试剂盒进行扩增。
1)引物设计
100k-gRNA-Foward：TAATACGACTCACTATAG AGGTGGCGTTCGTAG
100k-gRNA-Reverse：TTCTAGCTCTAAAAC TGCCTACGAACGCCACCT
protease-gRNA-Foward：TAATACGACTCACTATAG CCCCGGTAATAAGGT
protease-gRNA-Reverse：TTCTAGCTCTAAAAC TGAACCTTATTACCGGGG
2)扩增100k-gRNA和protease-gRNA的DNA模板
①准备0.3μM的100k-gRNA-Forward/Reverse引物混合工作液，包括10μM的100k-gRNA-Forward引物3ul，10μM的100k-gRNA-Reverse引物3ul，补充水至100ul。
②准备0.3μM的Aprotease-gRNA-Forward/Reverse引物混合工作液，包括10μM的protease-gRNA-Forward引物3ul，10μM的protease-gRNA-Reverse引物3ul，补充水至100ul。
③PCR反应体系
100k-gRNA的DNA模板扩增的PCR反应体系为：Phusion TM High-Fidelity PCR Master Mix(2X)12.5ul，Tracr Fragment+T7 Primer Mix 1ul，0.3μM 100k-gRNA-Forward/Reverse引物混合工作液1ul，补水至25ul。
protease-gRNA的DNA模板扩增的PCR反应体系为：Phusion TM High-Fidelity PCR Master Mix(2X)12.5ul，Tracr Fragment+T7 Primer Mix 1ul，0.3μM protease-gRNA-Forward/Reverse引物混合工作液1ul，补水至25ul。
④PCR程序
起始变性98℃，10sec，1循环；变性98℃，5sec；退火55℃，15sec，32循环；延伸72℃，1min，1循环；保持4℃。
3、体外转录获得100k-gRNA和protease-gRNA
使用TranscriptAid TM Enzyme Mix对模板DNA进行体外转录，获得100k-gRNA和protease-gRNA。
1)体外转录获得100k-gRNA、protease-gRNA
体外转录获得100k-gRNA的反应体系为：NTP mix 8ul，100k-gRNA DNA模板6ul，5X TranscriptAid TM Reaction Buffer 4ul，TranscriptAid TM Enzyme Mix 2ul。于37℃孵育4小时后加入1ul的DNase I，于37℃孵育15分钟。
体外转录获得protease-gRNA的反应体系为：NTP mix 8ul，protease-gRNA DNA模板6ul，5X TranscriptAid TM Reaction Buffer 4ul，TranscriptAid TM Enzyme Mix 2ul。于37℃孵育4小时后加入1ul的DNase I，于37℃孵育15分钟。
2)体外转录产物的纯化
将转录后的反应体系用无核酸酶水补充至200ul，加入100ul的Binding buffer，充分混匀，加入300ul的乙醇(>96％)，充分混匀，将混合液转移到the Gene JET TM RNA Purification Micro Column中，14000×g离心30-60秒，弃下液；加入700ul的Wash Buffer1(加入13mL乙醇)，14000×g离心30-60秒，弃下 液；加入700ul的Wash Buffer2(加入30mL乙醇)，14000×g离心30-60秒，弃下液，重复上述步骤一次。14000×g空离60秒，将所有的洗脱液完全去除，将空管放置于1.5mL的收集管中，在柱子中心加入10ul的无核酸酶水，14000×g离心60秒收集gRNA。
其中，Wash Buffer1和Wash Buffer2均为TranscriptAid TM Enzyme Mix试剂盒中的试剂，转录获得的100k-gRNA和protease-gRNA的RNA序列如下所示：
100k-gRNA:G AGGUGGCGUUCGUAGGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
protease-gRNA:G CCCCGGUAAUAAGGUUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU
4、CRISPR/Cas9“酶切”
使用100k-gRNA、protease-gRNA和cas9双酶切实施例2获得的缺失E1、E3和E4基因的腺病毒载体质粒，反应体系为Cas9蛋白3μg；100k-gRNA 6μg；protease-gRNA 6μg；实施例2获得的载体质粒3μg；NEB buffer 3.1 5ul；补充水至50ul。
上述酶切反应分别于37℃孵育过夜。取3ul样品进行琼脂糖凝胶验证，实验结果如图10所示。泳道1为100k-gRNA、protease-gRNA以及cas9“双酶切”载体质粒结果，出现了目的大小在1000-2500bp的片段，可见酶切结果正确。使用Axygen胶回收试剂盒将载体进行纯化。
5、获得含有部分敲除的100k、E4 ORF6/7表达框、Protease片段
1)部分敲除的100k、E4 ORF6/7表达框、Protease片段的扩增
①部分敲除的100k扩增引物：
100k-F：TGAGAATAGGTGGCGTTCGTAGGCAAGGCTGACATCCGCTATGG
100k-ORF6/7-R：TACAATTCCCAACACATACAAGTTTCCTTCTCCTATAGGCAGAA
扩增体系为：10μM 100k-F引物1ul；10μM 100k-ORF6/7-R引物1ul；模板pAd5△E4(100ng/ul)0.5ul；Q5高保真酶25ul；补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，20sec，35循环；延伸72℃，5min，1循环；保持4℃。
②E4 ORF6/7表达框扩增引物：
ORF6/7-F：ACTTGTATGTGTTGGGAATTGTA
ORF6/7-R：ATCGTTTGTGTTATGTTTCAACG
扩增体系为：ORF6/7-F引物1ul；10μM ORF6/7-R引物1ul；模板ORF6/7表达框基因(100ng/ul)0.5ul；Q5高保真酶25ul；补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，10sec，35循环；延伸72℃，5min，1循环；保持4℃。
③部分敲除的Protease片段的扩增
ORF6/7-Protease-F：CCCACCCTTGCCGTCTGCGCCGTATCGTTTGTGTTATGTTTCAACG
Protease-R：ATGGATCACAACCCCACCATGAACCTTATTACCGGGGTACCCA
扩增体系为：10μM ORF6/7-Protease-F引物1ul；10μM Protease-R引物1ul；模板pAd5△E4(100ng/ul)0.5ul；Q5高保真酶25ul；补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃， 10sec，35循环；延伸72℃，5min，1循环；保持4℃。
④100k、E4 ORF6/7表达框、protease PCR扩增结果如图11所示，其中泳道1为E4 ORF6/7表达框，泳道2为100k，M为15000bpMarker
可见扩增结果正确，使用Axygen胶回收试剂盒将片段分别进行胶回收纯化。
6、融合PCR获得100k、E4 ORF6/7表达框、Protease片段的融合片段
扩增体系为：10μM 100k-F引物1ul；10μM Protease-R引物1ul；模板100k胶回收产物(50ng/ul)1ul模板E4 ORF6/7表达框胶回收产物(50ng/ul)1ul模板E4 ORF6/7表达框胶回收产物(50ng/ul)1ul；Q5高保真酶25ul；补水至50ul。
PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，50sec，35循环；延伸72℃，5min，1循环；保持4℃。扩增结果如图12所示，其中泳道1为片段100k、E4 ORF6/7表达框、protease融合PCR产物，可见扩增结果正确。使用Axygen胶回收试剂盒将片段进行纯化。
7、载体连接
使用NEB的Gibson使100k、E4 ORF6/7表达框、protease融合PCR胶回收产物与步骤4中敲除E2a后的载体连接，连接体系如下所示：胶回收产物敲除E2a后的载体片段100ng，胶回收产物100k、E4 ORF6/7表达框、protease融合PCR片段50ng，Gibson预混液10ul，补水至20ul。于50℃，孵育40分钟。
8、转化
取出卡那抗性培养基平板，将制备好的NEB 10β感受态细胞放在冰上融化后，加入10ul连接产物，用移液器轻轻吸打均匀，在冰上放置30分钟；将离心管放置42℃水浴，热击90秒，利用卡那青霉素抗性筛选转化子。
9、菌落PCR进行转化子筛选
使用PCR扩增方式对转化子进行菌落PCR验证。
设计菌落PCR的下游引物
DBP-upsteam-F：GTTGGGCTCGCATGTGCCG
DBP-downsteam-R：ACTCCCATGGATCACAACCC
反应体系为：10μM DBP-upsteam-F引物1ul，10μM DBP-downsteam-R引物1ul，Q5高保真酶10ul，补水至20ul，挑取单克隆菌落于反应体系中。PCR程序为：起始变性98℃，10sec，1循环；变性98℃，5sec；退火60℃，30sec；延伸72℃，20sec，35循环；延伸72℃，5min，1循环；保持4℃。进行琼脂糖凝胶电泳验证，如图13所示，在9、18、21、24出现阳性条带。
10、质粒酶切验证
挑取9、18、21、24这4个阳性克隆菌落，提取质粒，进行XhoI酶切验证，酶切结果如图14所示，其中泳道1为9号阳性克隆XhoI酶切，泳道2为18号阳性克隆XhoI酶切，泳道3为21号阳性克隆XhoI酶切，泳道4为24号阳性克隆XhoI酶切，泳道5为对照质粒pAd5LCL3的XhoI酶切。从图14可以看出，质粒XhoI酶切结果均正确，同时测序结果正确，即获得了缺失E1、E3、E4和E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置的pAd5LCL3质粒，其载体图谱如图50所示。
实施例4非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-P72-IRES-B602L的构建
1、人类腺病毒5型载体E1区域穿梭质粒的构建
穿梭质粒pS5E1的骨架采用puc origin、amp等基本元素(2796bp)(pS5E1骨架由北京博迈德基因技术有限公司合成)，Ad5左臂ITR部分序列(355bp)，右臂PIX、PIVa2部分序列(2100bp)，以及CMV-MCS(Seq ID NO.12)(944bp)SV40 early polyA(Seq ID NO.13)(160bp)。
1)引物设计
puc-Ad5-right arm-F：TAATGCAGCTGGCTTATCGAAACGTGGAATGCGAGACCGTCT
Ad5-right arm-CMV-R：ACACACAAGCAGGGAGCAGATACAAGGGTGGGAAAGAATATATAAG
CMV-F：GTATCTGCTCCCTGCTTGTG
CMV-SV40-R：TAAACAAGTTGGGGTGGGCGAAGTGATCAGCGGGTTTAAACGGG
SV40-F：CTTCGCCCACCCCAACTTGT
SV40-R：AGAGGTCGACGGTATACAGAC
SV40-Ad5-left arm-F：TGTCTGTATACCGTCGACCTCTCCGAAAAACACCTGGGCGAGTCTCC
Ad5-left arm-puc-R：ACACTATAGAATACACGGAATTCTTAATTAAATCATCAATAATATACCTTATTTTG
puc-F：GAATTCCGTGTATTCTATAGTGT
puc-R：TTTCGATAAGCCAGCTGCATTA
2)目的片段的扩增
①以pCDNA3.1(+)为模板(该质粒购自赛默飞公司)，以CMV-F和CMV-SV40-R为引物，扩增pS5E1穿梭质粒的CMV启动子MCS片段；扩增体系：pCDNA3.1(+)质粒50ng，10uM CMV-F引物1ul，10uM CMV-SV40-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、1min，35个循环；72℃，5min。
②以pCDNA3.1(+)为模板(该质粒购自赛默飞公司)，以SV40-F和SV40-R为引物，扩增pS5E1穿梭质粒的SV40-earlypolyA片段；扩增体系：pCDNA3.1(+)质粒50ng，10uM SV40-F引物1ul，10uM SV40-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、10sec，35个循环；72℃，5min。
扩增产物琼脂糖验证如图15所示，其中泳道1为CMV-MCS片段，泳道2为SV40 earlypolyA片段，M为2000Marker。由图15可见，扩增结果正确。
③使用Axygen胶回收试剂盒进行纯化。
④以博迈德公司合成的pS5E1骨架质粒为模板，以puc-F和puc-R为引物，PCR扩增pS5E1穿梭质粒骨架，扩增体系：pS5E1骨架质粒50ng，10uM puc-F引物1ul，10uM puc-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、1min20sec，35个循环；72℃，5min。
⑤以pAd5LCL3质粒为模板，以SV40-Ad5-left arm-F和Ad5-left arm-puc-R为引物，扩增pS5E1穿梭质粒的左臂，扩增体系：pAd5LCL3质粒50ng，10uM SV40-Ad5-left arm-F引物1ul，10uM Ad5-left arm-puc-R引物1ul，Q5高保真酶20ul，补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。
⑥以pAd5LCL3质粒为模板，以puc-Ad5-right arm-F和Ad5-right arm-CMV-R为引物，扩增pS5E1穿梭质粒的右臂，扩增体系：pAd5LCL3质粒50ng，10uM puc-Ad5-right arm-F引物1ul，10uM Ad5-right arm-CMV-R引物1ul，Q5高保真酶20ul，补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、 30s，72℃、15s，35个循环；72℃，5min。
⑦以胶回收产物CMV-MCS为模板，以CMV-F和SV40-R为引物，扩增pS5E1穿梭质粒的CMV-MCS-SV40 earlypolyA片段，扩增体系：pAd5LCL3质粒50ng，10uM CMV-F引物1ul，10uM SV40-R引物1ul，Q5高保真酶20ul，补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40s，35个循环；72℃，5min。
扩增产物琼脂糖验证如图16所示，其中泳道1为CMV-MCS-SV40 earlypolyA融合片段，泳道2为PUC，泳道3为Ad5右臂，泳道4为Ad5左臂。
3)片段的连接转化
使用Axygen胶回收试剂盒将片段进行纯化，然后使用博迈德公司无缝克隆试剂盒将pS5E1骨架、Ad5左臂、Ad5右臂、CMV-MCS-SV40 earlypolyA这四个片段连接，连接体系为2×Smealess Cloning Mix 10ul、pS5E1骨架片段50ng、Ad5左臂50ng、Ad5右臂50ng、CMV-MCS-SV40 polyA 50ng、补水至20ul，50℃保温40分钟，获得连接产物质粒pS5E1。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
4)质粒的验证
①菌落PCR验证
挑选菌落进行琼脂糖凝胶验证，结果如图17所示，出现阳性条带。
②酶切验证
挑取阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行酶切验证，电泳结果如图18所示，其中左1-6为质粒pS5E1NcoI单酶切，右1-6为质粒pS5E1 PacI单酶切，M为15000bp Marker，酶切结果正确，成功构建人类腺病毒5型载体E1区域穿梭质粒pS5E1，其载体图谱如图51所示。
2、非洲猪瘟腺病毒5型载体穿梭质粒pS5E1-P72-IRES-B602L构建
1)pS5E1与IRES片段的连接
①引物合成
IRES-EcoRV-F：ccg GATATC TGTCGTCATCATCCTTATAGTCC
IRES-NotI-R：aaatat GCGGCCGC GGTTGTGGCCATTATCATCGTG
②扩增IRES片段
扩增体系：Q5酶25ul，10uM引物IRES-EcoRV-F 1ul，10uM引物IRES-NotI-R 1ul，模板IRES模板2ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。扩增结果电泳检测如图19所示，其中泳道1、2为IRES片段PCR扩增产物，M为15000bp ladder，可见扩增结果正确。
③使用Axygen PCR纯化试剂盒纯化IRES片段。
④目的片段IRES与pS5E1载体酶切
酶切反应体系：载体pS5E1、IRES片段～2ug，EcoRV和NotI各1ul；10×cutsmart buffer 5ul；补水至50ul；反应条件：37℃，30min；65℃，20min灭活；胶回收纯化。酶切产物电泳检测如图20所示，其中泳道1为片段IRES EcoRV，NotI酶切，泳道2为pS5E1 EcoRV，NotI酶切，M为15000bp ladder。
⑤pS5E1载体与IRES片段连接
连接体系：pS5E1(100ng)；IRES片段(载体：片段＝1：5,摩尔比)；T4 DNA连接酶1ul；10×ligase  buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物IRES-EcoRV-F 1ul，10uM引物IRES-NotI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。进行电泳验证，如图21所示，其中1-9号为菌落，M为Marker，由图21可见，2和6号出现阳性条带。
⑦质粒NotI、EcoRV酶切验证，选取2、6进行质粒提取，酶切验证，结果如图22所示，其中2号质粒NotI、EcoRV酶切鉴定，6号质粒NotI、EcoRV酶切鉴定，可见酶切结果正确。
2)pS5E1-IRES与P72片段的连接
①引物合成
P72-his-EcoRV-R：
cgGATATCTCAGTGGTGGTGGTGATGGTGGGTGCTGTATCTCAGCACGG
P72-BamHI-F：cgcGGATCCgccaccATGGCCAGCGGCGGAGCTTT
②PCR扩增P72片段
扩增体系：Q5酶25ul，10uM引物P72-BamHI-F 1ul，10uM引物P72-his-EcoRV-R 1ul，模板P72 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化P72片段。
④目的片段P72与pS5E1-IRES载体酶切
酶切反应体系：载体pS5E1-IRES、P72片段～2ug，EcoRV和BamHI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图23所示，其中泳道1为片段pS5E1-IRES、NotI酶切，泳道2为P72、NotI酶切，M为15000bp Marker。
⑤目的片段P72与pS5E1-IRES连接
连接体系：pS5E1-IRES(100ng)；P72片段(载体：片段＝1：5,摩尔比)；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物P72-BamHI-F 1ul，10uM引物P72-his-EcoRV-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。进行电泳验证，如图24所示，其中1-10号为菌落，M为Marker，由图24可见，2和5号出现阳性条带。
⑦质粒酶切验证(BamHI&EcoRV)，选取2、5进行质粒提取，酶切验证。结果如图25所示，其中5号质粒为阳性质粒。
3)pS5E1-P72-IRES与片段B602L的连接
①引物合成
B602L-NotI-F：aaatat GCGGCCGC ATGGCCGAATTCAATATTGATGAA
B602L-XhoI-R：cggCTCGAGTCAGTGGTGGTGGTGATGGTG GGCGTAATCGGGCACGTCGT
②PCR扩增B602L片段
扩增体系：Q5酶25ul，10uM引物B602L-NotI-F 1ul，10uM引物B602L-XhoI-R 1ul，模板P72 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化B602L片段。
④目的片段B602L与pS5E1-P72-IRES载体酶切
酶切反应体系：载体pS5E1-P72-IRES、B602L片段2ug，NotI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图26所示，其中泳道1为pS5E1-P72-IRES，NotI、XhoI酶切，泳道2为B602L片段，NotI、XhoI酶切，M为15000bp Marker。
⑤pS5E1-P72-IRES载体与B602L片段连接
连接体系：pS5E1-P72-IRES 100ng；B602L片段50ng；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物B602L-NotI-F 1ul，10uM引物B602L-XhoI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min；进行电泳验证，如图27所示，其中1-7号为菌落，M为Marker，由图27可见，出现阳性条带。
⑦质粒NotI、XhoI酶切验证，选取1、2、4、6进行质粒提取，酶切验证，结果如图28所示，其中1、2、4、6号泳道为1、2、4、6质粒NotI、XhoI酶切鉴定，M为15000bp Marker。由图28可见，酶切结果正确，成功构建非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-P72-IRES-B602L，其载体图谱如图52所示。
实施例5非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-P30-2A-P54的构建
1、人类腺病毒5型载体E4区域穿梭质粒的构建
穿梭质粒pS5E4的骨架采用puc origin、amp等基本元素，Ad5E4区域左臂ITR序列(370bp)，右臂部分fiber基因序列(1746bp)，以及EF1α-EGFP-HBV polyA基因。
1)基因合成
EF1α-EGFP-HBV polyA基因由博迈德公司合成。
2)引物设计
puc-Ad5E4-left arm-F：AGGTGACACTATAGAATACACGTTAATTAAATCATCAATAATATACCTTATTTTG
Ad5E4-left arm-EF1a-R：caatccccccttttcttttaaaaAACACCACTCGACACGGCAC
EF1α-F：ttttaaaagaaaaggggggattg
EF1α-R：TAGAGCCCCAGCTGGTTCTTT
EF1α-Ad5E4-right arm-F：GGAAAGAACCAGCTGGGGCTCTAGCAATTGAAAAATAAACACGTTGA
Ad5E4-right arm-puc-R：TAATACGACTCACTATAGGGAGACCCAAAATGTAACCACTGTGAG
puc-F：TCTCCCTATAGTGAGTCGTATT
puc-R：CGTGTATTCTATAGTGTCACCT
ORF6/7-Protease-F：CGTTGAAACATAACACAAACGATACGGCGCAGACGGCAAGGGTGGG
3)目的片段的扩增
①以EF1α-EGFP-HBV基因合成片段为模板，以EF1α-F和EF1α-R为引物，扩增pS5E4-EGFP穿梭质粒的EF1α-EGFP-HBV polyA片段；扩增体系：EF1α-EGFP-HBV基因合成片段50ng，10uM EF1α-F引物1ul，10uM EF1α-R引物1ul，Q5高保真酶20ul；补水至40ul。PCR程序为：98℃，10sec； 98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
②以pAd5LCL3为模板，以puc-Ad5E4-left arm-F和Ad5E4-left arm-EF1a-R为引物，扩增pS5E1穿梭质粒的左臂片段。扩增体系：pAd5LCL3质粒50ng，10uM puc-Ad5E4-left arm-F引物1ul，10uM Ad5E4-left arm-EF1a-R引物1ul，Q5高保真酶20ul；补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、10sec，35个循环；72℃，5min。
③以pAd5LCL3为模板，以EF1α-Ad5E4-right arm-F和Ad5E4-right arm-puc-R为引物，扩增pS5E4-EGFP穿梭质粒的右臂片段；扩增体系：pAd5LCL3质粒50ng，10uM EF1α-Ad5E4-right arm-F引物1ul，10uM Ad5E4-right arm-puc-R引物1ul，Q5高保真酶20ul；补水至40ul。
PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40sec，35个循环；72℃，5min。
④以pS5E1质粒为模板，以puc-F和puc-R为引物，PCR扩增pS5E4-EGFP穿梭质粒骨架；扩增体系：pS5E1骨架质粒50ng，10uM puc-F引物1ul，10uM puc-R引物1ul，Q5高保真酶20ul；补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、1min20sec，35个循环；72℃，5min。扩增产物琼脂糖验证如图29所示，其中泳道1为pS5E4-EGFP穿梭质粒左臂，泳道2为pS5E4-EGFP穿梭质粒右臂，泳道3为EF1α-EGFP-HBV，泳道4为pS5E4-EGFP穿梭质粒骨架，M为2000Marker。由图29可见，扩增结果正确。
4)使用Axygen胶回收试剂盒纯化目的片段。
5)片段的连接转化
使用博迈德公司无缝克隆试剂盒将pS5E4-EGFP穿梭质粒左臂、pS5E4-EGFP穿梭质粒右臂、EF1α-EGFP-HBV、pS5E4-EGFP穿梭质粒骨架这四个片段连接，连接体系为2×Smealess Cloning Mix 10ul、pS5E4-EGFP穿梭质粒左臂片段50ng、pS5E4-EGFP穿梭质粒右臂片段50ng、EF1α-EGFP-HBV片段50ng、pS5E4-EGFP穿梭质粒骨架片段50ng、补水至20ul，50℃保温40分钟；将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
6)质粒的验证
①菌落PCR验证
用引物puc-Ad5E4-left arm-F/ER1α-R为引物菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图30所示，出现阳性条带。
②酶切验证
挑取3、4、5、6号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行酶切验证，电泳结果如图31所示，其中1-4为3、4、5、6号阳性克隆PacI单酶切，5-8为3、4、5、6号阳性克隆HindIII单酶切，M1、M3：15000bp Marker；M2：2000bp Marker；酶切结果正确，测序正确；成功构建人类腺病毒5型载体E4区域穿梭质粒pS5E4-EGFP，其载体图谱如图53所示。
2、非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-P30-2A-P54构建
1)引物设计
P30-BamHI-F：cgcGGATCCGCCACC ATGGACTTCATCCTGAACATCA
P30-2A-R：CTCCGCTTCC GGCGTAGTCGGGCACGTCGTA
P2A-F：ACGACGTGCCCGACTACGCC GGAAGCGGAGCTACTAACTTC
P2A-R：CTGGAAGAACTCGCTGTCCAT AGGTCCAGGGTTCTCCTCCACGT
2A-P54-F：CCCTGGACCT ATGGACAGCGAGTTCTTCCAG
P54-XhoI-R：ccg CTCGAG TTAGAGGGAGTTTTCCAGGTC
2)目的片段P30、P54、2A的扩增
①以P30基因合成片段为模板，以P30-BamHI-F和P30-2A-R为引物，扩增P30片段；扩增体系：P30基因合成片段50ng，10uM P30-BamHI-F引物1ul，10uM P30-2A-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
②以P54基因合成片段为模板，以2A-P54-F和P54-XhoI-R为引物，扩增P54片段；扩增体系：P54基因合成片段50ng，10uM 2A-P54-F引物1ul，10uM P54-XhoI-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
③以2A基因合成片段为模板，以P2A-F和P2A-R为引物，扩增2A片段；扩增体系：2A基因合成片段50ng，10uM P2A-F引物1ul，10uM P2A-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
扩增结果如图32所示，其中泳道1为P30扩增片段，泳道2为P54扩增片段，泳道3为2A扩增片段，M1、M2为2000bp ladder。
3)使用Axygen胶回收试剂盒纯化目的片段。
4)融合PCR扩增P30-2A-P54片段
扩增体系：P30胶回收片段50ng、P54胶回收片段50ng、P2A胶回收片段50ng，10uM P30-BamHI-F引物1ul，10uM P54-XhoI-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、50sec，35个循环；72℃，5min。融合结果如图33所示，其中泳道1为P30-2A-P54片段，M为Maker。
5)目的片段P30-2A-P54与pS5E4-EGFP载体酶切
酶切反应体系：载体pS5E4-EGFP、P30-2A-P54片段2ug，BamHI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。Axygen试剂盒胶回收纯化。酶切结果如图34所示，其中泳道1、2为pS5E4-EGFP，BamHI、XhoI双酶切胶回收，3、4为片段P30-2A-P54，BamHI、XhoI双酶切胶回收，M为15000bp Marker。
6)pS5E4载体与P30-2A-P54片段连接与转化
连接体系：pS5E4(100ng)，P30-2A-P54片段(50ng)，T4 DNA连接酶1ul，10×ligase buffer 1ul，补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
7)质粒的验证
①菌落PCR验证
用引物P30-BamHI-F、P54-XhoI-R为引物，菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图35所示，其中1-20号为菌落，M为2000bp，由图35可见，2号、19号出现阳性条带。
②酶切验证
挑取2、19号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行BmHI、XhoI双酶切验证；酶切结果如图36所示，其中泳道2为2号阳性克隆BamHI、XhoI双酶切验证，泳道19为19号阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker。酶切结果正确，测序正确，成功构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-P30-2A-P54，其载体图谱如图 54所示。
实施例6穿梭质粒pS5E1-P72-IRES-B602L、pS5E4-P30-2A-P54与pAd5LCL3重组构建pAd5LCL3-P72-B602L-P30-P54质粒
1、穿梭质粒pS5E1-P72-IRES-B602L与腺病毒载体质粒pAd5LCL3的同源重组
1)PacI和SwaI对穿梭质粒pS5E1-P72-IRES-B602L和腺病毒载体质粒pAd5LCL3进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E1-P72-IRES-B602L 3μg；PacI 2ul；buffer cutsmart 4ul；补水至40ul。
B、腺病毒载体质粒pAd5LCL3 3ug；SwaI 2ul；Buffer 3.1 4ul；补水至40ul。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图37所示，其中泳道1为pAd5LCL3，泳道2为pS5E1-P72-IRES-B602L。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5ul；去磷酸化酶1ul；去磷酸化buffer 5ul；补水至50ul。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证；结果如图38所示，其中泳道1-7为pAd5LCL3-P72-IRES-B602L克隆，M15000Marker，从图38可以看出，1号和7号克隆酶切正确。
6)将1号阳性质粒转化至DH5α感受态，挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证，酶切结果如图39所示，其中1号泳道为pAd5LCL3-P72-IRES-B602L质粒XhoI酶切，2号泳道为pAd5LCL3-P72-IRES-B602L质粒PacI酶切，M为15000Marker，由图39可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-P72-IRES-B602L。
2、穿梭质粒pS5E4-P30-2A-P54与腺病毒载体质粒pAd5LCL3-P72-IRES-B602L同源重组获得pAd5LCL3-P72-B602L-P30-P54
1)PacI和I-sceI对穿梭质粒pS5E4-P30-2A-P54和腺病毒载体质粒pAd5LCL3-P72-IRES-B602L进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E4-P30-2A-P54 3μg；PacI 2ul；10×cutsmart buffer 4ul；补水至40ul。
B、腺病毒载体质粒pAd5LCL3-P72-IRES-B602L3ug；I-sceI 2ul；Buffer cutsmart 4ul；补水至40ul。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图40所示，其中泳道1为pAd5LCL3，泳道2为pS5E1-P72-IRES-B602L。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5ul；去磷酸化酶1ul；去磷酸化buffer 5ul；补水至50ul。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产 物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取8个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证，结果如图41所示，其中泳道1-8为菌落，M为15000Marker，从图41可以看出，4号质粒正确。
6)将4号阳性质粒转化至DH5α感受态；挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证；酶切结果如图42所示，其中1号泳道为pAd5LCL3-P72-B602L-P30-P54质粒XhoI酶切，2号泳道为pAd5LCL3-P72-B602L-P30-P54质粒PacI酶切，M为15000Marker，由图42可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-P72-B602L-P30-P54，其载体图谱如图55所示。
实施例7重组腺病毒的包装
使用293TD37细胞包装pAd5LCL3-P72-B602L-P30-P54质粒，操作步骤如下所示：
准备293TD37细胞：转染前一天准备细胞，将待转染的293TD37细胞接种到6孔板中，0.5×10 6活细胞/孔，于37℃，5％CO 2静置培养24小时，转染当天细胞有40-50％汇合率。
质粒pAd5LCL3-P72-B602L-P30-P54线性化：将待转染的质粒用PacI酶切，于37℃孵育40min后，65℃灭活20min。
转染：用100ul无血清培养基将线性化的2μg质粒和PEI分别稀释；向PEI稀释液中加入质粒稀释液，反复吸取5次或涡旋10秒钟混匀，室温下孵育10分钟后形成转染复合物。孵育过程中，从培养板上轻柔地吸出细胞培养液，加入新鲜的生长培养基2mL，10分钟后将转染复合物加到换了新鲜培养基的细胞中。
细胞培养：将转染后的293TD37细胞于37℃，5％CO 2培养箱中静置培养72-96小时；病毒质粒转染72-96小时后收集6孔板细胞悬液于1.5ml离心管中即TP0。
持续接毒：将收集的细胞悬液在-80℃反复冻融3次，4℃、2000g离心10分钟，取上清500ul感染293TD37细胞(293TD37细胞需要提前一天准备)，37℃、5％CO 2孵育60分钟，补充2mL的FBS的培养基，37℃、5％CO 2培养培养72小时，收集细胞悬液即TP1；重复之前的步骤，收集细胞悬液即TP2。持续接毒直至TP4细胞出现病变。
细胞病变：当293TD37细胞培养从TP0至TP4后，细胞逐渐病变，直至TP4时293TD37细胞完全病变。TP0至TP4引起的细胞病变情况分别如图43-47所示，TP4已完全病变。
实施例8非洲猪瘟多抗原重组腺病毒疫苗滴度的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，接种6孔板中(5×10 5/mL，每孔2ml)，于37℃，5％CO 2二氧化碳培养箱中静置培养。24小时后，待细胞贴壁生长成单层细胞，弃去培养基，用无血清DMEM维持液对重组的腺病毒作10 -3～10 -6倍连续稀释，每个稀释度接种2孔，每孔250uL，感染1小时后，弃上清，补充完全培养基，然后于37℃，5％二氧化碳培养箱中静置培养。24h后，弃上清，用PBS洗细胞，每孔1mL，弃PBS后，每孔加1mL冷甲醛固定，室温10min，弃甲醛，再用PBS冲洗细胞，每孔1mL，加腺病毒抗体-FITC，每孔1ml，室温1h后，再次用PBS冲洗细胞，每孔1mL，两遍后每孔加1mL PBS，荧光显微镜下计数(200倍,10个连续视野)。计算：病毒滴度(FFU/mL)＝平均数×1013×4×10 (-n)。pAd5LCL3-P72-B602L-P30-P54病毒的FFU为2×10 8FFU/mL，滴度较高。
实施例9非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54稳定性的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，将293TD37细胞种入6孔板中(5×10 5cells/mL，2mL/孔)，室温孵育1小时使其贴壁，孵育后镜检其贴壁程度。用pAd5LCL3-P72-B602L-P30-P54病毒颗粒进行感染，感染的滴度为5MOI/孔。293TD37细胞48小时后发生病变后，收集细胞，反复冻融3次后2000g离心，收集上清，将收集的上清检测FFU，后重新感染新的293TD37细胞，直至30代。将收集的第5、10、15、20、25、30代次的病毒液进行检测，发现病毒的基因组仍然完整，说明复制缺陷型pAd5LCL3-P72-B602L-P30-P54病毒能够在293TD37细胞中稳定包装。
实施例10非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54恢复突变(RCA)的检测
pAd5LCL3-P72-B602L-P30-P54病毒RCA检测，检测方法如下：
1、准备pAd5LCL3-P72-B602L-P30-P54病毒液，并测其病毒滴度，测定病毒颗粒浓度，病毒液加1％benzonase(Benzonase7.5～15units/mL病毒液)消化宿主细胞的DNA，37℃水浴40min。用300Kd的超滤离心管，经1000g，30min离心，1×PBS洗脱收集病毒颗粒，测A260，颗粒浓度＝A260×1.1×10^12VP/mL。
2、病毒感染，准备A549细胞的12孔板，每孔细胞为2.5×10 5/孔，弃培养基，PBS清洗一次，将腺病毒按照1×10 9VP/孔/0.5ml接种病毒，感染A549细胞，野生型腺病毒5型为阳性对照，37℃，5％CO 2，1h后，弃去病毒液，补足5％完全培养基，37℃，5％CO 2培养48h。
3、免疫染色，弃细胞上清，PBS表面冲洗细胞，用冰甲醛固定，放置-20℃，20min，1×PBS清洗三遍，每遍5min，每孔加入2ml 1％BSA-PBS溶液，放置摇床，孵育1h。弃去上清，加入腺病毒5型荧光抗体(1:500稀释)，孵育1h，1×PBS清洗三遍，每遍5min。
用10倍荧光显微镜观察，使用公式计算RCA
RCA＝(average positive cell field)×(374 field/well)×(dilution factor))/Total VPs in 0.5ml viral sample
判断标准为RCA的水平小于1RCA/3×10 10vp。经过统计，pAd5LCL3-P72-B602L-P30-P54的RCA的水平小于1RCA/3×10 10vp，说明本发明所制备的复制缺陷型pAd5LCL3-P72-B602L-P30-P54病毒在293TD37细胞中能够稳定包装，不会转化为野生型或转化为野生型的几率较低。
实施例11非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54蛋白表达检测
提前一天准备293TD37细胞，置于12孔细胞培养板中，使用非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54病毒感染293TD37细胞，48小时后细胞发生病变，收集全部的1ml细胞，使用PBS洗涤，制样，用于Western Blot检测；使用P30的抗体检测目的蛋白，P30的抗体是由原核表达的P30蛋白免疫的兔子血清。实验结果如图48所示，该疫苗可以清晰可见P30蛋白；同样使用P54、P72蛋白免疫的兔子血清，如图56所示：M，预染Makker；泳道1，P54抗体血清；泳道2，P72抗体血清；泳道3：293TD37细胞对照。由此可见非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54的目的蛋白有明显得表达。
实施例12非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54实在小鼠模型上的免疫学评价
12.1疫苗体液免疫反应检测
20只SPF级小鼠(6-8周龄)，随机分成4组，每组5只。根据表1所示分组情况对小鼠进行pAd5LCL3-P72-B602L-P30-P54免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表1：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000009
小鼠于免疫后14天采血，分离血清，使用间接ELISA法检测血清中针对非洲猪瘟目的蛋白P72和P30的IgG抗体滴度。检测结果如图57所示(ns,P≥0.05；*,P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001)，其中左为P72的IgG抗体滴度，右图为P30的IgG抗体滴度。
由图57可见，小鼠肌肉注射pAd5LCL3-P72-B602L-P30-P54后，针对P72、P30蛋白，均能够产生较高浓度的IgG抗体。P72抗体中，高剂量组抗体滴度平均值达10 5以上，中剂量组滴度平均值也达70000，与对照组有显著差异；P30抗体中，高剂量组与中剂量组均能诱导高滴度的抗体。
12.2细胞免疫反应检测
10只SPF级小鼠(6-8周龄)，随机分成2组，每组5只。根据表2所示分组情况对小鼠进行pAd5LCL3-P72-B602L-P30-P54免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表2：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000010
免疫后14天处死小鼠，分离脾淋巴细胞，使用穿梭质粒pS5E1-P72-IRES-B602L和pS5E4-P30-2A-P54转染的PK15细胞刺激培养6小时，同时加入蛋白分泌阻断剂阻断细胞因子分泌。6小时后，阻断Fc受体，对死细胞和细胞表面分子标志物进行染色，细胞经固定和穿孔之后，对细胞内细胞因子进行染色。细胞表面标志物包括CD4、CD8，细胞内细胞因子包括IFNγ、IL2。使用流式细胞仪(CyExpert)分析CD4+T细胞和CD8+T细胞经目的蛋白刺激后表达IFNγ、IL2的水平。
pAd5LCL3-P72-B602L-P30-P54诱导的CD8+T细胞和CD4+T细胞免疫反应如图58、图59所示，代表性结果如图60、图61所示，其中图60为肌肉注射pAd5LCL3-P72-B602L-P30-P54后细胞免疫反应代表图，图61为空白对照免疫反应代表图。结果表示：小鼠免疫后14天，脾细胞经目的蛋白刺激后，CD8+T细胞，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。CD4+T细胞经过刺激后，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。
12.3小鼠模型免疫原性评价小结
pAd5LCL3-P72-B602L-P30-P54重组腺病毒具有良好的免疫原性，可诱导小鼠产生高水平的血清IgG抗体。其中高剂量的1*10^8FFU和中剂量1*10^7FFU的免疫方式诱导的滴度都很高。由于P72和B602L抗原、P30和P54抗原分别由相同的表达元件调控表达同时，P72和P30血清IgG抗体可代表这四个抗原均能够较高的免疫原性。细胞免疫反应检测结果说明肌肉注射免疫1*10^7FFU的腺病毒载体疫苗，可诱导其免疫的小鼠产生特异性细胞免疫反应。
实施例13非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-P72-B602L-P30-P54在靶动物(三元猪)上的免疫学评价
13.1靶动物(三元猪)疫苗体液免疫反应检测
非洲猪瘟多抗原重组腺病毒pAd5LCL3-P72-B602L-P30-P54疫苗动物免疫：使用1*10^9FFU的pAd5LCL3-P72-B602L-P30-P54疫苗，免疫三元猪。四周后，采集猪血样，分离血清，使用IDVET非洲猪瘟检测试剂盒，对免疫后的血清样本进行检测。具体免疫方式如表3所示：
表3：疫苗免疫三元猪分组情况
Figure PCTCN2021104793-appb-000011
免疫实验组共有5组，空白对照组由2组，免疫实验结果如表4所示。
表4：实验检测结果
Figure PCTCN2021104793-appb-000012
其中，对于每个样品，计算其S/P百分比(S/P％)，S/P％＝(OD SAMPLE-OD NC)/(OD PC-OD NC)*100，每个样品算出S/P％，当S/P％≤30为阴性，30％＜S/P％＜40％为可疑，S/P％≥40％为阳性。
实验有效性判定：在下列情形下实验有效：
(1)阳性对照的平均净OD大于0.350；OD PC>0.350
(2)阳性对照与阴性对照平均净OD值比值大于3；OD PC/OD NC>3
实验结果说明：重组腺病毒pAd5LCL3-P72-B602L-P30-P54疫苗，在三元猪免疫试验中，能够诱导足够的免疫反应。
13.2非洲猪瘟多抗原重组腺病毒pAd5LCL3-P72-B602L-P30-P54疫苗诱导的细胞毒性T细胞(CTL)杀伤实验
非洲猪瘟多抗原重组腺病毒pAd5LCL3-P72-B602L-P30-P54疫苗动物免疫：使用1×10 8FFU的pAd5LCL3-P72-B602L-P30-P54疫苗，免疫三元猪，四周后，采集猪血样。猪外周血淋巴细胞分离：使用天津灏洋华科生物科技有限公司的猪外周血淋巴细胞分离试剂盒对采集的猪血样进行淋巴细胞分离，使用计数器对效应细胞计数。细胞毒性T细胞(CTL)杀伤实验：使用乳酸脱氢酶细胞毒性检测试剂盒(购自碧云天)检测细胞毒性T细胞(CTL)杀伤实验。具体步骤：1、提前一晚准备PK15细胞(细胞购自中国医学科学院基础医学研究所细胞资源中心)，并感染非洲猪瘟pAd5LCL3-P72-B602L-P30-P54疫苗和腺病毒载体对照疫苗(25MOI，提前18h)。
2、实验前将感染的PK15细胞用胰酶消化，用无血清培养基重悬稀释至1×10 5/ml作为靶细胞。在96孔底细胞培养板中加入靶细胞，每孔加100ul。3个效应细胞自然释放对照孔不加靶细胞，只加100ul培养液。
3、向各孔加100ul效应细胞，效应细胞与靶细胞的比例为50：1。自然释放孔不加效应细胞只加100ul培养液。同时设置最大释放对照孔，加细胞释放试剂。
4、置37℃，5％CO 2的二氧化碳培养箱中培养4小时。
5、离心培养板250g 10分钟。每孔吸出140ul上清液，对应加入另一块96孔酶联检测板中，按乳酸脱氢酶细胞毒性检测试剂盒操作说明书配制并加入60μ0。检测OD490吸光值。
杀伤活性(％)＝[(OD实验组－OD总自然释放)/(OD最大释放组－OD总自然释放)]×100％
实验结果如图49所示，经过多次试验，统计分析显示，非洲猪瘟疫苗pAd5LCL3-P72-B602L-P30-P54组与非相关抗原(相当于空白)腺病毒pAd5LCL3组相比CTL杀伤水平更高，且差异显著P<0.05。生理盐水组基本没有展现出杀伤水平，少量数据可能来自于误差或自然杀伤。对细胞毒性T细胞(CTL)的杀伤水平越高，则证明对非洲猪瘟病毒的特异性免疫反应越强，因此本实施例的非洲猪瘟疫苗pAd5LCL3-P72-B602L-P30-P54可显著增强对非洲猪瘟病毒较强的特异性免疫反应。
实施例14非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-C129Rubiqutin-IRES-MGF5L6L的构建
1、人类腺病毒5型载体E1区域穿梭质粒的构建按照实施例4所述的构建方法。
2、非洲猪瘟腺病毒5型载体穿梭质粒pS5E1-P72-IRES-B602L构建
1)pS5E1与IRES片段的连接
①引物合成
IRES-EcoRV-F：ccg GATATC TGTCGTCATCATCCTTATAGTCC
IRES-NotI-R：aaatat GCGGCCGC GGTTGTGGCCATTATCATCGTG
②扩增IRES片段
扩增体系：Q5酶25ul，10uM引物IRES-EcoRV-F 1ul，10uM引物IRES-NotI-R 1ul，模板IRES模板2ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。扩增结果电泳检测如图62所示，其中泳道1、2为IRES片段PCR扩增产物，M为15000bp Marker，可见扩增结果正确。
③使用Axygen PCR纯化试剂盒纯化IRES片段。
④目的片段IRES与pS5E1载体酶切
酶切反应体系：载体pS5E1、IRES片段～2ug，EcoRV和NotI各1ul；10×cutsmart buffer 5ul；补水至50ul；反应条件：37℃，30min；65℃，20min灭活；胶回收纯化。酶切产物电泳检测如图63所示，其中泳道1为片段IRES EcoRV、NotI酶切，泳道2为pS5E1 EcoRV、NotI酶切，M为15000bp Marker。
⑤pS5E1载体与IRES片段连接
连接体系：pS5E1(100ng)；IRES片段(载体：片段＝1：5,摩尔比)；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物IRES-EcoRV-F 1ul，10uM引物IRES-NotI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。进行电泳验证， 如图64所示，其中1-9号为菌落，M为Marker，由图64可见，2和6号出现阳性条带。
⑦质粒NotI、EcoRV酶切验证，选取2、6进行质粒提取，酶切验证，结果如图65所示，其中2号质粒NotI、EcoRV酶切鉴定，6号质粒NotI、EcoRV酶切鉴定，可见酶切结果正确。
2)pS5E1-IRES与MGF5L6L片段的连接
①引物合成
MGF5L6L-NotI-F：aaggaaaaaaGCGGCCGCgccaccATGCTGGTGATCTTCCTGGG
MGF5L6L-XhoI-R：catgCTCGAG TCAGGCGTAGTCAGGCACAT
②PCR扩增MGF5L6L片段
扩增体系：Q5酶25ul，10uM引物MGF5L6L-NotI-F 1ul，10uM引物MGF5L6L-XhoI-R 1ul，模板MGF5L6L 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化MGF5L6L片段。
④目的片段MGF5L6L与pS5E1-IRES载体酶切
酶切反应体系：载体pS5E1-IRES、MGF5L6L片段～2ug，NotI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图66所示，其中泳道1为pS5E1-IRES，NotI和XhoI双酶切，泳道2为片段MGF5L6L，NotI和XhoI双酶切，M为15000bp Marker。
⑤目的片段MGF5L6L与pS5E1-IRES连接
连接体系：pS5E1-IRES(100ng)；MGF5L6L片段(载体：片段＝1：3摩尔比)；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物MGF5L6L-NotI-F 1ul，10uM引物MGF5L6L-XhoI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、30s，35个循环；72℃，5min。进行电泳验证，如图67所示，其中1-12号为菌落，M为2000bp Marker。
⑦质粒酶切验证(NotI和XhoI)，选取2、9、11菌落进行质粒提取，酶切验证。结果如图68所示，均为阳性质粒。
3)pS5E1-IRES-MGF5L6L与片段CP129Rubiqutin的连接
①引物合成
CP129R-BamHI-F：cgcGGATCCgccaccATGGAGCACCCCAGCACAAA
CP129R-ubiqutin-R：GGGTTTTCACGAAAATCTGCATGGCGTAATCGGGCACGTCGTAA
ubiqutin-F：ATGCAGATTTTCGTGAAAACCC
ubiqutin-EcoRV-R：ccgGATATC TTACTTGTCTTCTGGTTTGTTGA
②PCR扩增CP129Rubiqutin片段
扩增CP129R片段
扩增体系：Q5酶25ul，引物CP129R-BamHI-F 1ul，引物CP129R-ubiqutin-R 1ul，模板C129R2ul，补水至50ul；反应条件：98℃30S；98℃10s，68℃30s，72℃15s，35个循环；72℃5min。
扩增ubiqutin片段
扩增体系：Q5酶25ul，引物ubiqutin-F 1ul，引物ubiqutin-EcoRV-R 1ul，模板ubiqutin 2ul，补水至50ul；反应条件：98℃30S；98℃10s，68℃30s，72℃15s，35个循环；72℃5min。
融合PCR扩增C129Rubiqutin片段
扩增体系：Q5酶25ul，上游引物CP129R-BamHI-F、下游引物ubiqutin-EcoRV-R，模板片段CP129R、片段ubiqutin各50ng，补水至50ul；反应条件：98℃；98℃5s，68℃30s，72℃30s，35个循环；72℃7min。
③使用Axygen PCR纯化试剂盒纯化CP129Rubiqutin片段。
④目的片段CP129Rubiqutin与pS5E1-IRES-MGF5L6L载体酶切
酶切反应体系：载体pS5E1-IRES-MGF5L6L、CP129Rubiqutin片段2ug，EcoRV和BamHI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图69所示，其中泳道1为pS5E1-IRES-MGF5L6L，EcoRV和BamHI酶切；泳道2为CP129Rubiqutin片段，EcoRV和BamHI酶切，M为15000bp Marker、2000bp Marker。
⑤pS5E1-IRES-MGF5L6L载体与CP129Rubiqutin片段连接
连接体系：pS5E1-IRES-MGF5L6L 100ng；CP129Rubiqutin片段50ng；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物CP129R-BamHI-F 1ul，10uM引物ubiqutin-EcoRV-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、30s，35个循环；72℃，5min；进行电泳验证，如图70所示，其中1-5号为菌落，M为2000bp Marker。由图70可见，1号和2号出现阳性条带。
⑦质粒BamHI、EcoRV酶切验证，选取1、2号菌落进行质粒提取，酶切验证，结果如图70所示，其中1、2号泳道为1、2号菌落质粒BamHI、EcoRV酶切鉴定，M为2000bp Marker。由图71可见，酶切结果正确，成功构建非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-CP129Rubiqutin-IRES-MGF5L6L，其载体图谱如图92所示。
实施例15非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-CP312R-2A-MGF5L6L的构建
1、人类腺病毒5型载体E4区域穿梭质粒的构建
穿梭质粒pS5E4的骨架采用puc origin、amp等基本元素，Ad5E4区域左臂ITR序列(370bp)，右臂部分fiber基因序列(1746bp)，以及EF1α-EGFP-HBV polyA基因。
1)基因合成
EF1α-EGFP-HBV polyA基因由博迈德公司合成。
2)引物设计
puc-Ad5E4-left arm-F：
AGGTGACACTATAGAATACACGTTAATTAAATCATCAATAATATACCTTATTTTG
Ad5E4-left arm-EF1α-R：caatccccccttttcttttaaaaAACACCACTCGACACGGCAC
EF1α-F：ttttaaaagaaaaggggggattg
EF1α-R：TAGAGCCCCAGCTGGTTCTTT
EF1α-Ad5E4-right arm-F：GGAAAGAACCAGCTGGGGCTCTAGCAATTGAAAAATAAACACGTTGA
Ad5E4-right arm-puc-R：TAATACGACTCACTATAGGGAGACCCAAAATGTAACCACTGTGAG
puc-F：TCTCCCTATAGTGAGTCGTATT
puc-R：CGTGTATTCTATAGTGTCACCT
ORF6/7-Protease-F：CGTTGAAACATAACACAAACGATACGGCGCAGACGGCAAGGGTGGG
3)目的片段的扩增
①以EF1α-EGFP-HBV基因合成片段为模板，以EF1α-F和EF1α-R为引物，扩增pS5E4-EGFP穿梭质粒的EF1α-EGFP-HBV polyA片段；扩增体系：EF1α-EGFP-HBV基因合成片段50ng，10uM EF1α-F引物1ul，10uM EF1α-R引物1ul，Q5高保真酶20ul；补水至40ul。PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
②以pAd5LCL3为模板，以puc-Ad5E4-left arm-F和Ad5E4-left arm-EF1α-R为引物，扩增pS5E1穿梭质粒的左臂片段。扩增体系：pAd5LCL3质粒50ng，10uM puc-Ad5E4-left arm-F引物1ul，10uM Ad5E4-left arm-EF1α-R引物1ul，Q5高保真酶20ul；补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、10sec，35个循环；72℃，5min。
③以pAd5LCL3为模板，以EF1α-Ad5E4-right arm-F和Ad5E4-right arm-puc-R为引物，扩增pS5E4-EGFP穿梭质粒的右臂片段；扩增体系：pAd5LCL3质粒50ng，10uM EF1α-Ad5E4-right arm-F引物1ul，10uM Ad5E4-right arm-puc-R引物1ul，Q5高保真酶20ul；补水至40ul。
PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40sec，35个循环；72℃，5min。
④以pS5E1质粒为模板，以puc-F和puc-R为引物，PCR扩增pS5E4-EGFP穿梭质粒骨架；扩增体系：pS5E1骨架质粒50ng，10uM puc-F引物1ul，10uM puc-R引物1ul，Q5高保真酶20ul；补水至40ul。PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、1min20sec，35个循环；72℃，5min。扩增产物琼脂糖验证如图72所示，其中泳道1为pS5E4-EGFP穿梭质粒左臂，泳道2为pS5E4-EGFP穿梭质粒右臂，泳道3为EF1α-EGFP-HBV，泳道4为pS5E4-EGFP穿梭质粒骨架，M为2000Marker。由图72可见，扩增结果正确。
4)使用Axygen胶回收试剂盒纯化目的片段。
5)片段的连接转化
使用博迈德公司无缝克隆试剂盒将pS5E4-EGFP穿梭质粒左臂、pS5E4-EGFP穿梭质粒右臂、EF1α-EGFP-HBV、pS5E4-EGFP穿梭质粒骨架这四个片段连接，连接体系为2×Smealess Cloning Mix 10μl、pS5E4-EGFP穿梭质粒左臂片段50ng、pS5E4-EGFP穿梭质粒右臂片段50ng、EF1α-EGFP-HBV片段50ng、pS5E4-EGFP穿梭质粒骨架片段50ng、补水至20μl，50℃保温40分钟；将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
6)质粒的验证
①菌落PCR验证
用引物puc-Ad5E4-left arm-F/EF1α-R为引物菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图73所示，出现阳性条带。
②酶切验证
挑取3、4、5、6号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行酶切验证，电泳结果如图74所示，其中1-4为3、4、5、6号阳性克隆PacI单酶切，5-8为3、4、5、6号阳性克隆HindIII单酶切，M1、M3：15000bp Marker；M2：2000bp Marker；酶切结果正确， 测序正确；成功构建人类腺病毒5型载体E4区域穿梭质粒pS5E4-EGFP，其载体图谱如图93所示。
2、非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-CP312R-2A-MGF5L6L构建
1)引物设计
PS5E4-CP312R-HamHI-F：ccaagctgtgaccggcgcctacGGATCCGCCACCATGACAACCCACAT
CP312R-2A-R：GAAGTTAGTAGCTCCGCTTCCGGCGTAATCAGGCACGTCGTA
CP312R-2A-F：TACGACGTGCCTGATTACGCCGGAAGCGGAGCTACTAACTTC
2A-MGF110-4L-R：GCCCAGAAACACCACCAGCATAGGTCCAGGGTTCTCCTCCA
MGF110-4L-F：ATGCTGGTGGTGTTTCTGGG
MGF110-4L-XhoI-R：CGGGTTTAAACGGGCCCTCTAGACTCGAGTCACAGGTCCTTCT
EF1α2(jd)-F：tggtgcctcctgaactgcgt
HBV(jd)-R：TAAGGGTCAATGTCCATGCC
2)目的片段CP312R、MGF110-4L、2A的扩增
①以CP312R基因合成片段为模板，以PS5E4-CP312R-HamHI-F和CP312R-2A-R为引物，扩增CP312R片段；扩增体系：CP312R基因合成片段50ng，10uM PS5E4-CP312R-HamHI-F引物1ul，10uM CP312R-2A-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
②以MGF110-4L基因合成片段为模板，以MGF110-4L-F和MGF110-4L-XhoI-R为引物，扩增MGF110-4L片段；扩增体系：MGF110-4L基因合成片段50ng，10uM MGF110-4L-F引物1ul，10uM MGF110-4L-XhoI-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
③以2A基因合成片段为模板，以CP312R-2A-F和2A-MGF110-4L-R为引物，扩增2A片段；扩增体系：2A基因合成片段50ng，10uM CP312R-2A-F引物1ul，10uM 2A-MGF110-4L-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
扩增结果如图75所示，其中泳道1为CP312R扩增片段，泳道2为2A扩增片段，泳道3为MGF110-4L扩增片段，M为2000bp Marker。
3)使用Axygen胶回收试剂盒纯化目的片段。
4)融合PCR扩增CP312R-2A-MGF110-4L片段
扩增体系：CP312R胶回收片段50ng、2A胶回收片段50ng、MGF110-4L胶回收片段50ng，10uM PS5E4-CP312R-HamHI-F引物1ul，10uM MGF110-4L-XhoI-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、50sec，35个循环；72℃，5min。融合结果如图76所示，其中泳道1为CP312R-2A-MGF110-4L片段，M为2000bp Marker。
5)pS5E4-EGFP载体酶切
酶切反应体系：载体pS5E4-EGFP 2ug，BamHI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。Axygen试剂盒胶回收纯化。胶回收结果如图77所示，其中泳道1为片段CP312R-2A-MGF110-4L胶回收，泳道2为载体pS5E4-EGFP，BamHI、XhoI双酶切胶回收，M为15000bp Marker。
6)pS5E4载体与CP312R-2A-MGF110-4L片段无缝克隆连接与转化
连接体系：pS5E4-EGFP胶回收产物(100ng)，CP312R-2A-MGF110-4L片段(50ng)，2×Smealess Cloning Mix 5ul，补水至10ul。反应条件：50℃，40min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
7)质粒的验证
①菌落PCR验证
用引物以EF1α2(jd)-F、HBV(jd)-R为引物，菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图78所示，其中1-12号为菌落，M为15000bp Marker，均为阳性条带。
②酶切验证
挑取1、2、3、4号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行BmHI、XhoI双酶切验证；酶切结果如图79所示，其中泳道1、2、3、4为阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker。酶切结果正确，测序正确，成功构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-CP312R-2A-MGF110-4L，其载体图谱如图94所示。
实施例16穿梭质粒pS5E1-CP129Rubiqutin-IRES-MGF5L6L、pS5E4-CP312R-2A-MGF110-4L与pAd5LCL3重组构建pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒
1、穿梭质粒pS5E1-C129Rubiqutin-IRES-MGF5L6L与腺病毒载体质粒pAd5LCL3的同源重组
1)PacI和SwaI对穿梭质粒pS5E1-C129Rubiqutin-IRES-MGF5L6L和腺病毒载体质粒pAd5LCL3进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E1-CP129Rubiqutin-IRES-MGF5L6L 3μg；PacI 2μl；buffer cutsmart 4μl；补水至40μl。
B、腺病毒载体质粒pAd5LCL3 3ug；SwaI 2μl；Buffer 3.1 4μl；补水至40μl。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图80所示，其中泳道1为pS5E1-C129Rubiqutin-IRES-MGF5L6L，泳道2为pAd5LCL3。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5μl；去磷酸化酶1μl；去磷酸化buffer 5μl；补水至50μl。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证；结果如图81所示，其中泳道1-5为pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L克隆，M：15000bp Marker，从图81可以看出，4号和5号克隆酶切正确。
6)将4号阳性质粒转化至DH5a感受态，挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证，酶切结果如图82所示，其中1、2号泳道为pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L质粒XhoI酶切，M为15000Marker，由图82可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L。
2、穿梭质粒pS5E4-CP312R-2A-MGF110-4L与腺病毒载体质粒pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L同源重组获得pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L
1)PacI和I-sceI对穿梭质粒pS5E4-CP312R-2A-MGF110-4L和腺病毒载体质粒pAd5LCL3-C129Rubiqutin-IRES-MGF5L6L进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E4-CP312R-2A-MGF110-4L 3μg；PacI 2μl；10×cutsmart buffer 4μl；补水至40μl。
B、腺病毒载体质粒pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L3ug；I-sceI 2μl；Buffer cutsmart 4μl；补水至40μl。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图83所示，其中泳道1为pS5E4-CP312R-2A-MGF110-4L，泳道2为pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5μl；去磷酸化酶1μl；去磷酸化buffer 5μl；补水至50μl。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取6个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证，结果如图84所示，其中泳道1-6为质粒，M为15000Marker，可以看出，3、4号质粒正确。
6)将3号阳性质粒转化至DH5a感受态；挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证；酶切结果如图85所示，其中1号泳道为pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒XhoI酶切，2号泳道为pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒PacI酶切，M为15000Marker，由图85可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L，其载体图谱如图95所示。
实施例17重组腺病毒的包装
使用293TD37细胞包装pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L质粒，操作步骤如下所示：
准备293TD37细胞：转染前一天准备细胞，将待转染的293TD37细胞接种到6孔板中，0.5×10 6/孔，于37℃，5％CO 2静置培养24小时，转染当天细胞有40-50％汇合率。
质粒pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L线性化：将待转染的质粒用PacI酶切，于37℃孵育40min后，65℃灭活20min。
转染：用100μl无血清培养基将线性化的2μg质粒和PEI分别稀释；向PEI稀释液中加入质粒稀释液，反复吸取5次或涡旋10秒钟混匀，室温下孵育10分钟后形成转染复合物。孵育过程中，从培养板上轻柔地吸出细胞培养液，加入新鲜的生长培养基2mL，10分钟后将转染复合物加到换了新鲜培养基的细胞中。
细胞培养：将转染后的293TD37细胞于37℃，5％CO 2培养箱中静置培养72-96小时；病毒质粒转染72-96小时后收集6孔板细胞悬液于1.5ml离心管中即TP0。
持续接毒：将收集的细胞悬液在-80℃反复冻融3次，4℃、2000g离心10分钟，取上清500μl感染293TD37细胞(293TD37细胞需要提前一天准备)，37℃、5％CO 2孵育60分钟，补充2mL的FBS的 培养基，37℃、5％CO 2培养培养72小时，收集细胞悬液即TP1；重复之前的步骤，收集细胞悬液即TP2。持续接毒直至细胞出现病变。
细胞病变：当293TD37细胞培养从TP0至TP4后，细胞逐渐病变，直至293TD37细胞完全病变。TP0至TP4引起的细胞病变情况分别如图86-90所示，TP4已完全病变。
实施例18非洲猪瘟多抗原重组腺病毒疫苗滴度的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，接种6孔板中(5×10 5/mL，每孔2ml)，于37℃，5％CO 2二氧化碳培养箱中静置培养。24小时后，待细胞贴壁生长成单层细胞，弃去培养基，用无血清DMEM维持液对重组的腺病毒作10 -3～10 -6倍连续稀释，每个稀释度接种2孔，每孔250uL，感染1小时后，弃上清，补充完全培养基，然后于37℃，5％二氧化碳培养箱中静置培养。24h后，弃上清，用PBS洗细胞，每孔1mL，弃PBS后，每孔加1mL冷甲醛固定，室温10min，弃甲醛，再用PBS冲洗细胞，每孔1mL，加腺病毒抗体-FITC，每孔1ml，室温1h后，再次用PBS冲洗细胞，每孔1mL，两遍后每孔加1mL PBS，荧光显微镜下计数(200倍,10个连续视野)。计算：病毒滴度(FFU/mL)＝平均数×1013×4×10 (-n)。pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒的FFU为1.9×10 8FFU/mL，滴度较高。
实施例19非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L稳定性的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，将293TD37细胞种入6孔板中(5×10 5cells/mL，2mL/孔)，室温孵育1小时使其贴壁，孵育后镜检其贴壁程度。用pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒颗粒进行感染，感染的滴度为5MOI/孔。293TD37细胞48小时后发生病变后，收集细胞，反复冻融3次后2000g离心，收集上清，将收集的上清检测FFU，后重新感染新的293TD37细胞，直至30代。将收集的第5、10、15、20、25、30代次的病毒液进行检测，发现病毒的基因组仍然完整，说明复制缺陷型pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒能够在293TD37细胞中稳定包装。
实施例20非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L恢复突变(RCA)的检测
pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒RCA检测，检测方法如下：
1、准备pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒液，并测其病毒滴度，测定病毒颗粒浓度，病毒液加1％Universal nuclease(Universal nuclease 7.5～15units/mL病毒液)消化宿主细胞的DNA，37℃水浴40min。用300Kd的超滤离心管，经1000g，30min离心，1×PBS洗脱收集病毒颗粒，测A260，颗粒浓度＝A260*1.1*10^12VP/mL。
2、病毒感染，准备A549细胞的6孔板，每孔细胞为2.5×10 5/孔，弃培养基，PBS清洗一次，将腺病毒按照1×10 9vp/孔接种病毒，感染A549细胞，野生型腺病毒5型为对照，37℃，5％CO 2，1h后，弃去病毒液，补足5％完全培养基，37℃，5％CO 2培养48h。
3、免疫染色，弃细胞上清，PBS表面冲洗细胞，用冰甲醛固定，放置-20℃，20min，1×PBS清洗三遍，每遍5min，每孔加入2ml 1％BSA-PBS溶液，放置摇床，孵育1h。弃去上清，加入腺病毒5型荧光抗体(1:500稀释)，孵育1h，1×PBS清洗三遍，每遍5min。
用10倍荧光显微镜观察，使用公式计算RCA
RCA＝(average positive cell field)×(374 field/well)×(dilution factor))/Total VPs in 0.5ml viral sample
判断标准为RCA的水平小于1RCA/3×10 10vp。经过统计，pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L的RCA的水平小于1RCA/3×10 10vp，说明本发明所制备的复制缺陷型pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒在293TD37细胞中能够稳定包装，不会转化为野生型或转化为野生型的几率较低。
实施例21非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L蛋白表达检测
提前一天准备293TD37细胞，置于12孔细胞培养板中，使用非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L病毒感染293TD37细胞，48小时后细胞发生病变，收集全部的1ml细胞，使用PBS洗涤，制样，用于Western Blot检测；使用HA的抗体检测目的蛋白，HA的抗体采购于Abcam。其中C129Rubiquitin、MGF5L6L、CP312R具有HA标签，其中C129Rubiquitin融合蛋白大小为34kda，MGF5L6L蛋白大小为25kda，CP312R蛋白大小为35kda。实验结果如图91所示，该疫苗可以清晰可见MGF5L6L蛋白，由此可见pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L的蛋白表达量较高。
实施例22非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L在小鼠模型上的免疫学评价
22.1细胞免疫反应检测
10只SPF级小鼠(6-8周龄)，随机分成2组，每组5只。根据表5所示分组情况对小鼠进行pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表5：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000013
免疫后14天处死小鼠，分离脾淋巴细胞，使用穿梭质粒pS5E1-C129Rubiqutin-MGF5L6L和pS5E4-CP312R-MGF110-4L转染的PK15细胞刺激培养6小时，同时加入蛋白分泌阻断剂阻断细胞因子分泌。6小时后，阻断Fc受体，对死细胞和细胞表面分子标志物进行染色，细胞经固定和穿孔之后，对细胞内细胞因子进行染色。细胞表面标志物包括CD4、CD8，细胞内细胞因子包括IFNγ、IL2。使用流式细胞仪(CyExpert)分析CD4+T细胞和CD8+T细胞经目的蛋白刺激后表达IFNγ、IL2的水平。
pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L诱导的CD8+T细胞和CD4+T细胞免疫反应如图96、97所示，代表性结果如图98至图99所示，其中图98为肌肉注射pAd5LCL3-C129Rubiqutin-MGF5L6L-CP312R-MGF110-4L后细胞免疫反应代表图，图99为空白对照免疫反应代表图。结果表示：小鼠免疫后14天，脾细胞经目的蛋白刺激后，CD8+T细胞，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。CD4+T细胞经过刺激后，其表达的IFNγ、 TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。细胞免疫反应检测结果说明肌肉注射免疫1*10^7FFU的腺病毒载体疫苗，可诱导其免疫的小鼠产生特异性细胞免疫反应。
实施例23非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L的构建
1、人类腺病毒5型载体E1区域穿梭质粒的构建，按照实施例4的方法进行构建得到穿梭质粒pS5E1。
2、非洲猪瘟腺病毒5型载体穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L构建
1)pS5E1与IRES片段的连接
按照实施例4提供的方法进行pS5E1与IRES片段的连接，构建得到pS5E1-IRES。
2)pS5E1-IRES与I215L片段的连接
①引物合成
I215L-NotI-F：aaggaaaaaaGCGGCCGCgccaccATGGTGAGCAGGTTTCTGATC
I215L-XhoI-R：catgCTCGAG TCAGGCGTAATCGGGCACAT
②PCR扩增I215L片段
扩增体系：Q5酶25ul，10uM引物I215L-NotI-F 1ul，10uM引物I215L-XhoI-R 1ul，模板I215L 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化I215L片段。
④目的片段I215L与pS5E1-IRES载体酶切
酶切反应体系：载体pS5E1-IRES、I215L片段～2ug，NotI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图100所示，其中泳道载体为pS5E1-IRES NotI和XhoI双酶切，泳道I215L为片段I215L NotI和XhoI双酶切，M为15000bp、2000bp Marker。
⑤目的片段I215L与pS5E1-IRES连接
连接体系：pS5E1-IRES(100ng)；I215L片段(载体：片段＝1：3摩尔比)；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM通用引物CMV-F 1ul，10uM引物I215L-XhoI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、30s，35个循环；72℃，5min。进行电泳验证，如图101所示，其中1-11号为菌落，M为2000bp Marker。由图101可见，1、2、5、6、7、8、11为阳性菌落。
⑦质粒酶切验证(NotI和XhoI)，选取5、6、7、8菌落进行质粒提取，酶切验证。结果如图102所示，均为阳性质粒。
3)pS5E1-IRES-MGF5L6L与片段L8Lubiqutin的连接
①引物合成
L8L-BamHI-F：cgcGATCCgccaccATGGGCAACAGACTGATCAAG
L8L-ubiqutin-R：AAGGGTTTTCACGAAAATCTGCATGGCGTAGTCGGGCACGTCGT
ubiqutin-F：ATGCAGATTTTCGTGAAAACCC
ubiqutin-EcoRV-R：ccgGATATC TTACTTGTCTTCTGGTTTGTTGA
②PCR扩增L8Lubiqutin片段
扩增L8L片段
扩增体系：Q5酶25ul，引物L8L-BamHI-F 1ul，引物L8L-ubiqutin-R 1ul，模板L8L 2ul，补水至50ul；反应条件：98℃30S；98℃10s，68℃30s，72℃15s，35个循环；72℃5min。
扩增ubiqutin片段
扩增体系：Q5酶25ul，引物ubiqutin-F 1ul，引物ubiqutin-EcoRV-R 1ul，模板ubiqutin 2ul，补水至50ul；反应条件：98℃30S；98℃10s，68℃30s，72℃15s，35个循环；72℃5min。
使用Axygen胶回收纯化试剂盒纯化L8L、ubiqutin片段；
融合PCR扩增L8Lubiqutin片段
扩增体系：Q5酶25ul，上游引物L8L-BamHI-F、下游引物ubiqutin-EcoRV-R模板片段L8L、片段ubiqutin各50ng，补水至50ul；反应条件：98℃；98℃5s，68℃30s，72℃30s，35个循环；72℃7min。
③使用Axygen PCR纯化试剂盒纯化L8Lubiqutin片段，融合后的L8Lubiqutin片段如图103所示。
④目的片段L8Lubiqutin与pS5E1-IRES-I215L载体酶切
酶切反应体系：载体pS5E1-IRES-I215L、L8Lubiqutin片段～2ug，EcoRV和BamHI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图104所示，其中泳道1为pS5E1-IRES-I215L质粒；泳道2为pS5E1-IRES-I215L质粒EcoRV和BamHI酶切；泳道3为L8Lubiqutin片段EcoRV和BamHI酶切，M为15000bp Marker。
⑤pS5E1-IRES-I215L载体与L8Lubiqutin片段连接
连接体系：pS5E1-IRES-I215L 100ng；L8Lubiqutin片段50ng；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM通用引物CMV-F 1ul，10uM引物ubiqutin-EcoRV-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、40s，35个循环；72℃，5min；进行电泳验证，如图105所示，其中1-24号为菌落，M为2000bp Marker。
⑦质粒BamHI、EcoRV酶切验证，选取4、6、9、14、17、18菌落进行质粒提取，酶切验证，结果如图106所示，BamHI、EcoRV酶切鉴定，M为15000bp Marker。由图106可见，酶切结果正确，成功构建非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L质粒，其载体图谱如图123所示。
实施例24非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-I73Rhbsag-2A-E146L的构建
1、人类腺病毒5型载体E4区域穿梭质粒的构建
按照实施例5提供的方法，成功构建人类腺病毒5型载体E4区域穿梭质粒pS5E4-EGFP。
2、非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-I73Rhbsag-2A-E146L构建
1)引物设计
pS5E4-I73R-BamHI-F：ccaagctgtgaccggcgcctacGGATCCgccaccATGGAGACACAGAAG
I73R-hbsag-R：AGCCGCTGGTGGTGTTCTCCATGGCGTAGTCAGGCACATCGTA
hbsag-F：ATGGAGAACACCACCAGCGGC
hbsag-2A-R：TGAAGTTAGTAGCTCCGCTTCCGATGTACACCCAGAGGCAGAA
2A-F：GGAAGCGGAGCTACTAACTTC
2A-E146L-R：ACAAAGTCTGTTGTTCCGCCCATAGGTCCAGGGTTCTCCTCCA
E146L-F：ATGGGCGGAACAACAGACTTT
E146L-pS5E4-XhoI-R：CGGGTTTAAACGGGCCCTCTAGACTCGAGTTAGATGATTCTCTGC
2)目的片段I73R、hbsag、2A、E146L的扩增
①以I73R基因合成片段为模板，以pS5E4-I73R-BamHI-F和I73-hbsag-R为引物，扩增I73R片段；扩增体系：I73基因合成片段50ng，10uM pS5E4-I73R-BamHI-F引物1ul，10uM I73-hbsag-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
②以hbsag基因合成片段为模板，以hbsag-F和hbsag-2A-R为引物，扩增hbsag片段；扩增体系：hbsag基因合成片段50ng，10uM hbsag-F引物1ul，10uM hbsag-2A-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
③以2A基因合成片段为模板，以2A-F和2A-E146L-R为引物，扩增2A片段；扩增体系：2A基因合成片段50ng，10uM 2A-F引物1ul，10uM 2A-E146L-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
④以E146L基因合成片段为模板，以E146L-F和E146L-pS5E4-XhoI-R为引物，扩增E146L片段；扩增体系：E146L基因合成片段50ng，10uM E146L-F引物1ul，10uM E146L-pS5E4-XhoI-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、30sec，35个循环；72℃，5min。
使用Axygen胶回收试剂盒纯化目的片段。
4)融合PCR扩增I73Rhbsag片段
扩增体系：I73R胶回收片段50ng、hbsag胶回收片段50ng，10uM pS5E4-I73R-HamHI-F引物1ul，10uM hbsag-2A-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
5)融合PCR扩增2A-E146L片段
扩增体系：2A胶回收片段50ng、E146L胶回收片段50ng，10uM 2A-F引物1ul，10uM E146L-pS5E4-XhoI-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、30sec，35个循环；72℃，5min。融合结果如图107所示，其中泳道1为I73Rhbsag片段，泳道2为2A-E146L片段，M为2000bp Marker。
6)pS5E4-EGFP载体酶切
酶切反应体系：载体pS5E4-EGFP 2ug，BamHI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。Axygen试剂盒胶回收纯化。
7)使用Axygen胶回收试剂盒纯化载体片段；
胶回收结果如图108所示，其中泳道1为片段pS5E4-EGFPBamHI、XhoI双酶切胶回收，M为15000bp Marker。
8)pS5E4-EGFP胶回收载体与I73Rhbsag片段、2A-E146L无缝克隆连接与转化
连接体系：pS5E4-EGFP胶回收产物(100ng)，I73Rhbsag片段(50ng)，2A-E146L片段(50ng)，2×Smealess Cloning Mix 5ul，补水至10ul。反应条件：50℃，40min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
9)质粒的验证
①菌落PCR验证
用引物以EF1α2(jd)-F、HBV(jd)-R为引物，菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图109所示，其中1-12号为菌落，M为15000bp Marker，均为阳性条带。
②酶切验证
挑取1、2、3号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行BmHI、XhoI双酶切验证；酶切结果如图110所示，其中泳道1、2、3为阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker。酶切结果正确，测序正确，成功构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-I73Rhbsag-2A-E146L，其载体图谱如图124所示。
实施例25穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L、pS5E4-I73Rhbsag-2A-E146L与pAd5LCL3重组构建pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L质粒
1、穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L与腺病毒载体质粒pAd5LCL3的同源重组
1)PacI和SwaI对穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L和腺病毒载体质粒pAd5LCL3进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E1-L8Lubiqutin-IRES-I215L 3μg；PacI 2ul；buffer cutsmart 4ul；补水至40ul。
B、腺病毒载体质粒pAd5LCL3 3ug；SwaI 2ul；Buffer 3.1 4ul；补水至40ul。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图111所示，其中，泳道1为pAd5LCL3，泳道2为pS5E1-L8Lubiqutin-IRES-I215L。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5ul；去磷酸化酶1ul；去磷酸化buffer 5ul；补水至50ul。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证；结果如图112所示，其中泳道1-7为pAd5LCL3-L8Lubiqutin-IRES-I215L克隆，M：15000bp Marker，从图112可以看出，6号和7号克隆酶切正确。
6)将6号阳性质粒转化至DH5a感受态，挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证，酶切结果如图113所示，其中1号泳道为pAd5LCL3-L8Lubiqutin-IRES-I215L质粒XhoI酶切，M为15000bp Marker，由图113可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-L8Lubiqutin-IRES-I215L。
2、穿梭质粒pS5E4-I73Rhbsag-2A-E146L与腺病毒载体质粒pAd5LCL3-L8Lubiqutin-IRES-I215L同源重组获得pAd5LCL3-L8Lubiqutin-I215L-I73HbsAg-E146L
1)PacI和I-sceI对穿梭质粒pS5E4-I73Rhbsag-2A-E146L和腺病毒载体质粒pAd5LCL3-L8Lubiqutin-IRES-I215L进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E4-I73Rhbsag-2A-E146L 3μg；PacI 2ul；10×cutsmart buffer 4ul；补水至40ul。
B、腺病毒载体质粒pAd5LCL3-L8Lubiqutin-IRES-I215L 3ug；I-sceI 2ul；Buffer cutsmart 4ul；补水 至40ul。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图114所示，其中泳道1为pS5E4-I73Rhbsag-2A-E146L，泳道2为pAd5LCL3-L8Lubiqutin-IRES-I215L。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5ul；去磷酸化酶1ul；去磷酸化buffer 5ul；补水至50ul。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取6个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证，结果如图115所示，其中泳道1-8为质粒，M为15000bp Marker，可以看出，1-8质粒均正确。
6)将2号阳性质粒转化至DH5a感受态；挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证；酶切结果如图116所示，2号泳道为pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L质粒XhoI酶切，M为15000Marker，由图116可见，pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L构建成功，酶切结果正确，其载体图谱如图125所示。
实施例26重组腺病毒的包装
使用293TD37细胞包装pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L质粒，操作步骤如下所示：
准备293TD37细胞：转染前一天准备细胞，将待转染的293TD37细胞接种到6孔板中，0.5×10 6/孔，于37℃，5％CO 2静置培养24小时，转染当天细胞有40-50％汇合率。
质粒pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L线性化：将待转染的质粒用PacI酶切，于37℃孵育40min后，65℃灭活20min。
转染：用100ul无血清培养基将线性化的2μg质粒和PEI分别稀释；向PEI稀释液中加入质粒稀释液，反复吸取5次或涡旋10秒钟混匀，室温下孵育10分钟后形成转染复合物。孵育过程中，从培养板上轻柔地吸出细胞培养液，加入新鲜的生长培养基2mL，10分钟后将转染复合物加到换了新鲜培养基的细胞中。
细胞培养：将转染后的293TD37细胞于37℃，5％CO 2培养箱中静置培养72-96小时；病毒质粒转染72-96小时后收集6孔板细胞悬液于1.5ml离心管中即TP0。
持续接毒：将收集的细胞悬液在-80℃反复冻融3次，4℃、2000g离心10分钟，取上清500ul感染293TD37细胞(293TD37细胞需要提前一天准备)，37℃、5％CO 2孵育60分钟，补充2mL的FBS的培养基，37℃、5％CO 2培养培养72小时，收集细胞悬液即TP1；重复之前的步骤，收集细胞悬液即TP2。持续接毒直至细胞出现病变。
细胞病变：当293TD37细胞培养从TP0至TP4后，细胞逐渐病变，直至TP4时293TD37细胞完全病变。TP0至TP4引起的细胞病变情况分别如图117-121所示。
实施例27非洲猪瘟多抗原重组腺病毒疫苗滴度的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，接种6孔板中 (5×105/mL，每孔2ml)，于37℃，5％CO 2二氧化碳培养箱中静置培养。24小时后，待细胞贴壁生长成单层细胞，弃去培养基，用无血清DMEM维持液对重组的腺病毒作10 -3～10 -6倍连续稀释，每个稀释度接种2孔，每孔250uL，感染1小时后，弃上清，补充完全培养基，然后于37℃，5％二氧化碳培养箱中静置培养。24h后，弃上清，用PBS洗细胞，每孔1mL，弃PBS后，每孔加1mL冷甲醛固定，室温10min，弃甲醛，再用PBS冲洗细胞，每孔1mL，加腺病毒抗体-FITC，每孔1ml，室温1h后，再次用PBS冲洗细胞，每孔1mL，两遍后每孔加1mL PBS，荧光显微镜下计数(200倍,10个连续视野)。计算：病毒滴度(FFU/mL)＝平均数×1013×4×10 (-n)。pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒的FFU为2.2×10 8FFU/mL，滴度较高。
实施例28非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L稳定性的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，将293TD37细胞种入6孔板中(5×10 5cells/mL，2mL/孔)，室温孵育1小时使其贴壁，孵育后镜检其贴壁程度。用pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒颗粒进行感染，感染的滴度为5MOI/孔。293TD37细胞48小时后发生病变后，收集细胞，反复冻融3次后2000g离心，收集上清，将收集的上清检测FFU，后重新感染新的293TD37细胞，直至30代。将收集的第5、10、15、20、25、30代次的病毒液进行检测，发现病毒的基因组仍然完整，说明复制缺陷型pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒能够在293TD37细胞中稳定包装。
实施例29非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L恢复突变(RCA)的检测
pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒RCA检测，检测方法如下：
1、准备pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒液，并测其病毒滴度，测定病毒颗粒浓度，病毒液加1％Universal nuclease(Universal nuclease 7.5～15units/mL病毒液)消化宿主细胞的DNA，37℃水浴40min。用300Kd的超滤离心管，经1000g，30min离心，1×PBS洗脱收集病毒颗粒，测A260，颗粒浓度＝A260*1.1*10^12VP/mL。
2、病毒感染，准备A549细胞的6孔板，每孔细胞为2.5×10 5/孔，弃培养基，PBS清洗一次，将腺病毒按照1×10 9vp/孔接种病毒，感染A549细胞，野生型腺病毒5型为对照，37℃，5％CO 2，1h后，弃去病毒液，补足5％完全培养基，37℃，5％CO 2培养48h。
3、免疫染色，弃细胞上清，PBS表面冲洗细胞，用冰甲醛固定，放置-20℃，20min，1×PBS清洗三遍，每遍5min，每孔加入2ml 1％BSA-PBS溶液，放置摇床，孵育1h。弃去上清，加入腺病毒5型荧光抗体(1:500稀释)，孵育1h，1×PBS清洗三遍，每遍5min。
用10倍荧光显微镜观察，使用公式计算RCA
RCA＝(average positive cell field)×(374 field/well)×(dilution factor))/Total VPs in 0.5ml viral sample
判断标准为RCA的水平＜1RCA/3×10 10vp。经过统计pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L的RCA的水平小于1RCA/3×10 10vp，说明本发明所制备的复制缺陷型pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒在293TD37细胞中能够稳定包装，不会转化为野生型或转化为野生型的几率较低。
实施例30非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L蛋白表达检测
提前一天准备293TD37细胞，置于12孔细胞培养板中，使用非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L病毒感染293TD37细胞，48小时后细胞发生病变，收集全部的1ml细胞，使用PBS洗涤，制样，用于Western Blot检测；使用HA的抗体检测目的蛋白，HA的抗体采购于Abcam。其中L8Lubiquitin融合蛋白，I215L蛋白具有HA标签，蛋白大小为32kda、26kda。实验结果如图122所示，泳道1为293空白细胞，泳道2、3为293TD37细胞感染pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L的样品；可以清晰可见L8Lubiquitin融合蛋白与I215L蛋白有正常表达，由此可见pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L疫苗能在293细胞中正常表达目的蛋白。
实施例31非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L小鼠模型上的免疫学评价
疫苗细胞免疫反应检测
10只SPF级小鼠(6-8周龄)，随机分成2组，每组5只。根据表6所示分组情况对小鼠进行pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表6：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000014
免疫后14天处死小鼠，分离脾淋巴细胞，使用穿梭质粒pS5E1-L8Lubiqutin-I215L和pS5E4-I73Rhbsag-E146L转染的PK15细胞刺激培养6小时，同时加入蛋白分泌阻断剂阻断细胞因子分泌。6小时后，阻断Fc受体，对死细胞和细胞表面分子标志物进行染色，细胞经固定和穿孔之后，对细胞内细胞因子进行染色。细胞表面标志物包括CD4、CD8，细胞内细胞因子包括IFNγ、IL2。使用流式细胞仪(CyExpert)分析CD4+T细胞和CD8+T细胞经目的蛋白刺激后表达IFNγ、IL2的水平。
pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L诱导的CD8+T细胞和CD4+T细胞免疫反应如图126和图127所示，代表性结果如图128至图129所示，其中图128为肌肉注射pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L后细胞免疫反应代表图，图129为空白对照免疫反应代表图。结果表示：小鼠免疫后14天，脾细胞经目的蛋白刺激后，CD8+T细胞，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。CD4+T细胞经过刺激后，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。细胞免疫反应检测结果说明肌肉注射免疫1*10^7FFU的腺病毒载体疫苗疫苗，可诱导其免疫的小鼠产生特异性细胞免疫反应。
实施例32非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-EP402R-IRES-EP153R的构建
1、人类腺病毒5型载体E1区域穿梭质粒的构建
按照实施例4提供的方法成功构建人类腺病毒5型载体E1区域穿梭质粒pS5E1。
2、非洲猪瘟腺病毒5型载体穿梭质粒pS5E1-EP402R-IRES-EP153R构建
1)pS5E1与IRES片段的连接
按照实施例4提供的方法成功构建pS5E1-IRES。
2)pS5E1-IRES与EP402R片段的连接
①引物合成
EP402R-BamHI-F：cgc GGATCC gccaccATGATCATCATCGTGATCTTCC
EP402R-EcoRV：ccg GATATC ttaAGCGTAGTCTGGGACGTCGT
②PCR扩增EP402R片段
扩增体系：Q5酶25ul，10uM引物EP402R-BamHI-F 1ul，10uM引物EP402R-EcoRV 1ul，模板EP402R 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、45s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化EP402R片段。
④目的片段EP402R与pS5E1-IRES载体酶切
酶切反应体系：载体pS5E1-IRES、EP402R片段～2ug，EcoRV和BamHI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图23所示，其中泳道1为片段EP402R，BamHI、EcoRV双酶切，泳道2为pS5E1-IRES，BamHI、EcoRV双酶切，M为15000bp Marker。
⑤目的片段EP402R与pS5E1-IRES连接
连接体系：pS5E1-IRES(100ng)；EP402R片段(载体：片段＝1：3摩尔比)；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物EP402R-BamHI-F 1ul，10uM引物EP402R-EcoRV-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、1min，35个循环；72℃，5min。进行电泳验证，如图130所示，其中1-6号为菌落，M为2000bp Marker。
⑦质粒酶切验证(BamHI&EcoRV)，选取1、2、4菌落进行质粒提取，酶切验证。结果如图131所示，均为阳性质粒。
3)pS5E1-EP402R-IRES与片段EP153R的连接
①引物合成
EP153R-NotI-F：ATAAGAAT GCGGCCGCgccaccATGTTCAGCAACAAGAAGTACAT
EP153R-XhoI-R：AAACTCGAGTCACTTGCTACAGATGTACAG
②PCR扩增EP153R片段
扩增体系：Q5酶25ul，10uM引物EP153R-NotI-F 1ul，10uM引物EP153R-XhoI-R 1ul，模板EP153R 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化EP153R片段。
④目的片段EP153R与pS5E1-EP402R-IRES载体酶切
酶切反应体系：载体pS5E1-EP402R-IRES、EP153R片段～2ug，NotI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图132所示，其中泳道1为pS5E1-EP402R-IRES，NotI、XhoI酶切，泳道2为EP153R片段，NotI、XhoI酶切，M为2000bp Marker。
⑤pS5E1-EP402R-IRES载体与EP153R片段连接
连接体系：pS5E1-EP402R-IRES 100ng；EP153R片段50ng；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM通用引物CMV-F 1ul，10uM引物EP153R-XhoI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、2min30s，35个循环；72℃，5min；进行电泳验证，如图133所示，其中1-14号为菌落，M为5000bp Marker。
⑦质粒BamHI、XhoI酶切验证，选取1、2、3、11、13进行质粒提取，酶切验证，结果如图134所示，其中1、2、4、6号泳道为1、2、4、6质粒BamHI、XhoI酶切鉴定，M为15000bp Marker。由图134可见，酶切结果正确，成功构件非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-EP402R-IRES-EP153R，其载体图谱如图150所示。
实施例33非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-I177L-2A-K205Rubiqutin构建
1、人类腺病毒5型载体E4区域穿梭质粒的构建
按照实施例5的方法，成功构建人类腺病毒5型载体E4区域穿梭质粒pS5E4-EGFP。
2、非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-I177L-2A-K205Rubiqutin构建
1)引物设计
EF1α-BamHI-I177L-F：ccaagctgtgaccggcgcctacGGATCCGCCACCATGTGGAAGGTGAA
I177L-R：GGCGTAATCGGGCACGTCG
I177L-2A-F：CTACGACGTGCCCGATTACGCCGGAAGCGGAGCTACTAACTTC
2A-K205R-R：AACTGCTCTCTGGGCTCCACCATAGGTCCAGGGTTCTCCTCCA
K205R-F：ATGGTGGAGCCCAGAGAGCA
K205R-ubiqutin-R：GGGTTTTCACGAAAATCTGCATGGCGTAATCGGGCACATCGT
ubiqutin-F：ATGCAGATTTTCGTGAAAACCC
ubiquitin-XhoI-HBV-R：GGGTTTAAACGGGCCCTCTAGACTCGAGTTACTTGTCTTCTGGTTTGTTGA
2)目的片段I177L-K205Rubiqutin的扩增
①以I177L基因合成片段为模板，以EF1α-BamHI-I177L-F和I177L-R为引物，扩增I177L片段；扩增体系：I177L基因合成片段50ng，10uM EF1α-BamHI-I177L-F引物1ul，10uM I177L-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、30sec，35个循环；72℃，5min。
②以2A基因合成片段为模板，以I177L-2A-F和2A-K205R-R为引物，扩增2A片段；扩增体系：2A基因合成片段50ng，10uM I177L-2A-F引物1ul，10uM 2A-K205R-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
③以K205R基因合成片段为模板，以K205R-F和K205R-ubiqutin-R为引物，扩增K205R片段；扩增体系：K205R基因合成片段50ng，10uM K205R-F引物1ul，10uM K205R-ubiqutin-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、30sec，35个循环；72℃，5min。
④以ubiqutin基因合成片段为模板，以ubiqutin-F和ubiquitin-XhoI-HBV-R为引物，扩增ubiqutin片段；扩增体系：ubiqutin基因合成片段50ng，10uM ubiqutin-F引物1ul，10uM ubiquitin-XhoI-HBV-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
3)使用Axygen胶回收试剂盒纯化目的片段。
4)融合PCR扩增I177L-2A片段、K205Rubiqutin片段
扩增体系：I177L胶回收片段50ng、2A胶回收片段50ng，10uM EF1α-BamHI-I177L-F引物1ul，10uM 2A-K205R-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
扩增体系：K205R胶回收片段50ng、ubiqutin胶回收片段50ng，10uM K205R-F引物1ul，10uM 2A-K205R-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
PCR产物电泳检测如图135所示，其中泳道1为片段K205R；泳道2为片段ubiqutin；泳道3为片段K205Rubiqutin，M为2000bp Marker；泳道4为片段2A；泳道5为片段I177L；泳道6为片段I177L-2A，M为2000bp Marker。
5)pS5E4-EGFP载体酶切
酶切反应体系：载体pS5E4-EGFP 2ug，BamHI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。Axygen试剂盒胶回收纯化。
6)使用Axygen胶回收试剂盒纯化载体片段
胶回收结果如图136所示，其中泳道1为片段pS5E4-EGFP，BamHI、XhoI双酶切胶回收，M为15000bp Marker。
7)pS5E4-EGFP胶回收载体与I177L-2A片段、K205Rubiqutin无缝克隆连接与转化
连接体系：pS5E4-EGFP胶回收产物(100ng)，I177L-2A片段(50ng)，K205Rubiqutin片段(50ng)，2×Smealess Cloning Mix 5ul，补水至10ul。反应条件：50℃，40min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
8)质粒的验证
①菌落PCR验证
用引物以EF1α2(jd)-F、HBV(jd)-R为引物，菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图137所示，其中1-3号为菌落，M为15000bp Marker。
②酶切验证
挑取1、2、3号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行BmHI、XhoI双酶切验证；酶切结果如图138所示，其中泳道1、2、3为阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker。酶切结果正确，测序正确，成功构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-I177L-2A-K205Rubiqutin，测序正确，其载体图谱如图151所示。
实施例34穿梭质粒pS5E1-EP402R-IRES-EP153R、pS5E4-I177L-2A-K205Rubiqutin与pAd5LCL3重组构建pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒
1、穿梭质粒pS5E1-EP402R-IRES-EP153R与腺病毒载体质粒pAd5LCL3的同源重组
1)PacI和SwaI对穿梭质粒pS5E1-EP402R-IRES-EP153R和腺病毒载体质粒pAd5LCL3进行酶切， 酶切反应体系如下：
A、穿梭质粒pS5E1-EP402R-IRES-EP153R 3μg；PacI 2μl；buffer cutsmart 4μl；补水至40μl。
B、腺病毒载体质粒pAd5LCL3 3ug；SwaI 2μl；Buffer 3.1 4μl；补水至40μl。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图139所示，其中，泳道1为pAd5LCL3，泳道2为pS5E1-EP402R-IRES-EP153R。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5μl；去磷酸化酶1μl；去磷酸化buffer 5μl；补水至50μl。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证；结果如图140所示，其中泳道1-8为pAd5LCL3-EP402R-IRES-EP153R克隆，M：15000bp Marker，从图140可以看出，2、3、8号克隆酶切正确。
6)将2号阳性质粒转化至DH5α感受态，挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证，酶切结果如图141所示，由图141可知，其中1号泳道为pAd5LCL3-EP402R-IRES-EP153R质粒XhoI酶切，2号泳道为pAd5LCL3-EP402R-IRES-EP153R质粒PacI酶切，3号泳道为pAd5LCL3-EP402R-IRES-EP153R质粒BamHI酶切，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-EP402R-IRES-EP153R。
2、穿梭质粒pS5E4-I177L-2A-K205Rubiqutin与腺病毒载体质粒pAd5LCL3-EP402R-IRES-EP153R同源重组获得pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin
1)PacI和I-sceI对穿梭质粒pS5E4-I177L-2A-K205Rubiqutin和腺病毒载体质粒pAd5LCL3-EP402R-IRES-EP153R进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E4-I177L-2A-K205Rubiqutin 3μg；PacI 2μl；10×cutsmart buffer 4μl；补水至40μl。
B、腺病毒载体质粒pAd5LCL3-EP402R-IRES-EP153R 3ug；I-sceI 2μl；Buffer cutsmart 4μl；补水至40μl。
反应条件37℃，1h；65℃，20min灭活。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5μl；去磷酸化酶1μl；去磷酸化buffer 5μl；补水至50μl。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取6个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证，结果如图142所示，其中泳道1-7为质粒，M为15000Marker，可以看出，1、7号质粒正确。
6)将1号阳性质粒转化至DH5α感受态；挑取1个菌落于5mL含有Kan的LB液体培养基中，37℃ 振荡培养12～16h，并提取质粒再次进行XhoI酶切验证；酶切结果如图143所示，其中泳道1、2为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒XhoI酶切，泳道3为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒BamHI酶切，泳道4为pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒PacI酶切，M为15000Marker，由图143可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin，酶切结果正确，其载体图谱如图152所示。
实施例35重组腺病毒的包装
使用293TD37细胞包装pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin质粒，操作步骤如下所示：
准备293TD37细胞：转染前一天准备细胞，将待转染的293TD37细胞接种到6孔板中，0.5×10 6/孔，于37℃，5％CO 2静置培养24小时，转染当天细胞有40-50％汇合率。
质粒pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin线性化：将待转染的质粒用PacI酶切，于37℃孵育40min后，65℃灭活20min。
转染：用100μl无血清培养基将线性化的2μg质粒和PEI分别稀释；向PEI稀释液中加入质粒稀释液，反复吸取5次或涡旋10秒钟混匀，室温下孵育10分钟后形成转染复合物。孵育过程中，从培养板上轻柔地吸出细胞培养液，加入新鲜的生长培养基2mL，10分钟后将转染复合物加到换了新鲜培养基的细胞中。
细胞培养：将转染后的293TD37细胞于37℃，5％CO 2培养箱中静置培养72-96小时；病毒质粒转染72-96小时后收集6孔板细胞悬液于1.5ml离心管中即TP0。
持续接毒：将收集的细胞悬液在-80℃反复冻融3次，4℃、2000g离心10分钟，取上清500μl感染293TD37细胞(293TD37细胞需要提前一天准备)，37℃、5％CO 2孵育60分钟，补充2mL的FBS的培养基，37℃、5％CO 2培养培养72小时，收集细胞悬液即TP1；重复之前的步骤，收集细胞悬液即TP2。持续接毒直至细胞出现病变。
细胞病变：当293TD37细胞培养从TP0至TP4后，细胞逐渐病变，直至TP4293TD37细胞完全病变。TP0至TP4引起的细胞病变情况分别如图144-148所示，TP4已完全病变。
实施例36非洲猪瘟多抗原重组腺病毒疫苗滴度的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，接种6孔板中(5×10 5/mL，每孔2ml)，于37℃，5％CO 2二氧化碳培养箱中静置培养。24小时后，待细胞贴壁生长成单层细胞，弃去培养基，用无血清DMEM维持液对重组的腺病毒作10 -3～10 -6倍连续稀释，每个稀释度接种2孔，每孔250uL，感染1小时后，弃上清，补充完全培养基，然后于37℃，5％二氧化碳培养箱中静置培养。24h后，弃上清，用PBS洗细胞，每孔1mL，弃PBS后，每孔加1mL冷甲醛固定，室温10min，弃甲醛，再用PBS冲洗细胞，每孔1mL，加腺病毒抗体-FITC，每孔1ml，室温1h后，再次用PBS冲洗细胞，每孔1mL，两遍后每孔加1mL PBS，荧光显微镜下计数(200倍,10个连续视野)。计算：病毒滴度(FFU/mL)＝平均数×1013×4×10 (-n)。pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒的FFU为1.8×10 8FFU/mL，滴度较高。
实施例37非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin稳定性的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，将293TD37细胞种入6孔板中(5×10 5cells/mL，2mL/孔)，室温孵育1小时使其贴壁，孵育后镜检其贴壁程度。用pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒颗粒进行感染，感染的滴度为5MOI/孔。293TD37细胞48小时后发生病变后，收集细胞，反复冻融3次后2000g离心，收集上清，将收集的上清检测FFU，后重新感染新的293TD37细胞，直至30代。将收集的第5、10、15、20、25、30代次的病毒液进行检测，发现病毒的基因组仍然完整，说明复制缺陷型pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒能够在293TD37细胞中稳定包装。
实施例38非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin恢复突变(RCA)的检测
pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒RCA检测，检测方法如下：
1、准备pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒液，并测其病毒滴度，测定病毒颗粒浓度，病毒液加1％Universal nuclease(Universal nuclease 7.5～15units/mL病毒液)消化宿主细胞的DNA，37℃水浴40min。用300Kd的超滤离心管，经1000g，30min离心，1×PBS洗脱收集病毒颗粒，测A260，颗粒浓度＝A260*1.1*10^12VP/mL。
2、病毒感染，准备A549细胞的6孔板，每孔细胞为2.5×10 5/孔，弃培养基，PBS清洗一次，将腺病毒按照1×10 9vp/孔接种病毒，感染A549细胞，野生型腺病毒5型为对照，37℃，5％CO 2，1h后，弃去病毒液，补足5％完全培养基，37℃，5％CO 2培养48h。
3、免疫染色，弃细胞上清，PBS表面冲洗细胞，用冰甲醛固定，放置-20℃，20min，1×PBS清洗三遍，每遍5min，每孔加入2ml 1％BSA-PBS溶液，放置摇床，孵育1h。弃去上清，加入腺病毒5型荧光抗体(1:500稀释)，孵育1h，1×PBS清洗三遍，每遍5min。
用10倍荧光显微镜观察，使用公式计算RCA
RCA＝(average positive cell field)×(374 field/well)×(dilution factor))/Total VPs in 0.5ml viral sample
判断标准为RCA的水平小于1RCA/3×10 10vp。经过统计pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin的RCA的水平小于1RCA/3×10 10vp，说明本发明所制备的复制缺陷型pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒在293TD37细胞中能够稳定包装，不会转化为野生型或转化为野生型的几率较低。
实施例39非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin蛋白表达检测
提前一天准备293TD37细胞，置于12孔细胞培养板中，使用非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin病毒感染293TD37细胞，48小时后细胞发生病变，收集全部的1ml细胞，使用PBS洗涤，制样，用于Western Blot检测；使用本公司制备的EP153R鼠多抗血清检测目的蛋白，EP153R鼠多抗血清是由大肠杆菌系统表达的EP153R蛋白免疫小鼠获得。EP153R蛋白的大小为15kda。实验结果如图148所示，泳道1为293TD37细胞感染pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin的样品；可以清晰可见EP153R蛋白有正常表达，由此可见pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin疫苗能够在293细胞中表达目的蛋白。
同时，使用本公司制备的EP153R鼠多抗血清检测目的蛋白，EP153R鼠多抗血清是由大肠杆菌系 统表达的EP153R蛋白免疫小鼠获得。EP153R蛋白的大小为15kda。
实施例40非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin在小鼠模型上的免疫学评价
40.1疫苗体液免疫反应检测
20只SPF级小鼠(6-8周龄)，随机分成4组，每组5只。根据表7所示分组情况对小鼠进行pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表7：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000015
小鼠于免疫后14天采血，分离血清，使用间接ELISA法检测血清中针对非洲猪瘟目的蛋白EP402R(EP402R蛋白是由本公司在昆虫细胞中制备并免疫小鼠后获得)的IgG抗体滴度。检测结果如图153所示(ns,P≥0.05；*,P<0.05；**,P<0.01；***,P<0.001；****,P<0.0001)：小鼠肌肉注射pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin后，针对EP402R蛋白，能够产生较高浓度的IgG抗体。高剂量组抗体滴度平均值达70000以上，中剂量组的滴度平均值也达50000，与对照组有显著差异。
40.2细胞免疫反应检测
10只SPF级小鼠(6-8周龄)，随机分成2组，每组5只。根据表8所示分组情况对小鼠进行pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表8：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000016
免疫后14天处死小鼠，分离脾淋巴细胞，使用穿梭质粒pS5E1-EP402R-IRES-EP153R和pS5E4-I177L-2A-K205Rubiqutin转染的PK15细胞刺激培养6小时，同时加入蛋白分泌阻断剂阻断细胞因子分泌。6小时后，阻断Fc受体，对死细胞和细胞表面分子标志物进行染色，细胞经固定和穿孔之后，对细胞内细胞因子进行染色。细胞表面标志物包括CD4、CD8，细胞内细胞因子包括IFNγ、IL2。使用流式细胞仪(CyExpert)分析CD4+T细胞和CD8+T细胞经目的蛋白刺激后表达IFNγ、IL2的水平。
pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin诱导的CD8+T细胞和CD4+T细胞免疫反应如图154、图155所示，代表性结果如图156至图157所示，其中图156为肌肉注射pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin后细胞免疫反应代表图，图157为空白对照免疫反应代表图。结果表示：小鼠免疫后14天，脾细胞经目的蛋白刺激后，CD8+T细胞，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。CD4+T细胞经过刺激后，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。
40.3小鼠模型免疫原性评价小结
pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin重组腺病毒具有良好的免疫原性，可诱导小鼠产生高水平的血清IgG抗体。其中高剂量的1*10^8FFU和中剂量1*10^7FFU的免疫方式诱导的滴度都很高。细胞免疫反应检测结果说明肌肉注射免疫1*10^7FFU的腺病毒载体疫苗，可诱导其免疫的小鼠产生特异性细胞免疫反应。
实施例41非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-F317L-IRES-A151R的构建
1、人类腺病毒5型载体E1区域穿梭质粒的构建
按照实施例4提供的方法，成功构建人类腺病毒5型载体E1区域穿梭质粒pS5E1。
2、非洲猪瘟腺病毒5型载体穿梭质粒pS5E1-F317L-IRES-A151R构建
1)pS5E1与IRES片段的连接
按照实施例4提供的方法，成功构建pS5E1-IRES。
2)pS5E1-IRES与F317L片段的连接
①引物合成
F317L-BamHI-F：cgc GGATCC gccaccATGGTGGAGACCCAGATGGACA
F317L-EcoRV-R：ccg GATATC TCAGTGGTGGTGGTGGTGGTG
②PCR扩增F317L片段
扩增体系：Q5酶25ul，10uM引物F317L-BamHI-F 1ul，10uM引物F317L-EcoRV-R 1ul，模板F317L 1ul，补水至50ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。
③使用Axygen PCR纯化试剂盒纯化F317L片段。
④目的片段F317L与pS5E1-IRES载体酶切
酶切反应体系：载体pS5E1-IRES、F317L片段～2ug，BamHI和EcoRV各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图158所示，其中泳道1为pS5E1-IRES，BamHI和EcorV双酶切，泳道2为片段F317L，BamHI和EcoRV双酶切，M为15000bp Marker。
⑤目的片段F317L与pS5E1-IRES连接
连接体系：pS5E1-IRES(100ng)；F317L片段(载体：片段＝1：3摩尔比)；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物F317L-BamHI-F 1ul，10uM引物IRES-NotI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min。进行电泳验证，如图159所示，其中1-24号为菌落，M为2000bp Marker。由图159可见，9、10号为阳性菌落。
⑦质粒酶切验证(BamHI和EcoRV)，选取9、10菌落进行质粒提取，酶切验证。结果如图160所示，均为阳性质粒。
3)pS5E1-F317L-IRES与片段A151R的连接
①引物合成
A151R-NotI-F：aaatat GCGGCCGC ATGAACAAGAAGATCATCGTGATG
A151R-6His-XhoI-R：
cggCTCGAGTCAGTGGTGGTGGTGATGGTGCTGGAAGATGTTGGGGGACATGA
②PCR扩增A151R片段
扩增体系：Q5酶25ul，引物A151R-NotI-F 1ul，引物A151R-6His-XhoI-R 1ul，模板A151R，补水至50ul；反应条件：98℃30S；98℃10s，68℃30s，72℃30s，35个循环；72℃5min。
③使用Axygen PCR纯化试剂盒纯化A151R片段。
④目的片段A151R与pS5E1-F317L-IRES载体酶切
酶切反应体系：载体pS5E1-F317L-IRES、A151R片段～2ug，NotI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。胶回收纯化。酶切产物电泳检测如图161所示，其中泳道1为pS5E1-F317L-IRES，NotI和XhoI酶切；泳道2、3为A151R片段，NotI和XhoI酶切，M为15000bp Marker。
⑤pS5E1-F317L-IRES载体与A151R片段连接
连接体系：pS5E1-F317L-IRES 100ng；A151R片段50ng；T4 DNA连接酶1ul；10×ligase buffer 1ul；补水至10ul。反应条件：室温，30min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
⑥菌落PCR验证
扩增体系：Q5酶10ul，10uM引物IRES-ECoRV-F 1ul，10uM引物A151R-6His-XhoI-R 1ul，补水至20ul；PCR程序为：98℃，10s；98℃、5s，60℃、30s，72℃、20s，35个循环；72℃，5min；进行电泳验证，如图162所示，其中1-24号为菌落，其中M为2000bp Marker。
⑦质粒BamHI、XhoI酶切验证，选取4、15、23、24菌落进行质粒提取，酶切验证，结果如图28所示，BamHI、EcoRV酶切鉴定，M为2000bp Marker。由图163可见，酶切结果正确，成功构件非洲猪瘟腺病毒5型载体E1区域穿梭质粒pS5E1-F317L-IRES-A151R，测序正确，其载体图谱如图51所示。
实施例5非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-P34-2A-pp62的构建
1、人类腺病毒5型载体E4区域穿梭质粒的构建
按照实施例5提供的方法，成功构建人类腺病毒5型载体E4区域穿梭质粒pS5E4-EGFP。
2、非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-P34-2A-pp62构建
1)引物设计
EF1α-BamHI-P34-F：tccaagctgtgaccggcgcctacGGATCCGCCACCATGGGGAATCGCGGGTCTTCT
P34-2A-R：GCCCTTTTTGGCGCAGCTGTT
P34-2A-F：AGAACAGCTGCGCCAAAAAGGGCGGAAGCGGAGCTACTAACTTC
2A-pp62-R：AACTGCTTCATGTTGCTGGGCATAGGTCCAGGGTTCTCCTCCA
pp62-F：ATGCCCAGCAACATGAAGCAG
pp62-XhoI-pS5E4-R：GGGTTTAAACGGGCCCTCTAGACTCGAGttaCAGCAGCTTCA
GGATCTCGTT
2)目的片段P34、2A、pp62的扩增
①以P34基因合成片段为模板，以EF1α-BamHI-P34-F和P34-2A-R为引物，扩增P34片段；扩增体系：P34基因合成片段50ng，10uM EF1α-BamHI-P34-F引物1ul，10uM P34-2A-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
②以2A基因合成片段为模板，以P34-2A-F和2A-pp62-R为引物，扩增2A片段；扩增体系：2A基因合成片段50ng，10uM P34-2A-F引物1ul，10uM 2A-pp62-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、20sec，35个循环；72℃，5min。
③以pp62基因合成片段为模板，以pp62-F和pp62-XhoI-pS5E4-R为引物，扩增2A片段；扩增体系：pp62基因合成片段50ng，10uM pp62-F引物1ul，10uM pp62-XhoI-pS5E4-R引物1ul，Q5高保真酶20ul；补水至40ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。
PCR产物电泳检测如图164所示，其中泳道1为片段p34；2为片段2A；3为片段pp62，M为15000bp Marker。
3)使用Axygen胶回收试剂盒纯化目的片段。
4)融合PCR扩增P34-2A片段
扩增体系：P34胶回收片段50ng、2A胶回收片段50ng，10uM EF1α-BamHI-P34-F引物1ul，10uM 2A-pp62-R引物1ul，Q5高保真酶25ul；补水至50ul；PCR程序为：98℃，10sec；98℃、5sec，60℃、30sec，72℃、40sec，35个循环；72℃，5min。融合结果如图165所示，其中泳道1为P34-2A片段，M为2000bp Marker
5)pS5E4-EGFP载体酶切
酶切反应体系：载体pS5E4-EGFP 2ug，BamHI和XhoI各1ul；10×cutsmart buffer 5ul；补水至50ul。反应条件：37℃，30min；65℃，20min灭活。Axygen试剂盒胶回收纯化。
6)使用Axygen胶回收试剂盒纯化载体片段；
胶回收结果如图166所示，其中泳道1为片段pS5E4-EGFP，BamHI、XhoI双酶切胶回收，M为15000bp Marker。
7)pS5E4-EGFP胶回收载体与P34-2A片段、pp62无缝克隆连接与转化
连接体系：pS5E4-EGFP胶回收产物(100ng)，P34-2A片段(50ng)，pp62片段(50ng)，2×Smealess Cloning Mix 5ul，补水至10ul。反应条件：50℃，40min。将连接产物转化至DH5α感受态细胞，涂布于含有氨苄抗性的平板上，37℃培养12-16小时。
8)质粒的验证
①菌落PCR验证
用引物以EF1α2(jd)-F、HBV(jd)-R为引物，菌落PCR扩增目的片段，琼脂糖凝胶验证，结果如图167所示，其中1-12号为菌落，M为15000bp Marker。
②酶切验证
挑取1、2、9、11号阳性克隆置于5mL含有氨苄抗性LB液体培养基中培养12-15小时，提取质粒进行BmHI、XhoI双酶切验证；酶切结果如图168所示，其中泳道1、2、9、11为阳性克隆BamHI、XhoI双酶切验证，M为15000bp Marker。酶切结果正确，测序正确，成功构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒pS5E4-P34-2A-pp62，其载体图谱如图53所示。
实施例43穿梭质粒pS5E1-F317L-IRES-A151R、pS5E4-P34-2A-pp62与pAd5LCL3重组构建pAd5LCL3-F317L-A151R-P34-pp62质粒
1、穿梭质粒pS5E1-F317L-IRES-A151R与腺病毒载体质粒pAd5LCL3的同源重组
1)PacI和SwaI对穿梭质粒pS5E1-F317L-IRES-A151R和腺病毒载体质粒pAd5LCL3进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E1-F317L-IRES-A151R 3μg；PacI 2μl；buffer cutsmart 4μl；补水至40μl。
B、腺病毒载体质粒pAd5LCL3 3ug；SwaI 2μl；Buffer 3.1 4μl；补水至40μl。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图169所示，其中，泳道1为pAd5LCL3，泳道2为pS5E1-F317L-IRES-A151R。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5μl；去磷酸化酶1μl；去磷酸化buffer 5μl；补水至50μl。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证；结果如图170所示，其中泳道1-7为pAd5LCL3-F317L-IRES-A151R克隆，M：15000bp Marker，从图38可以看出，3号克隆酶切正确。
6)将3号阳性质粒转化至DH5α感受态，挑取一个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证，酶切结果如图171所示，由图171可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-F317L-IRES-A151R。
2、穿梭质粒pS5E4-P34-2A-pp62与腺病毒载体质粒pAd5LCL3-F317L-IRES-A151R同源重组获得pAd5LCL3-F317L-A151R-P34-pp62
1)PacI和I-sceI对穿梭质粒pS5E4-P34-2A-pp62和腺病毒载体质粒pAd5LCL3-F317L-IRES-A151R进行酶切，酶切反应体系如下：
A、穿梭质粒pS5E4-P34-2A-pp62 3μg；PacI 2μl；10×cutsmart buffer 4μl；补水至40μl。
B、腺病毒载体质粒pAd5LCL3-F317L-IRES-A151R 3ug；I-sceI 2μl；Buffer cutsmart 4μl；补水至40μl。
反应条件37℃，1h；65℃，20min灭活。
取2ul琼脂糖凝胶验证，验证结果如图172所示，其中泳道1为pS5E4-P34-2A-pp62，泳道2为pAd5LCL3-F317L-IRES-A151R。
2)酶切产物去磷酸化
反应体系：酶切反应液37.5μl；去磷酸化酶1μl；去磷酸化buffer 5μl；补水至50μl。反应条件37℃，1h；65℃，5min灭活。
3)使用OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段。
4)取100ng纯化后的穿梭质粒和100ng纯化后的腺病毒载体共转化BJ5183感受态细胞，转化产物涂布含有Kan的LB平板，37℃培养12～16h。
5)挑取6个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒进行XhoI酶切验证，结果如图173所示，其中泳道1-4为质粒，M为15000Marker，可以看出，2号质粒正确。
6)将2号阳性质粒转化至DH5α感受态；挑取1个菌落于5mL含有Kan的LB液体培养基中，37℃振荡培养12～16h，并提取质粒再次进行XhoI酶切验证；酶切结果如图174所示，其中1号泳道为pAd5LCL3-F317L-A151R-P34-2A-pp62质粒XhoI酶切，M为15000Marker，由图174可知，酶切结果正确，成功构建腺病毒载体质粒pAd5LCL3-F317L-A151R-P34-pp62，其载体图谱如图54所示。
实施例44重组腺病毒的包装
使用293TD37细胞包装pAd5LCL3-F317L-A151R-P34-pp62质粒，操作步骤如下所示：
准备293TD37细胞：转染前一天准备细胞，将待转染的293TD37细胞接种到6孔板中，0.5×10 6/孔，于37℃，5％CO 2静置培养24小时，转染当天细胞有40-50％汇合率。
质粒pAd5LCL3-F317L-A151R-P34-pp62线性化：将待转染的质粒用PacI酶切，于37℃孵育40min后，65℃灭活20min。
转染：用100μl无血清培养基将线性化的2μg质粒和PEI分别稀释；向PEI稀释液中加入质粒稀释液，反复吸取5次或涡旋10秒钟混匀，室温下孵育10分钟后形成转染复合物。孵育过程中，从培养板上轻柔地吸出细胞培养液，加入新鲜的生长培养基2mL，10分钟后将转染复合物加到换了新鲜培养基的细胞中。
细胞培养：将转染后的293TD37细胞于37℃，5％CO2培养箱中静置培养72-96小时；病毒质粒转染72-96小时后收集6孔板细胞悬液于1.5ml离心管中即TP0。
持续接毒：将收集的细胞悬液在-80℃反复冻融3次，4℃、2000g离心10分钟，取上清500μl感染293TD37细胞(293TD37细胞需要提前一天准备)，37℃、5％CO 2孵育60分钟，补充2mL的FBS的培养基，37℃、5％CO 2培养培养72小时，收集细胞悬液即TP1；重复之前的步骤，收集细胞悬液即TP2。持续接毒直至细胞出现病变。
细胞病变：当293TD37细胞培养从TP0至TP4后，细胞逐渐病变，直至TP4时293TD37细胞完全病变。TP0至TP4引起的细胞病变情况分别如图175-179所示，TP4已完全病变。
实施例45非洲猪瘟多抗原重组腺病毒疫苗滴度的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，接种6孔板中(5×10 5/mL，每孔2ml)，于37℃，5％CO 2二氧化碳培养箱中静置培养。24小时后，待细胞贴壁生长成单层细胞，弃去培养基，用无血清DMEM维持液对重组的腺病毒作10 -3～10 -6倍连续稀释，每个稀释度接种2孔，每孔250uL，感染1小时后，弃上清，补充完全培养基，然后于37℃，5％二氧化碳培养箱中静置培养。24h后，弃上清，用PBS洗细胞，每孔1mL，弃PBS后，每孔加1mL冷甲醛固定，室温10min，弃甲醛，再用PBS冲洗细胞，每孔1mL，加腺病毒抗体-FITC，每孔1ml，室温1h后，再次用PBS冲洗细胞，每孔1mL，两遍后每孔加1mL PBS，荧光显微镜下计数(200倍,10个连续视野)。计算：病毒滴度(FFU/mL)＝平均数×1013×4×10 (-n)。pAd5LCL3-F317L-A151R-P34-pp62病毒的FFU为 2.4×10 8FFU/mL，滴度较高。
实施例46非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-F317L-A151R-P34-pp62稳定性的检测
准备293TD37细胞，取T75培养瓶中生长良好的细胞，弃上清，PBS洗细胞，用0.25％胰蛋白酶消化，再加入含有10％胎牛血清的DMEM新鲜培养基10mL终止消化，然后吹打混匀，将293TD37细胞种入6孔板中(5×10 5cells/mL，2mL/孔)，室温孵育1小时使其贴壁，孵育后镜检其贴壁程度。用pAd5LCL3-F317L-A151R-P34-pp62病毒颗粒进行感染，感染的滴度为5MOI/孔。293TD37细胞48小时后发生病变后，收集细胞，反复冻融3次后2000g离心，收集上清，将收集的上清检测FFU，后重新感染新的293TD37细胞，直至30代。将收集的第5、10、15、20、25、30代次的病毒液进行检测，发现病毒的基因组仍然完整，说明复制缺陷型pAd5LCL3-F317L-A151R-P34-pp62病毒能够在293TD37细胞中稳定包装。
实施例47非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-F317L-A151R-P34-pp62恢复突变(RCA)的检测
pAd5LCL3-F317L-A151R-P34-pp62病毒RCA检测，检测方法如下：
1、准备pAd5LCL3-F317L-A151R-P34-pp62病毒液，并测其病毒滴度，测定病毒颗粒浓度，病毒液加1％Universal nuclease(Universal nuclease 7.5～15units/mL病毒液)消化宿主细胞的DNA，37℃水浴40min。用300Kd的超滤离心管，经1000g，30min离心，1×PBS洗脱收集病毒颗粒，测A260，颗粒浓度＝A260*1.1*10^12VP/mL。
2、病毒感染，准备A549细胞的6孔板，每孔细胞为2.5×10 5/孔，弃培养基，PBS清洗一次，将腺病毒按照1×10 9vp/孔接种病毒，感染A549细胞，野生型腺病毒5型为对照，37℃，5％CO 2，1h后，弃去病毒液，补足5％完全培养基，37℃，5％CO 2培养48h。
3、免疫染色，弃细胞上清，PBS表面冲洗细胞，用冰甲醇固定，放置-20℃，20min，1×PBS清洗三遍，每遍5min，每孔加入2ml 1％BSA-PBS溶液，放置摇床，孵育1h。弃去上清，加入腺病毒5型荧光抗体(1:500稀释)，孵育1h，1×PBS清洗三遍，每遍5min。
用10倍荧光显微镜观察，使用公式计算RCA
RCA＝(average positive cell field)×(374 field/well)×(dilution factor))/Total VPs in 0.5ml viral sample
判断标准为RCA的水平小于1RCA/3×10 10vp。经过统计pAd5LCL3-F317L-A151R-P34-pp62的RCA的水平小于1RCA/3×10 10vp，说明本发明所制备的复制缺陷型pAd5LCL3-F317L-A151R-P34-pp62病毒在293TD37细胞中能够稳定包装，不会转化为野生型或转化为野生型的几率较低。
实施例48非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-F317L-A151R-P34-pp62蛋白表达检测
提前一天准备293TD37细胞，置于12孔细胞培养板中，使用非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-F317L-A151R-P34-pp62病毒感染293TD37细胞，48小时后细胞发生病变，收集全部的1ml细胞，使用PBS洗涤，制样，用于Western Blot检测；使用本公司制备的pp62鼠多抗血清检测目的蛋白，pp62鼠多抗血清是由昆虫SF9系统表达的pp62蛋白免疫小鼠获得。pp62蛋白的大小为60kda；
实验结果如图180所示，泳道4为293TD37细胞感染pAd5LCL3-F317L-A151R-P34-pp62的样品；可以清晰可见pp62蛋白有正常表达，由此可见pAd5LCL3-F317L-A151R-P34-pp62疫苗的蛋白能够在293细胞中正常表达。
实施例49非洲猪瘟多抗原重组腺病毒疫苗pAd5LCL3-F317L-A151R-P34-pp62实在小鼠模型上的 免疫学评价
49.1疫苗体液免疫反应检测
20只SPF级小鼠(6-8周龄)，随机分成4组，每组5只。根据表9所示分组情况对小鼠进行pAd5LCL3-F317L-A151R-P34-pp62免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表9：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000017
小鼠于免疫后14天采血，分离血清，使用间接ELISA法检测血清中针对非洲猪瘟目的蛋白pp62抗体滴度(pp62蛋白由本公司自行制备，昆虫细胞中表达)。检测结果如图55所示：小鼠肌肉注射pAd5LCL3-F317L-A151R-P34-pp62后，针对pp62蛋白，能够产生较高浓度的IgG抗体。高剂量组抗体滴度平均值达10 5以上，中剂量组滴度平均值也达70000，与对照组有显著差异。
49.2细胞免疫反应检测
10只SPF级小鼠(6-8周龄)，随机分成2组，每组5只。根据表10所示分组情况对小鼠进行pAd5LCL3-F317L-A151R-P34-pp62免疫。注射方式为：后大腿内侧肌肉注射；注射剂量：100ul。
表10：疫苗免疫检测小鼠分组情况
Figure PCTCN2021104793-appb-000018
免疫后14天处死小鼠，分离脾淋巴细胞，使用穿梭质粒pS5E1-P72-IRES-B602L和pS5E4-P30-2A-P54转染的PK15细胞刺激培养6小时，同时加入蛋白分泌阻断剂阻断细胞因子分泌。6小时后，阻断Fc受体，对死细胞和细胞表面分子标志物进行染色，细胞经固定和穿孔之后，对细胞内细胞因子进行染色。细胞表面标志物包括CD4、CD8，细胞内细胞因子包括IFNγ、IL2。使用流式细胞仪(CyExpert)分析CD4+T细胞和CD8+T细胞经目的蛋白刺激后表达IFNγ、IL2的水平。
pAd5LCL3-P72-B602L-P30-P54诱导的CD8+T细胞和CD4+T细胞免疫反应如图184、图185所示，代表性结果如图186至图187所示，其中图186为肌肉注射pAd5LCL3-P72-B602L-P30-P54后细胞免疫反应代表图，图187为空白对照免疫反应代表图。结果表示：小鼠免疫后14天，脾细胞经目的蛋白刺激后，CD8+T细胞，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。CD4+T细胞经过刺激后，其表达的IFNγ、TNFα和IL2水平均显著高于Ad5载体对照组(Control)(P<0.05)。
12.3小鼠模型免疫原性评价小结
pAd5LCL3-F317L-A151R-P34-pp62重组腺病毒具有良好的免疫原性，可诱导小鼠产生高水平的血清IgG抗体。其中高剂量的1*10^8FFU和中剂量1*10^7FFU的免疫方式诱导的滴度都很高。细胞免疫反应检测结果说明肌肉注射免疫1*10^7FFU的腺病毒载体疫苗疫苗，可诱导其免疫的小鼠产生特异 性细胞免疫反应。
虽然本发明披露如上，但本发明并非限定于此。任何本领域技术人员，在不脱离本发明的精神和范围内，均可作各种更动与修改，因此本发明的保护范围应当以权利要求所限定的范围为准。

Claims (23)

  1. 一种重组腺病毒载体pAd5LCL3，其特征在于，缺失E1、E3、E4和E2a基因，具有可分别同时表达一个或多个外源的抗原基因的E1区和E4区。
  2. 如权利要求1所述的重组腺病毒载体pAd5LCL3，其特征在于，E4基因缺失的ORF6/7位于E2a区，所述的ORF6/7具有如序列表中Seq ID NO.7所示的核苷酸序列。
  3. 如权利要求2所述的重组腺病毒载体pAd5LCL3，其特征在于，所述E1区预置了Swa Ⅰ酶切位点；所述E4区预置了I-sceI酶切位点。
  4. 如权利要求4所述的重组腺病毒载体pAd5LCL3，其特征在于，所述pAd5LCL3具有如序列表Seq ID NO.5所示的核苷酸序列。
  5. 如权利要求1-4任一项所述的重组腺病毒载体pAd5LCL3的构建方法，其特征在于，利用CRISPR/cas9敲除腺病毒环状载体质粒的E1、E3、E4和E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置。
  6. 如权利要求5所述的构建方法，其特征在于，包含以下步骤：
    1)利用CRISPR/cas9敲除腺病毒环状载体质粒的E1基因，引入Swa Ⅰ酶切位点，融合后的片段与载体无缝克隆，再利用CRISPR/cas9敲除E3基因，再使用无缝克隆方式连接，获得缺失E1和E3基因的腺病毒载体质粒pAd5。
    2)再利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5的E4基因，使用PCR扩增并引入I-sceI酶切位点，再使用无缝克隆方法，获得缺失E1、E3和E4基因的腺病毒载体质粒pAd5△E4。
    3)利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5△E4的E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置，再使用无缝克隆方法，获得缺失E1、E3、E4和E2a基因的腺病毒载体质粒pAd5LCL3。
  7. 一种重组腺病毒疫苗，其特征在于，在pAd5LCL3的E1区和E4区包含目的基因。
  8. 如权利要求7所述的疫苗，其特征在于，所述目的基因为病毒、细菌、肿瘤的基因或基因片段。
  9. 如权利要求8所述的疫苗，其特征在于，所述目的基因为非洲猪瘟病毒基因。
  10. 一种非洲猪瘟病毒疫苗，其特征在于，所述疫苗为通过构建一种非洲猪瘟病毒的四种抗原基因共表达的重组腺病毒载体，再经293TD37细胞包装而获得。
  11. 如权利要求10所述的疫苗，其特征在于，所述非洲猪瘟病毒的四种抗原基因共表达的重组腺病毒载体需通过由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的293TD37细胞进行重组腺病毒包装，293TD37细胞的细胞株保藏编号为：CCTCC NO:C201996，保藏于中国典型培养物保藏中心。
  12. 如权利要求11所述的疫苗，其特征在于，所述四种抗原基因分别为以下五组抗原基因中的任意一组：第一组：P72、B602L、P30和P54；第二组：C129Rubiqutin、MGF5L6L、CP312R和MGF110-4L；第三组：L8Lubiqutin、I215L、I73Rhbsag和E146L；第四组：EP402R、EP153R、I177L和K205Rubiqutin；第五组：F317L、A151R、P34和pp62。
  13. 如权利要求12所述的疫苗，其特征在于，所述第一组中，P72和B602L表达在E1区，P30和P54表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-P72-B602L-P30-P54；所述第二组中， CP129Rubiqutin是在CP129R上添加分子佐剂ubiqutin获得，CP129Rubiqutin和MGF5L6L表达在E1区，CP312R和MGF110-4L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；所述第三组中，L8Lubiqutin是在L8L上添加分子佐剂ubiqutin获得，I73Rhbsag是在173R上添加分子佐剂hbsag获得，L8Lubiqutin和I215L表达在E1区，I73Rhbsag和E146L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；所述第四组中，K205Rubiqutin是在K205R上添加分子佐剂ubiqutin获得，EP402R和EP153R表达在E1区，I177L和K205Rubiqutin表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；所述第五组中，F317L和A151R表达在E1区，P34和pp62表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-F317L-A151R-P34-pp62。
  14. 如权利要求13所述的疫苗，其特征在于，所述P72、B602L、P30、P54和pAd5LCL3-P72-B602L-P30-P54分别具有如序列表中Seq ID NO.1、Seq ID NO.2、Seq ID NO.3、Seq ID NO.4、Seq ID NO.6所示的核苷酸序列；所述CP129R、ubiqutin、MGF5L6L、CP312R、MGF110-4L、pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L分别具有如序列表中Seq ID NO.14、Seq ID NO.15、Seq ID NO.16、Seq ID NO.17、Seq ID NO.18、Seq ID NO.19所示的核苷酸序列；所述L8L、ubiqutin、I215L、I73R、hbsag、E146L、pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L分别具有如序列表中Seq ID NO.20、Seq ID NO.15、Seq ID NO.22、Seq ID NO.23、Seq ID NO.24、Seq ID NO.25、Seq ID NO.26所示的核苷酸序列；所述EP402R、EP153R、I177L、K205R、ubiqutin、pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin分别具有如序列表中Seq ID NO.27、Seq ID NO.28、Seq ID NO.29、Seq ID NO.30、Seq ID NO.31、Seq ID NO.32所示的核苷酸序列；所述F317L、A151R、P34、pp62、pAd5LCL3-F317L-A151R-P34-pp62分别具有如序列表中Seq ID NO.33、Seq ID NO.34、Seq ID NO.36、Seq ID NO.36、Seq ID NO.37所示的核苷酸序列。
  15. 一种如权利要求10～14任一项所述的一种非洲猪瘟病毒疫苗的构建方法，其特征在于，包含以下步骤：
    1)利用CRISPR/cas9敲除腺病毒环状载体质粒的E1基因，引入Swa Ⅰ酶切位点，融合后的片段与载体无缝克隆，再利用CRISPR/cas9敲除E3基因，再使用无缝克隆方式连接，获得缺失E1和E3基因的腺病毒载体质粒pAd5；
    2)再利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5的E4基因，使用PCR扩增并引入I-sceI酶切位点，再使用无缝克隆方法，获得缺失E1、E3和E4基因的腺病毒载体质粒pAd5△E4；
    3)利用CRISPR/cas9敲除腺病毒环状载体质粒pAd5△E4的E2a基因，并将E4区的ORF6/7表达框放在敲除了E2a区的序列位置，再使用无缝克隆方法，获得缺失E1、E3、E4和E2a基因的腺病毒载体质粒pAd5LCL3；
    4)构建腺病毒E1区域穿梭质粒，pS5E1通过DNA连接酶分别与第一组的P72、IRES、B602L、或第二组的CP129Rubiqutin、IRES、MGF5L6L、或第三组的L8Lubiqutin、IRES、I215L、或第四组的EP402R、 IRES、EP153R、或第五组的F317L、IRES、A151R基因片段连接，构建非洲猪瘟腺病毒5型载体E1区域穿梭质粒，分别为第一组：pS5E1-P72-IRES-B602L；第二组：pS5E1-CP129Rubiqutin-IRES-MGF5L6L；第三组pS5E1-L8Lubiqutin-IRES-I215L；第四组：pS5E1-EP402R-IRES-EP153R；第五组：pS5E1-F317L-IRES-A151R。
    5)构建腺病毒E4区域穿梭质粒，分别与第一组的P30、2A、P54；或第二组的CP312R、2A、MGF5L6L；或第三组的I73Rhbsag、2A、E146L；或第四组的I177L、2A、K205Rubiqutin；或第五组的P34、2A、pp62基因通过融合PCR技术得到基因片段，分别为P30-2A-P54、CP312R-2A-MGF5L6L、I73Rhbsag-2A-E146L、I177L-2A-K205Rubiqutin、P34-2A-pp62，通过对穿梭质粒pS5E4-EGFP酶切，敲除EGFP，并通过DNA连接酶与基因片段连接，构建非洲猪瘟腺病毒5型载体E4区域穿梭质粒，分别为第一组：pS5E4-P30-2A-P54；第二组：pS5E4-CP312R-2A-MGF5L6L；第三组：pS5E4-I73Rhbsag-2A-E146L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pS5E4-P34-2A-pp62。
    6)将分别将E1区域穿梭质粒pS5E1-P72-IRES-B602L、或pS5E1-CP129Rubiqutin-IRES-MGF5L6L、或pS5E1-L8Lubiqutin-IRES-I215L、或pS5E1-EP402R-IRES-EP153R、或pS5E1-F317L-IRES-A151R与腺病毒载体质粒pAd5LCL3同源重组，得到腺病毒载体质粒第一组：pAd5LCL3-P72-IRES-B602L；第二组pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L；第三组：pAd5LCL3-L8Lubiqutin-IRES-I215L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pAd5LCL3-F317L-IRES-A151R。
    7)分别将E4区域穿梭质粒第一组：pS5E4-P30-2A-P54；第二组：pS5E4-CP312R-2A-MGF5L6L；第三组：pS5E4-I73Rhbsag-2A-E146L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pS5E4-P34-2A-pp62与腺病毒载体质粒第一组：pAd5LCL3-P72-IRES-B602L；第二组pAd5LCL3-CP129Rubiqutin-IRES-MGF5L6L；第三组：pAd5LCL3-L8Lubiqutin-IRES-I215L；第四组：pS5E4-I177L-2A-K205Rubiqutin；第五组：pAd5LCL3-F317L-IRES-A151R同源重组，获得四种抗原基因共表达的重组腺病毒疫苗第一组：pAd5LCL3-P72-B602L-P30-P54；第二组：pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；第三组：pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；第四组：pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；第五组：pAd5LCL3-F317L-A151R-P34-pp62。
  16. 根据权利要求15所述的方法，其特征在于，步骤1)所述的腺病毒环状载体质粒来源于在A549细胞中扩增野生型人腺病毒5型病毒，收集并浓缩病毒液，采用HirtViral DNA Extract方法提取腺病毒5型基因组，使用cosmid方法将线性的腺病毒5型基因组构建成环状的腺病毒环状载体质粒。
  17. 根据权利要求15或16所述的方法，其特征在于，步骤3)所述的ORF6/7表达框基因具有如序列表中Seq ID NO.7所示的核苷酸序列；步骤4)所述的IRES具有如序列表中Seq ID NO.8所示的核苷酸序列；步骤5)所述的2A具有如序列表中Seq ID NO.9所示的核苷酸序列。
  18. 根据权利要求15～17任一项所述的方法，其特征在于，步骤4)所述的穿梭质粒pS5E1骨架采用puc origin、amp基本元素，Ad5左臂ITR部分序列，右臂PIX、PIVa2部分序列，以及CMV-MCS SV40 early polyA；步骤5)所述的E4区域穿梭质粒pS5E4-EGFP的骨架采用puc origin、amp基本元素，Ad5E4区域 左臂ITR序列，右臂部分fiber基因序列，以及EF1α-EGFP-HBV polyA基因；其中puc origin、amp基本元素具有如序列表可见中Seq ID NO.10所示的核苷酸序列，EF1α-EGFP-HBV polyA基因具有如序列表可见中Seq ID NO.11所示的核苷酸序列。
  19. 根据权利要求15～18任一项所述的方法，其特征在于，步骤6)所述E1区域穿梭质粒穿梭质粒与腺病毒载体质粒pAd5LCL3同源重组，是通过PacI和SwaI对穿梭质粒和腺病毒载体质粒pAd5LCL3进行酶切，酶切产物去磷酸化，OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段，转化产物涂布平板，挑取菌落，进行XhoI酶切验证。
  20. 根据权利要求15～19任一项所述的方法，其特征在于，步骤7)所述E4区域穿梭质粒与腺病毒载体质粒同源重组，是通过PacI和I-sceI对E4区域穿梭质粒和腺病毒载体质粒进行酶切，酶切产物去磷酸化，OMEGA Ultra-Sep Gel Extraction Kit进行胶回收载体和片段，转化产物涂布平板，挑取菌落，进行XhoI酶切验证。
  21. 一种重组腺病毒载体的包装方法，其特征在于，分别将所述的重组腺病毒疫苗第一组：pAd5LCL3-P72-B602L-P30-P54；第二组：pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；第三组：pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；第四组：pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；第五组：pAd5LCL3-F317L-A151R-P34-pp62；用PacI酶切，线性化后的质粒用于转染；转染由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的293TD37细胞，收集细胞悬液。
  22. 根据权利要求7所述的一种重组腺病毒载体的包装方法，其特征在于，由以下步骤制得：
    1)将分别将所述的重组腺病毒疫苗第一组：pAd5LCL3-P72-B602L-P30-P54；第二组：pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；第三组：pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；第四组：pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；第五组：pAd5LCL3-F317L-A151R-P34-pp62；用PacI酶切，线性化后的质粒用于转染；使用PEI转染试剂转染293TD37细胞；
    2)转染后的293TD37细胞于37℃，5％CO 2培养箱中培养72-96小时后收集细胞悬液，即为TP0代腺病毒；
    3)TP0代腺病毒感染293TD37细胞于37℃，5％CO 2培养箱中培养72小时，收集细胞悬液即TP1代腺病毒；
    4)重复3)，收集细胞悬液即TP2代腺病毒；
    5)持续接毒直至细胞发生病变。
  23. 293TD37细胞用于包装非洲猪瘟病毒四种抗原基因共表达的重组腺病毒载体的用途，其特征在于，所述四种抗原基因分别为第一组：P72、B602L、P30和P54；第二组：C129Rubiqutin、MGF5L6L、CP312R和MGF110-4L；第三组：L8Lubiqutin、I215L、I73Rhbsag和E146L；第四组：EP402R、EP153R、I177L和K205Rubiqutin；第五组：F317L、A151R、P34和pp62；其中第一组中，P72和B602L表达在E1区，P30和P54表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-P72-B602L-P30-P54；所述 第二组中，CP129Rubiqutin是在CP129R上添加分子佐剂ubiqutin获得，CP129Rubiqutin和MGF5L6L表达在E1区，CP312R和MGF110-4L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-CP129Rubiqutin-MGF5L6L-CP312R-MGF110-4L；所述第三组中，L8Lubiqutin是在L8L上添加分子佐剂ubiqutin获得，I73Rhbsag是在173R上添加分子佐剂hbsag获得，L8Lubiqutin和I215L表达在E1区，I73Rhbsag和E146L表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-L8Lubiqutin-I215L-I73Rhbsag-E146L；所述第四组中，K205Rubiqutin是在K205R上添加分子佐剂ubiqutin获得，EP402R和EP153R表达在E1区，I177L和K205Rubiqutin表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-EP402R-EP153R-I177L-K205Rubiqutin；所述第五组中，F317L和A151R表达在E1区，P34和pp62表达在E4区，构成四种抗原基因共表达的重组腺病毒载体pAd5LCL3-F317L-A151R-P34-pp62；
    其中，所述293TD37细胞是由pcDNA3.1+(hyg)-ORF6-IRES-DBP构建的，细胞株保藏编号为：CCTCC NO:C201996，保藏于中国典型培养物保藏中心。
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