WO2022068247A1 - 一种腺病毒四价疫苗 - Google Patents

一种腺病毒四价疫苗 Download PDF

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WO2022068247A1
WO2022068247A1 PCT/CN2021/097796 CN2021097796W WO2022068247A1 WO 2022068247 A1 WO2022068247 A1 WO 2022068247A1 CN 2021097796 W CN2021097796 W CN 2021097796W WO 2022068247 A1 WO2022068247 A1 WO 2022068247A1
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gene
plasmid
adenovirus type
replication
adenovirus
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陈凌
杨臣臣
冯立强
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广州恩宝生物医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the technical field of virus immunology, in particular to an adenovirus quadrivalent vaccine.
  • Adenovirus (Adenovirus, Ad) is a double-stranded DNA virus with a genome length of about 35-40 kb. It is known that human adenoviruses are divided into 7 subgroups (A ⁇ G), including more than 50 serotypes and more than 90 genotypes. After infecting patients, they mainly cause acute respiratory diseases (adenovirus B and C subgroups), conjunctivitis ( Adenovirus B and D subgroups) and gastroenteritis (adenovirus F subgroups 41 and 42, G subgroup 52). Respiratory tract infections caused by adenovirus are mostly caused by adenovirus types 3, 4 and 7.
  • Ad4 and Ad7 are mainly concentrated in the military, schools and other places where youth and adolescents gather, and even lead to the death of patients. However, there is no specific drug for the treatment of adenovirus infection, and only supportive treatment can be taken in clinical practice.
  • the vaccine is an oral live virus vaccine in the form of enteric-coated capsules prepared by the wild-type Ad4 and Ad7 on human embryonic kidney diploid fibroblasts, which are prepared by freezing and dehydration, mixing with cellulose lactose and the like.
  • the use of the vaccine effectively controlled the outbreak of adenovirus infection in the US military.
  • the Ad4 and Ad7 vaccines used by the U.S. military have great risks.
  • the vaccines are mainly low-dose wild-type adenoviruses with poor safety. There is a risk of contaminating the living environment after the residual live virus is excreted from the intestinal tract, which can easily cause viral infections. secondary pollution, so it cannot be widely used in the general population. Therefore, it is very necessary to develop a replication-defective adenovirus vaccine with high safety and protection against strong virus strains.
  • adenovirus vectors have been widely used in vaccine development, gene therapy and other fields. They are not only safe, but also have strong immune responses in vivo. Studies have shown that the E1 gene of adenovirus is an essential gene for its replication and proliferation, and the E3 gene plays a key role in resisting the host's immune system. After knocking out the E1 and E3 genes, adenovirus loses its ability to replicate in normal humans, and has an attenuated phenotype in this regard. At the same time, the main surface antigens of Ad3, Ad4, Ad7 and Ad55, Hexon, Fiber, etc., are not affected and will not affect the immunogenicity of the vaccine.
  • replication-deficient adenoviruses as vaccines can effectively increase the completeness and use of vaccines.
  • studies have found that many adenoviruses, especially non-subgroup C adenoviruses, have low yields in production cell lines after knocking out the E1 and E3 genes. The interaction can not effectively inhibit the nuclear export of host cell mRNA, and can not increase the expression of virus late protein.
  • These adenoviruses require the 293 cell line or other cell lines expressing the corresponding E1 gene for production. Therefore, replication-deficient Ad3, Ad4, and Ad7 that only knock out E1 and E3 genes are difficult to produce in vaccine-producing cell lines 293 or PerC6. Therefore, improving the production capacity of replication-deficient adenovirus in these cell lines is a technical bottleneck problem that needs to be solved.
  • the object of the present invention is to provide a preparation method and application of replication-deficient recombinant Ad3, Ad4, Ad7 and Ad55 that can be amplified on a large scale in vaccine production cell lines, and replication-deficient recombinant Ad3 in order to overcome the defects of the prior art , Ad4, Ad7 and Ad55 are mixed in a certain proportion to prepare a tetravalent vaccine.
  • the vaccine can effectively stimulate the body to produce specific humoral and cellular immune responses, and produce specific Ad3, Ad4, Ad7 and Ad55 neutralizing antibodies. Prevention of infection by Ad3, Ad4, Ad7 and Ad55 pathogens.
  • a composition comprising replication deficient human adenovirus type 3, adenovirus type 4, adenovirus type 7 and adenovirus type 55.
  • the E1 and E3 genes of the replication-deficient human adenovirus type 3, adenovirus type 4, adenovirus type 7 and adenovirus type 55 are deleted, and part of the coding frame of the E4 gene is replaced with the corresponding coding frame of the human adenovirus type 5 E4 gene .
  • the coding frame of the E4 gene includes Orf2, Orf3, Orf4 Orf6 coding frame.
  • the E1 gene region of at least one of the replication-defective human adenovirus type 3, adenovirus type 4, adenovirus type 7, and adenovirus type 55 integrates an exogenous gene expression cassette.
  • the replication-defective human adenovirus type 3, adenovirus type 4, adenovirus type 7 and adenovirus type 55 are mixed in a ratio of 1:1:1:1.
  • An adenovirus tetravalent vaccine formulation comprising the composition described above.
  • the formulation further comprises a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • the present invention replaces the partial coding frames of the E4 genes of Ad3, Ad4, Ad7 and Ad55 with the corresponding coding frames of the Ad5E4 gene, greatly improving the replication-deficient Ad3, Ad4, Ad7 and Safety of Ad55 and its ability to replicate in producer cell lines.
  • Ad3, Ad4, Ad7 and Ad55 tetravalent vaccine prepared by the present invention can effectively induce Ad3, Ad4, Ad7 and Ad55 specific humoral immunity and cellular immunity in experimental animals after primary immunization and booster immunization.
  • the quadrivalent vaccine of the present invention greatly improves the safety, protection range and range of use.
  • Figure 1 is a flow chart of the construction of pAd3 ⁇ E1 ⁇ E3 plasmid.
  • Figure 2 is a flow chart of plasmid construction of pAd3 ⁇ E1 ⁇ E3 (Orf2-6).
  • Figure 3 is a flow chart of the construction of pAd3 ⁇ E1 ⁇ E3(Orf2-6)-EGFP plasmid.
  • FIG. 4 is a diagram showing the results of restriction enzyme digestion identification of pAd3 ⁇ E1 ⁇ E3 plasmid, pAd3 ⁇ E1 ⁇ E3(Orf2-6) plasmid, and pAd3 ⁇ E1 ⁇ E3(Orf2-6)-EGFP.
  • Figure 5 is a graph showing the results of production and purification of a replication-defective Ad3 vector.
  • Figure 6 shows the results of plaque formation experiments of replication-deficient Ad3 vectors in HEK293 and A549 cells.
  • Figure 7 is a flow chart of pAd4 plasmid construction.
  • Figure 8 is a flow chart of the construction of the pAd4 ⁇ E3 plasmid.
  • Figure 9 is a flow chart of the construction of the pAd4 ⁇ E1 ⁇ E3 plasmid.
  • Figure 10 is a flow chart of the construction of the pAd4 ⁇ E1 ⁇ E3 (Orf2-6) plasmid.
  • Figure 11 is a flow chart of the construction of pAd4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP plasmid.
  • Fig. 12 is a graph showing the results of restriction enzyme digestion identification of pAd4 plasmid, pAd4 ⁇ E3 plasmid, pAd4 ⁇ E1 ⁇ E3 plasmid, pAd4 ⁇ E1 ⁇ E3(Orf2-6) plasmid, and pAd4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP.
  • Figure 13 shows the results of production and purification of replication-defective Ad4 vectors.
  • Figure 14 shows the results of plaque formation experiments of replication-deficient Ad4 vectors in HEK293 and A549 cells.
  • Figure 15 is a flow chart (A) of the construction of the pAd7 plasmid and a result of restriction enzyme digestion (B).
  • Fig. 16 is a flow chart (A) of the construction of pAd7 ⁇ E3 plasmid and a result of restriction enzyme digestion (B).
  • Fig. 17 is a flow chart (A) of the construction of pAd7 ⁇ E1 ⁇ E3 plasmid and a result of restriction enzyme digestion (B).
  • Fig. 18 is a flow chart (A) of the construction of plasmid pAd7 ⁇ E1 ⁇ E3 (Orf2-6) and a result of restriction enzyme digestion (B).
  • Fig. 19 is a flow chart (A) of the construction of pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP plasmid and a result of restriction enzyme digestion (B).
  • Figure 20 is a graph showing the results of production and purification of a replication-defective Ad7 vector.
  • Figure 21 shows the results of plaque formation experiments of replication-deficient Ad7 vectors in HEK293 and A549 cells.
  • Fig. 22 is a diagram showing the construction process of pAd55 ⁇ E1 ⁇ E3-Kana plasmid and the result of restriction enzyme digestion.
  • Fig. 23 is a diagram showing the construction process of pAd55 ⁇ E1 ⁇ E3 plasmid and the result of restriction enzyme digestion.
  • Fig. 24 is a diagram showing the construction process of the pAd55 ⁇ E1 ⁇ E3 (Orf2-6) plasmid and the results of restriction enzyme digestion.
  • Fig. 25 is a diagram showing the construction process of pAd55 ⁇ E1 ⁇ E3(Orf2-6)-EGFP plasmid and the result of restriction enzyme digestion.
  • Figure 26 is a graph showing the results of production and purification of a replication-defective Ad55 vector.
  • Figure 27 shows the results of plaque formation experiments with replication-deficient Ad55 vectors in HEK293 and A549 cells.
  • Figure 28 shows the results of the determination of Ad3, Ad4, Ad7 and Ad55 neutralizing antibody levels in cynomolgus monkey serum.
  • Figure 29 shows the results of determination of Ad2, Ad11 and Ad14 cross-neutralizing antibody levels in cynomolgus monkey serum.
  • Figure 30 shows the results of the rhesus monkey PMBC ELISPOT experiment.
  • human adenovirus type 3 human, human type 4 adenovirus, human type 7 adenovirus, human type 55 adenovirus refers to the type 3 adenovirus, type 4 adenovirus, type 7 adenovirus known to those of ordinary skill in the art Adenovirus and adenovirus type 55, the adenovirus genomes used in the examples are also derived from these known human adenoviruses.
  • the replication-defective human adenovirus type 3, human adenovirus type 4, human adenovirus type 7 and human adenovirus type 55 vectors of the present invention are not limited to the specific clinical isolates used in the examples.
  • PCR amplification was performed using pAd3 ⁇ E3 as a template to obtain recombinant arms L-delE1 and R-delE1.
  • L-delE1 F GATTATTGACTAGAGTATACAGTGCCACCTGACGTCTAAGAAA (SEQ ID NO. 1);
  • L-delE1 R GATATCGTTTAAACACTAGTCACACCTCATTTTTACGTCACCTTT (SEQ ID NO. 2).
  • PCR program 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delE1 F ACTAGTGTTTAAACGATATCAGCCGGTGTGCGTGGATGTG (SEQ ID NO. 3);
  • R-delE1 R CCCAGTAGAAGCGCCGGTGCGAGACCGATGGTCCAGGGC (SEQ ID NO. 4).
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 80 seconds; 25 cycles.
  • L-delE1 and L-delE1 were ligated into pVax vector digested by homologous recombinase (Exnase) to obtain p3SE1LR.
  • pVax-delE1(L+R) was linearized with BstZ17+SgrAI, it was co-transformed into BJ5183 competent cells (Stratagene) with pAd3 ⁇ E3 linearized by single enzyme digestion with PmeI; after screening for ampicillin resistance, the plasmid was manually extracted and further transformed into XL -Blue competent cells; the plasmid was manually extracted to obtain the pAd3 ⁇ E1 ⁇ E3 plasmid.
  • the technical process is shown in Figure 1, and the identification results of enzyme digestion are shown in Figure 4.
  • the original E1 region of the adenovirus genome in the obtained pAd3 ⁇ E1 ⁇ E3 plasmid was introduced into a PmeI restriction site to facilitate subsequent cloning.
  • PCR amplification was performed using the Ad3 genome as a template to obtain recombinant arms 3E4L and 3E4R.
  • 3E4RF GATTATTGACTAGAGTATACTGTCTAATGGTGGTGCGGCTGA (SEQ ID NO. 5);
  • 3E4R R CGCGTACAGACTAGAATTC AAGGAATTTCAATAAAAAATGTTGAACTTT (SEQ ID NO. 6).
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • 3E4RF GAATTCTAGTCTGTACGCGTCATATCATAGTAGCCTGTCGAACA (SEQ ID NO. 7);
  • 3E4R R CCCAGTAGAAGCGCCGGTG ATGGCTAATGAGGCTTTGTATGTGT (SEQ ID NO. 8).
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • the modified shuttle plasmid p3 ⁇ E4-(L+R) of Ad3E4 gene was obtained by ligating to pVax vector with homologous recombinase.
  • the Orf2-6 of Ad5 adenovirus E4 gene was obtained by PCR amplification with Ad5 genome as template.
  • Orf2-6 primer sequence :
  • Orf2-6 F TTTTATTGAAATTCCTT CTACATGGGGGTAGAGTCATAATC (SEQ ID NO. 9);
  • Orf2-6 R CAGGCTACTATGATATGA ATGCAGAAAACCCGCAGACATGTTT (SEQ ID NO. 10).
  • PCR program 95°C, 30 seconds; 65°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Homologous recombinase was used to ligate into the MluI linearized p3 ⁇ E4-(L+R) vector to obtain the modified shuttle plasmid p3SE4-Orf2-6 of the Ad3E4 gene.
  • p3SE4-Orf2-6 was linearized with BstZ17I+SgrAI double digestion, it was co-transformed into BJ5183 competent cells with pAd3 ⁇ E1 ⁇ E3 linearized by MluI digestion; after ampicillin resistance screening, the plasmid was manually extracted and further transformed into XL-Blue competent cells cells (Beijing Spechui Biotechnology Co., Ltd.); the plasmid was manually extracted to obtain the pAd3 ⁇ E1 ⁇ E3 (Orf2-6) plasmid, the technical process is shown in Figure 2, and the enzyme digestion identification results are shown in Figure 4.
  • the CMV-EGFP-BGH expression cassette was obtained by PCR with the following primers.
  • PCR program 95°C, 30 seconds; 66°C, 30 seconds; 72°C, 30s; 25 cycles.
  • the CMV-EGFP-BGH expression cassette double-digested by SpeI and EcoRV was connected with the p3SE1LR vector double-digested by SpeI+EcoRV using SoultionI to obtain the shuttle plasmid pGK31-EGFP carrying the recombination arm.
  • the pGK31-EGFP plasmid was cut with BstZ17I+SgrAI and recovered by ethanol precipitation; pAd3 ⁇ E1 ⁇ E3 (Orf2-6) was linearized with PmeI and then recovered by ethanol precipitation; co-transformed BJ5183 and homologous recombination to obtain pAd3 ⁇ E1 ⁇ E3 (Orf2-6) carrying the exogenous gene expression cassette -EGFP plasmid, the technical process is shown in Figure 3, and the identification results of enzyme digestion are shown in Figure 1-4.
  • pAd3 ⁇ E1 ⁇ E3(Orf2-6) and pAd3 ⁇ E1 ⁇ E3(Orf2-6)-EGFP were linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection.
  • 5% fetal bovine serum in DMEM medium incubate for 7-10 days, and observe cytopathic changes; after poisoning, collect cells and culture supernatant, freeze and thaw 3 times in a 37-degree water bath and liquid nitrogen, and centrifuge to remove cell debris.
  • the growth ability of replication-deficient Ad3 virus in helper cells HEK293 and non-helper cells A549 was identified by plaque formation assay according to routine experimental methods. After the 293 or A549 cells in the six-well plate were 90% full, they were infected with Ad3 ⁇ E1 ⁇ E3(Orf2-6)-EGFP, and the infection titer was 1 ⁇ 10 7 Vp/well. Four hours after infection, the medium was aspirated and plated on a 1% agarose gel (containing 1% agarose, 1% BSA, 1 x MEM medium). After being placed in a 37°C incubator for 9-12 days, the formation of virus clones was observed under a fluorescence microscope and photographed for recording. The results are shown in Figure 6.
  • Replication-deficient Ad3 ⁇ E1 ⁇ E3(Orf2-6)-EGFP can only form plaques in HEK293 cells, but not in A549 cells. This indicates that the replication-deficient Ad3 vector can efficiently proliferate in E1 gene-complemented HEK293 cells, but is not replication competent in non-helper cells such as A549 cells, with an attenuated phenotype. At the same time, the results also show that the replication-defective human adenovirus type 3 vector can carry the reporter gene into the target cells, so it can be used in the reporter tracking system.
  • Ad4 genome was used as a template for PCR amplification to obtain recombinant arms Ad4-L and Ad4-R.
  • Ad4-L primer sequence
  • Ad4-L Fw ATAGAATTCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 13);
  • Ad4-L RwR TTTACTAGTGTTTAAACGTAATCGAAAACCTCCACGTAATGG (SEQ ID NO. 14).
  • PCR program 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Ad4-R primer sequence
  • Ad4-R Fw ACTAGTAGCTGGATCCAAGCCTCGAGGCACTACAATG (SEQ ID NO. 15);
  • Ad4-R Rw CCTGCCGTTCGACGATGCGATCGCCATCATCAATAATATACCTTATAGATGG (SEQ ID NO. 16).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 80 seconds; 25 cycles.
  • Homologous recombinase was used to ligate into pSIMPLE 19 (EcoRV) vector (TaKaRa) to obtain the Ad4 genome circularization shuttle plasmid pT-Ad4(L+R).
  • L-delE3 F GACATTGATTATTGACTAGTTTCAACACCTGGACCACTGCC (SEQ ID NO. 17);
  • L-delE3 R ATTTAAATTGGAATTCAAGGTCAGAGACTGGTTGAAGGATG (SEQ ID NO. 18).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delE3 F GAATTCCAATTTAAATAGCAGTCTGGCGATACCAAGG (SEQ ID NO. 19);
  • R-delE3 R GTTTAAACGGGCCCTCTAGACATTCTTGGTGGTGACAGGGTC (SEQ ID NO. 20).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • the E3 gene knockout shuttle plasmid pVax-delE3(L+R) was obtained by ligating to pVax vector with homologous recombinase.
  • the recombinant arms L-delE1 and R-delE1 were obtained by PCR amplification using the Ad4 genome as a template.
  • L-delE1 (or L-delK) primer sequence:
  • L-delE1 F CCAGATATACGCGTGTATACCATCATCAATAATATACCTTATAGATGG (SEQ ID NO. 21);
  • L-delE1 R GATATCAAGTTAATTAAAATCGAAAACCTCCACGTAAAC (SEQ ID NO. 22).
  • PCR program 95°C, 30 seconds; 50°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delE1 (or R-delK) primer sequence:
  • R-delE1 F TTAATTAACTTGATATCGTGTGGATGTGACGGAGGAC (SEQ ID NO. 23);
  • R-delE1 R GCCCAGTAGAAGCGCCGGTGCGGGATTATTAGTGGAACTTGAG (SEQ ID NO. 24).
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Homologous recombinase was used to ligate to pVax vector (Invitrogen) to obtain the shuttle plasmid pVax-delE1 (L+R) knocking out the E1 gene.
  • pVax-delE1(L+R) was linearized by double digestion with BstZ17I+SgrAI, it was co-transformed into BJ5183 competent cells (Stratagene) with pAd4 ⁇ E3 linearized by digestion with PsiI; after screening for ampicillin resistance, the plasmid was manually extracted and further XL-Blue competent cells (Beijing Spechui Biotechnology Co., Ltd.) were transformed; plasmids were manually extracted to obtain pAd4 ⁇ E1 ⁇ E3 plasmids. The original E1 region of the adenovirus genome in the obtained pAd4 ⁇ E1 ⁇ E3 plasmid was introduced with a PacI restriction site to facilitate subsequent cloning.
  • PCR amplification was performed using the Ad4 genome as a template to obtain recombinant arms 4E4L and 4E4R.
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Homologous recombinase was used to ligate into pVax vector (Invitrogen) to obtain an engineered shuttle plasmid pGK143-(L+R) to the Ad4E4 gene.
  • the Orf2-6 of Ad5 adenovirus E4 gene was obtained by PCR amplification with Ad5 genome as template.
  • Orf2-6 primer sequence :
  • Orf2-6 F TCCTCGGTGGTTGGAATCACAGCTACATGGGGGTAGAGTCATAATCG (SEQ ID NO. 29);
  • Orf2-6 R CCAAAAACACTAACCATGCTGGAATGCAGAAAACCCGCAGACATGTTTGAG (SEQ ID NO. 30).
  • PCR program 95°C, 30 seconds; 65°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • Homologous recombinase was used to connect to the BamHI linearized pGK143-(L+R) vector to obtain the modified shuttle plasmid pGK143-Orf2-6 of the Ad4 E4 gene.
  • pGK143-Orf2-6 was linearized by double digestion with BstZ17I+SgrAI, it was co-transformed into BJ5183 competent cells (Stratagene) with the linearized pAd4 ⁇ E1 ⁇ E3 by SwaI; after screening for ampicillin resistance, the plasmid was manually extracted and further transformed into XL- Blue competent cells (Beijing Spechui Biotechnology Co., Ltd.); the plasmid was manually extracted to obtain the pAd4 ⁇ E1 ⁇ E3 (Orf2-6) plasmid.
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • PCR program 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute, 30 seconds; 25 cycles.
  • the homologous recombination arms 4SE1L and 4SE1R in the E1 region were connected to the pVax vector by homologous recombinase (Vazyme) ligation to obtain the shuttle plasmid pGK41-(L+R) carrying the recombination arms.
  • Vazyme homologous recombinase
  • the CMV-EGFP-BGH expression cassette was obtained by PCR with the following primers.
  • PCR program 95°C, 30 seconds; 66°C, 30 seconds; 72°C, 30s; 25 cycles.
  • Homologous recombinase (Vazyme) was used to connect the CMV-EGFP-BGH expression cassette to the pGK41-(L+R) vector to obtain the shuttle plasmid pGK41-EGFP carrying the recombination arm.
  • the pGK41-EGFP plasmid was cut with BstZ17I+SgrAI and recovered by ethanol precipitation; pAd4 ⁇ E1 ⁇ E3(Orf2-6) was linearized with PacI and then recovered by ethanol precipitation; co-transformed BJ5183, and homologously recombined to obtain pAd4 ⁇ E1 ⁇ E3(Orf2-6) carrying the exogenous gene expression cassette -EGFP plasmid, the technical process is shown in Figure 11. See Figure 12 for the identification results of double-enzyme digestion.
  • pAd4 ⁇ E1 ⁇ E3(Orf2-6) and pAd4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP were linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection.
  • 5% fetal bovine serum in DMEM medium incubate for 7-10 days, and observe cytopathic changes; after poisoning, collect cells and culture supernatant, freeze and thaw 3 times in a 37-degree water bath and liquid nitrogen, and centrifuge to remove cell debris.
  • the growth ability of replication-deficient Ad4 virus in helper cells HEK293 and non-helper cells A549 was identified by plaque formation assay according to routine experimental methods. After the 293 or A549 cells in the six-well plate were 90% full, they were infected with Ad4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP, and the infection titer was 1X107Vp/well. Four hours after infection, the medium was aspirated and plated on a 1% agarose gel (containing 1% agarose, 1% BSA, 1 x MEM medium). After being placed in a 37°C incubator for 9-12 days, the formation of virus clones was observed under a fluorescence microscope and photographed for recording. The results are shown in Figure 14.
  • the replication-deficient Ad4 ⁇ E1 ⁇ E3(Orf2-6)-EGFP can only form plaques in HEK293 cells, but not in A549 cells. This indicates that the replication-deficient Ad4 vector can efficiently proliferate in E1 gene-complemented HEK293 cells, but is not replication competent in non-helper cells such as A549 cells, with an attenuated phenotype. At the same time, the results also show that the replication-defective human adenovirus type 4 vector can carry the reporter gene into the target cells, so it can be used in the reporter tracking system.
  • the left arm (L-Ad7) and the right arm (R-Ad7) of the Ad7 genome were obtained by PCR.
  • L-Ad7-F ACTGCGATCGCCTCTCTATTTAATATACCTTATAGATGG (SEQ ID NO. 37);
  • L-Ad7-R ACATGGATCCTCACTGAAGATAATCTCCTGTGG (SEQ ID NO. 38).
  • PCR conditions 95°C, 3 min; 95°C, 30s; 56°C, 30s; 72°C, 40s; cycles 30; 72°C, 5min; stored at 12°C.
  • R-Ad7-F AGCTGGATCCGAACCACCAGTAATATCATCAAAG (SEQ ID NO. 39);
  • R-Ad7-R TGAGCGATCGCCTCTCTATATAATATACCTTATAGATGGAA (SEQ ID NO. 40).
  • PCR conditions 95°C, 3min; 95°C, 30s; 56°C, 30s; 72°C, 1min; cycles 30; 72°C, 5min; storage at 12°C.
  • the PCR product and T vector were ligated into three fragments using Exnase recombinase to obtain pT-Ad7(L+R).
  • pT-Ad7(L+R) was digested and linearized with BamHI, and then co-transformed with the genome of Ad7 into BJ5183 competent cells for recombination.
  • the ampicillin resistance plate was used for resistance screening.
  • the monoclonal obtained by screening was amplified and extracted.
  • the plasmid was transformed into XL-Blue chemically competent cells, and the plasmid was extracted to obtain pAd7. Different digestion methods were used for identification. Two AsisI digestion sites were introduced on both sides of the genome to facilitate subsequent linearization of the modified Ad7 genome. for virus rescue.
  • the specific construction process is shown in Figure 15.
  • E3 knockout shuttle plasmid pVax- ⁇ E3(L+R) Using the genome of Ad7 as a template, the left arm (L- ⁇ E3) and the right arm (R- ⁇ E3) of the E3 gene were obtained by PCR.
  • L- ⁇ E3-F CATACTAGTCTGGTCTACTTCAACCCCTTCTCCG (SEQ ID NO. 41);
  • L- ⁇ E3-R GCAGAATTCATTTAAATGGAGGAAGGGTCTGGGTCTTCTG (SEQ ID NO. 42).
  • PCR conditions 95°C, 3min; 95°C, 30s; 63°C, 30s; 72°C, 30s; cycles 30; 72°C, 5min;
  • R- ⁇ E3-F GCAGATATCATTTAAATAGACCCTATGCGGCCTAAGAGAC (SEQ ID NO. 43);
  • R- ⁇ E3-R ACATCTAGAGACAGTTGGCTCTGGTGGGGT (SEQ ID NO. 44).
  • PCR conditions 95°C, 3 min; 95°C, 30s; 61°C, 30s; 72°C, 40s; cycles 30; 72°C, 5min; stored at 12°C.
  • L- ⁇ E3 was digested with SpeI+EcoRI, it was ligated into pVax vector digested with the same enzyme to obtain pVax-L- ⁇ E3.
  • R- ⁇ E3 was digested with EcoRV+XbaI and ligated to the pVax-L- ⁇ E3 backbone with the same restriction enzyme to obtain pVax- ⁇ E3(L+R).
  • pVax- ⁇ E3(L+R) was linearized with SpeI+XbaI
  • pAd7 was linearized with EcoRI
  • the plasmid was extracted by hand, and then continued to transform XL-Blue Competent cells were extracted by hand and identified by restriction enzyme digestion.
  • a genomic plasmid pAd7 ⁇ E3 was obtained, which knocked out the E3 gene and introduced a unique single-enzyme cleavage site SwaI in the E3 region.
  • the insertion of SwaI restriction site facilitates linearization in the E3 gene region.
  • the schematic diagram of the construction of the shuttle plasmid and the pAd7 ⁇ E3 plasmid and the identification results of the digestion of the large plasmid are shown in Figure 16 .
  • E1 knockout shuttle plasmid pT-Ad7 ⁇ E1(L+R) Using the genome of Ad7 as a template, the left arm (L- ⁇ E1) and the right arm (R- ⁇ E1) of the E1 gene were obtained by PCR.
  • L- ⁇ E1-F ACTCACCGGCGGCGATCGCCTCTCTATTTAATATACCTTATAGATGG (SEQ ID NO. 45);
  • L- ⁇ E1-R ATCACAATTGAATTCGTTTAAACGTAATCGAAAACCTCCACGTAA (SEQ ID NO. 46).
  • PCR conditions 95°C, 3min; 95°C, 30s; 54°C, 30s; 72°C, 30s; cycles 30; 72°C, 5min; storage at 12°C.
  • R-SE1-F ATAGAATTC ACTAGTGAGGCCCGATCATTTGGTGCT (SEQ ID NO. 47);
  • R-SE1-R ACGTATAC CTATCATTATGGATGAGTGCATGG (SEQ ID NO. 48).
  • PCR conditions 95°C, 3min; 95°C, 30s; 61°C, 30s; 72°C, 1min 10s; cycles 30; 72°C, 5min;
  • the PCR product and T vector were ligated into three fragments using Exnase recombinase to obtain pT-Ad7(L+R).
  • pT-Ad7- ⁇ E1(L+R) was linearized with Bstz17I, and pAd7 was linearized with AatII.
  • BJ5183 competent cells were co-transformed, plated on ampicillin-resistant plates, and plasmids were extracted by hand, and then transformed into XL-Blue Competent cells were extracted by hand and identified by restriction enzyme digestion.
  • the genomic plasmid pAd7 ⁇ E1 ⁇ E3 which knocked out the E1 gene and introduced a single restriction site PmeI in the E1 region.
  • the insertion of PmeI restriction site facilitates linearization in the E1 gene region.
  • the schematic diagram of the construction of the shuttle plasmid and the pAd7 ⁇ E1 ⁇ E3 plasmid and the identification results of the digestion of the large plasmid are shown in Figure 17 .
  • L-SE4-F CGCGGATCTTCCAGAGATGTTTAAACAACCAGTTACTCCTAGAACAGTCAGC (SEQ ID NO. 49);
  • L-SE4-R ACGCGTATGGATTTAAAT CGATGCAGGCGAGAGTCTATTC (SEQ ID NO. 50).
  • R-SE4-F ATTTAAATCCATACGCGTGGAGTTCTTATTAAGTGCGGATGG(SEQ ID NO.51)
  • R-SE4-R GCCTGCCGTTCGACGATGTTTAAAC CAGCTGGCACGACAGGTTTC (SEQ ID NO. 52).
  • L-SE4 and R-SE4 fragments obtained by PCR were subjected to three-fragment ligation with the blunt-ended T vector to obtain p7SE4.
  • Ad5 Orf2-6-F TCACAGTCCAACTGCT CCTACATGGGGGTAGAGTCATAATCG (SEQ ID NO. 53);
  • Ad5 Orf2-6-R GCGCGGTAACCTATTG CATGCAGAAAACCCGCAGACATG (SEQ ID NO. 54).
  • the backbone sequence was obtained by PCR using p7SE4 as the template:
  • p7SE4-F CAATAGGTTACCGCGCTGCG (SEQ ID NO. 55);
  • P7SE4-R AGCAGTTGGACTGTGAAAGCGC (SEQ ID NO. 56).
  • the fragment obtained by above-mentioned PCR utilizes Exnase enzyme to carry out double fragment ligation to obtain p7SE4 (Orf2-6);
  • p7SE4 (Orf2-6) was digested and linearized by PmeI
  • pAd7 ⁇ E1 ⁇ E3 was digested and linearized by SwaI
  • the above two digested products were recovered by ethanol precipitation.
  • BJ5183 competent cells were co-transformed for recombination, and the ampicillin plate was used for resistance Screening, amplify the obtained monoclonal and then extract its plasmid to transform into XL-Blue competent cells, extract the plasmid to obtain pAd7 ⁇ E1 ⁇ E3 (Orf2-6), extract the plasmid for restriction digestion identification, the specific construction process and large plasmid of pAd7 ⁇ E1 ⁇ E3 (Orf2-6) See Figure 18 for the identification results.
  • the left arm SE1L and the right arm SE1R of the shuttle plasmid in the E1 gene region were obtained by PCR using the genome of Ad7 as a template.
  • SE1L-F CCAGATATACGCGTGTATACTTAATTAACGGCATCAGAGCAGATTGTACTG (SEQ ID NO. 57);
  • SE1L-R GTTTAAACAAGATTTAAATGTAATCGAAAACCTCCACGTAAACG (SEQ ID NO. 58).
  • SE1R-F ATTTAAATCTTGTTTAAACGAATTCACTAGTGAGGCCCGATC (SEQ ID NO. 59);
  • SE1R-R GCCCAGTAGAAGCGCCGGTGTTAATTAACAAGTAGCTTGTCCTCAGCCAGG (SEQ ID NO. 60).
  • the expression cassette of CMV-EGFP-BGH was obtained by PCR amplification using the pGA1-EGFP plasmid stored in the laboratory as a template.
  • CMV-EGFP-BGH-F ACTAGTGAATTCGTTTACTAGTTATTAATAGTAATCAATTACGGG (SEQ ID NO. 61);
  • pSE1LR was linearized with PmeI digestion, and then ligated with the CMV-EGFP-BGH expression cassette obtained by PCR amplification using Exnase enzyme to obtain pGK71-EGFP.
  • pGK71-EGFP was linearized with PacI
  • pAd7 ⁇ E1 ⁇ E3 (Orf2-6) was linearized with PmeI digestion
  • the above two digestion products were recovered by ethanol precipitation
  • BJ5183 competent cells were co-transformed for recombination
  • the ampicillin plate was used for resistance Screening, amplify the obtained monoclonal and then extract its plasmid to transform into XL-Blue competent cells, extract the plasmid to obtain pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP, extract the plasmid for identification by enzyme digestion, and construct pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP specifically See Figure 19 for the process and identification of the megaplasmid.
  • pAd7 ⁇ E1 ⁇ E3(Orf2-6) and pAd7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP were linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic liposome transfection.
  • the production and purification results of the replication-defective Ad7 vector are shown in FIG. 20 .
  • Plaque assays were used to determine the replication ability of the replication-deficient Ad7 vector in helper 293 and non-helper A549 cells. Inoculate 293 or A549 cells in a 6-well cell plate, and when the cell density is close to 100%, the harvested P1 generation Ad7 ⁇ E1 ⁇ E3(Orf2-6)-EGFP virus stock solution was serially diluted and infected with 293 or A549 cells, respectively. concentration in duplicate. After 2 hours of virus infection of cells, the medium was aspirated, and about 2 ml of agarose gel (containing 1 ml of 1.4% agarose, 1 ml of 1 ⁇ MEM medium, 200ul of BSA, 1 ⁇ penicillin antibiotics) was placed on each well.
  • agarose gel containing 1 ml of 1.4% agarose, 1 ml of 1 ⁇ MEM medium, 200ul of BSA, 1 ⁇ penicillin antibiotics
  • Ad14 ⁇ E1 ⁇ E3(Orf2-6)-EGFP deletes both E1 and E3 genes, and can only form viral plaques in helper cells 293, but cannot form viral plaques in normal human A549 cells.
  • the above results indicate that the replication-deficient Ad7 vector can replicate in helper cells 293, but cannot replicate in normal human cells such as A549 cells, with an attenuated phenotype.
  • replication-deficient Ad7 vectors can express the carried reporter gene in infected cells for use in biotracer systems.
  • L-delE1 F ATAGAATTCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 63);
  • L-delE1 R TTTACTAGTGTTTAAACGTAATCGAAAACCTCCACGTAATGG (SEQ ID NO. 64).
  • PCR program 95°C, 30 seconds; 62°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delE1 F ATTTCTAGAGTTTAAACGAGACCGGATCATTTGGTTATTG (SEQ ID NO. 65);
  • R-delE1 R: AAAGAATTCGGGAAATGCAAATCTGTGAGGG (SEQ ID NO. 66).
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 80 seconds; 25 cycles.
  • L-delE1 was digested with SpeI+EcoRI and then ligated to the same digested pVax vector to obtain pVax-L-delE1; R-delE1 was digested with EcoRI+XbaI and then ligated to the same digested pVax-L-delE1 to be knocked out
  • the shuttle plasmid pVax-delE1(L+R) of the E1 gene was digested with SpeI+EcoRI and then ligated to the same digested pVax vector to obtain pVax-L-delE1;
  • pVax-delE1(L+R) was linearized with EcoRI, it was co-transformed into BJ5183 competent cells with pAd55 ⁇ E3 which was linearized by single enzyme digestion with PacI; after ampicillin and kanamycin double resistance screening, the plasmid was manually extracted and further transformed XL-Blue competent cells; the plasmid was manually extracted to obtain the pAd55 ⁇ E1 ⁇ E3-Kana plasmid. Enzyme cleavage identification was performed with different enzyme cleavage combinations, as shown in Figure 22. The original E1 region of the adenovirus genome in the obtained pAd55 ⁇ E1 ⁇ E3-Kana plasmid was introduced with two PmeI restriction sites to facilitate subsequent cloning.
  • the recombinant arms L-delK and R-delK were obtained by PCR amplification using the Ad55 genome as a template.
  • L-delK F ATAACTAGTGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 67);
  • L-delK R TTTGAATTCGTTTAAACGTAATCGAAAACCTCCACGTAATGG (SEQ ID NO. 68).
  • PCR program 95°C, 30 seconds; 61°C, 30 seconds; 72°C, 20 seconds; 25 cycles.
  • R-delK F ATCGTTTAAACGAGACCGGATCATTTGGTTATTG (SEQ ID NO. 69);
  • R-delK R ATCTCTAGAGGGAAATGCAAATCTGTGAGGG (SEQ ID NO. 70).
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 80 seconds; 25 cycles.
  • L-delK was digested with SpeI+EcoRI and then ligated to the same digested pVax vector to obtain pVax-L-delK; R-delK was digested with EcoRI+XbaI and then ligated to the same digested pVax-L-delE3 to be knocked out Kana gene shuttle plasmid pVax-delK (L+R).
  • pVax-delK(L+R) was linearized with SpeI+XbaI
  • pAd55 ⁇ E1 ⁇ E3-Kana was linearized with PmeI, recovered by ethanol precipitation, and co-transformed into BJ5183 competent cells, spread on ampicillin-resistant plates, manually extracted plasmids, and continued to transform XL -Blue competent cells, plasmids were manually extracted and identified by restriction enzyme digestion.
  • a genomic plasmid pAd55 ⁇ E1 ⁇ E3 was obtained in which the Kana resistance gene was removed and a unique restriction site PmeI was introduced in the E1 region.
  • the schematic diagram of the construction of the shuttle plasmid and the pAd55 ⁇ E1 ⁇ E3 plasmid and the identification results of enzyme digestion are shown in FIG. 23 .
  • R-delE3 Mlu GATCACGCGTGGACTAAGAGACCTGCTACCCATG (SEQ ID NO. 71);
  • L-delK R TGTAGATCTGTTTAAACCTTTAGCCCCATTACGTCAGTTTAG (SEQ ID NO. 72).
  • PCR program 95°C, 30 seconds; 61°C, 30 seconds; 72°C, 4 minutes; 25 cycles.
  • Sap-p55E4 R TTACGCTCTTCCTAGCCGTGATCCAGACTCCGG (SEQ ID NO. 73);
  • Sap-p55E4orf2 F ATAGCTCTTCCCATTGTTAGTTTTGAATGAGTCTGCA (SEQ ID NO. 74);
  • PCR program 95°C, 30 seconds; 61.5°C, 30 seconds; 72°C, 4 minutes; 25 cycles.
  • Ad5 E4 Orf(2-6) with SapI site added at the end was obtained by PCR amplification with Ad5 genome as template.
  • Sap-5ORF2-6F AATAGCTCTTCCCTACATGGGGGTAGAGTCATAATCG (SEQ ID NO. 75)
  • Sap-5ORF2-6R ATATGCTCTTCCATGCAGAAAACCCGCAGACATG (SEQ ID NO. 76)
  • PCR program 95°C, 30 seconds; 61°C, 30 seconds; 72°C, 2 minutes; 25 cycles.
  • p55E4 (Orf2-6) was linearized with MluI+PmeI, and pAd55 ⁇ E1 ⁇ E3 was linearized with PsiI.
  • BJ5183 competent cells were co-transformed, and the recombinant plasmid pAd55 ⁇ E1 ⁇ E3 (Orf2-6) was obtained with the deletion of E1 and E3 genes and the modified E4 gene.
  • the specific construction process and identification results are shown in Figure 24.
  • SE1L F AATGGTACCGGGGTGGAGTGTTTTTGCAAG (SEQ ID NO. 77);
  • SE1LR ATCGTAATCGAAAACCTCCACGTAATGG (SEQ ID NO. 78).
  • PCR program 95°C, 30 seconds; 61°C, 30 seconds; 72°C, 30 seconds; 25 cycles.
  • SE1R F AACACTAGTGAGACCGGATCATTTGGTTATTG (SEQ ID NO. 79);
  • PCR program 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 1 minute, 30 seconds; 25 cycles.
  • pSE3LR was cut with KpnI+EcoRV, and SE1L was cut with the same enzyme, and pSE1L was obtained by ligation; pSE1L was cut with SpeI+MluI, and SE1R was cut with the same enzyme, and pSE1LR was obtained by ligation.
  • the CMV-EGFP-BGH expression cassette was obtained by PCR with the following primers.
  • PCR program 95°C, 30 seconds; 66°C, 30 seconds; 72°C, 1 minute, 45 seconds; 25 cycles.
  • pSE1LR was cut with SpeI+EcoRV
  • CMV-EGFP-BGH was cut with SpeI
  • ligated to obtain the target shuttle plasmid pGK551-EGFP.
  • the pGK551-EGFP plasmid was cut with BstZ17I+SgrAI and recovered by ethanol precipitation; pAd55 ⁇ E1 ⁇ E3 (Orf2-6) was linearized with PmeI and then recovered by ethanol precipitation; BJ5183 was co-transformed and homologous recombination was used to obtain pAd55 ⁇ E1 ⁇ E3 (Orf2-6) carrying the exogenous gene expression cassette. -EGFP plasmid.
  • the specific construction process and identification results are shown in Figure 25.
  • pAd55 ⁇ E1 ⁇ E3(Orf2-6) and pAd55 ⁇ E1 ⁇ E3(Orf2-6)-EGFP were linearized with AsiSI, recovered by ethanol precipitation, and transfected into 293 cells by cationic lipofection.
  • virus concentration OD260 ⁇ dilution factor ⁇ 36/genome length (Kb); the virus stock solution is frozen at -80°C.
  • the production and purification results of the replication-defective Ad55 vector are shown in FIG. 26 .
  • the growth ability of replication-deficient Ad55 vectors in helper cells 293 and non-helper cells A549 was identified by plaque formation assays according to conventional methods. After the 293 or A549 cells in the six-well plate were 90% full, they were infected with Ad55 ⁇ E1 ⁇ E3(Orf2-6)-EGFP, and the infection titer was 1 ⁇ 10 7 Vp/well. Four hours after infection, the medium was aspirated and plated on a 1% agarose gel (containing 1% agarose, 5% fetal bovine serum, 1 x MEM medium). After being placed in a 37°C incubator for 10-12 days, the formation of virus clones was observed under a fluorescence microscope and photographed. The results are shown in Figure 27.
  • the replication-deficient Ad55 ⁇ E1 ⁇ E3(Orf2-6)-EGFP can only form plaques in 293 cells, but not in A549 cells. This indicates that the replication-deficient Ad55 vector can efficiently proliferate in 293 cells, but is not replication-competent in normal human cells such as A549 cells, with an attenuated phenotype. At the same time, the results also show that the replication-deficient human adenovirus type 55 vector can carry the reporter gene into the target cells, so it can be used in the reporter tracking system.
  • the replication-deficient Ad3, Ad4, Ad7 and Ad55 vaccines purified by cesium chloride density gradient force centrifugation were diluted to 8 ⁇ 10 11 vp/ml respectively, and were individually divided into 500ul/tube and stored at -80°C.
  • Ad3, Ad4, Ad7 and Ad55 quadrivalent vaccines (Ad3: 2 ⁇ 10 10 vp/ml; Ad4: 2 ⁇ 10 10 vp/ml, Ad7: 2 ⁇ 10 10 vp/ml; Ad4: 2 ⁇ 10 10 vp/ml ): Transfer 250ul (1 tube) of each of the aliquoted Ad3, Ad4, Ad7 and Ad55 vaccines to a 15ml centrifuge tube, dilute 10-fold, mix well, and store at -80°C for later use.
  • a protocol for evaluating the immunogenicity of Ad3, Ad4, Ad7 and Ad55 tetravalent vaccines in rhesus monkeys was designed. As shown in Table 1, the immunogenicity of Ad3, Ad4, Ad7 and Ad55 tetravalent vaccines was evaluated according to the designed immunization protocol.

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Abstract

本发明公开了一种腺病毒四价疫苗，其包括复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒，所述复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒的E1、E3基因缺失，E4基因的部分编码框置换为人5型腺病毒E4基因的相应编码框。本发明的腺病毒四价疫苗能够有效刺激机体产生体液免疫应答和细胞免疫应答，产生高效价的特异性中和抗体，用于预防病原体的感染。

Description

一种腺病毒四价疫苗 技术领域
本发明属于病毒免疫学技术领域，具体涉及一种腺病毒四价疫苗。
背景技术
腺病毒(Adenovirus，Ad)是双链DNA病毒，其基因组长度约35-40kb。已知人腺病毒分为7个亚群(A～G)，包括50多个血清型及90多个基因型，感染患者后主要引起急性呼吸道疾病(腺病毒B和C亚群)、结膜炎(腺病毒B和D亚群)和胃肠炎(腺病毒F亚群41型和42型，G亚群52型)。腺病毒导致的呼吸道感染多由腺病毒3型、4型和7型引起，近年来腺病毒11型和14型导致的上呼吸道感染和肺炎逐渐增多，而且病毒常常发生变异，导致呼吸道疾病暴发流行。2006年保定市解放军252医院收治了一些呼吸道感染发热病人，确诊为腺病毒55型引起的呼吸道感染。腺病毒55型也是由腺病毒11型和14型基因重组而来的变异病毒。Ad4和Ad7主要集中在部队、学校等青年和青少年聚集的地方爆发，甚至导致病人的死亡。但是，尚无治疗腺病毒感染的特效药物，临床上只能采取支持性治疗。
目前，抗腺病毒感染的疫苗仅在美国军队获得使用。该疫苗为野生型Ad4、Ad7在人胚肾二倍体成纤维细胞上传代，经冷冻脱水、混和纤维素乳糖等制成的肠溶型胶囊形式的口服活病毒疫苗。该疫苗的使用有效控制了美军腺病毒感染疫情的爆发。但是，美军使用的Ad4和Ad7苗存在极大的风险，该疫苗主要是低剂量野生型腺病毒，安全性较差，存在残余活病毒从肠道排出后污染生活环境的风险，极易造成病毒的二次污染，因而不能广泛应用于普通人群。所以，研制安全性高并能预防强病毒株的复制缺陷型腺病毒疫苗是十分必要的。
复制缺陷型腺病毒载体已经被广泛的应用于疫苗研发、基因治疗等领域，其不仅安全性好，并且在生物体内又发生较强的免疫反应。已有研究显示，腺病毒E1基因是其复制增殖的必需基因，E3基因在抵抗宿主的免疫系统中起关键性作用。敲除E1、E3基因后，腺病毒在正常人体内丧失复制能力，在此方面具有减毒表型。同时，Ad3、Ad4、Ad7和Ad55主要的表面抗原Hexon、Fiber等则不受影响，不会影响疫苗的免疫原性。因此，采用复制缺陷的腺病毒作为疫苗可有效增加疫苗的全性和使用范围。但是，研究发现很多腺病毒，尤其是非C亚群腺病毒，敲除E1、E3基因后在生产细胞株中产量较低，其主要原因是Ad5 E1B 55K不能与B亚型腺病毒E4 Orf6蛋白产生相互作用，不能有效抑制宿主细胞mRNA的出核，不能提高病毒晚期蛋白的表达。这些腺病毒需要表达相应E1基因的293细胞株或其他细胞株才能生产。因此，仅敲除E1、E3基因的复制缺陷型Ad3、Ad4和Ad7在疫苗生产细胞株293或PerC6种难以生产。因此，提升复制缺陷型腺病毒在这些细胞株中的生产能力是当前需要解决的瓶颈技术问题。
发明内容
本发明的目的在于为了克服现有技术缺陷，提供一种可在疫苗生产细胞株中大规模扩增的复制缺陷型重组Ad3、Ad4、Ad7和Ad55的备方法和应用，及复制缺陷型重组Ad3、Ad4、Ad7和Ad55以一定的比例混合制备四价疫苗，该疫苗免疫机体可以有效刺激机体产生特异性体液免疫和细胞免疫反应，产生特异性Ad3、Ad4、Ad7和Ad55中和抗体，用于预防Ad3、Ad4、Ad7和Ad55的病原体的感染。
本发明所采取的技术方案是：
一种组合物，其包括复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒。
所述复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒的E1、E3基因缺失，E4基因的部分编码框置换为人5型腺病毒E4基因的相应编码框。
所述E4基因的编码框包括Orf2、Orf3、Orf 4 Orf6编码框。
作为优选地，所述复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒、55型腺病毒中至少有一种的E1基因区域整合外源基因表达框。
进一步优选地，所述复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒颗粒数按比1：1：1：1的比例混合。
以上所述的组合物在制备疫苗或药物中的应用。
一种腺病毒四价疫苗制剂，其包括以上所述的组合物。
作为优选地，所述制剂还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
本发明的有益效果是：
(1)本发明在敲除E1和E3基因的基础上，将Ad3、Ad4、Ad7和Ad55的E4基因部分编码框置换为Ad5E4基因的相应编码框，大大提升复制缺陷型Ad3、Ad4、Ad7和Ad55的安全性及其在生产细胞株中的复制能力。
(2)本发明制备得到的Ad3、Ad4、Ad7和Ad55四价疫苗经初免和加强免后，能够有效在实验动物体内诱导产生Ad3、Ad4、Ad7和Ad55特异性体液免疫和细胞免疫，产生特异性Ad3、Ad4、Ad7和Ad55中和抗体，用于预防Ad3、Ad4、Ad7和Ad55的病原体的感染。与市面上Ad4和Ad7二价活疫苗(美国)相比较，本发明的四价疫苗在保留了Ad3、Ad4、Ad7和Ad55的免疫原性的条件下大大提高了疫苗的安全性、保护范围和使用范围。
附图说明
图1为pAd3ΔE1ΔE3质粒构建流程图。
图2为pAd3ΔE1ΔE3(Orf2-6)质粒构建流程图。
图3为pAd3ΔE1ΔE3(Orf2-6)-EGFP质粒构建流程图。
图4为pAd3ΔE1ΔE3质粒、pAd3ΔE1ΔE3(Orf2-6)质粒、pAd3ΔE1ΔE3(Orf2-6)-EGFP的酶切鉴定结果图。
图5为复制缺陷型Ad3载体的生产及纯化结果图。
图6为复制缺陷型Ad3载体在HEK293及A549细胞中的噬斑形成实验结果。
图7为pAd4质粒构建流程图。
图8为pAd4ΔE3质粒的构建流程图。
图9为pAd4ΔE1ΔE3质粒的构建流程图。
图10为pAd4ΔE1ΔE3(Orf2-6)质粒的构建流程图。
图11为pAd4ΔE1ΔE3(Orf2-6)-EGFP质粒的构建流程图。
图12为pAd4质粒、pAd4ΔE3质粒、pAd4ΔE1ΔE3质粒、pAd4ΔE1ΔE3(Orf2-6)质粒、pAd4ΔE1ΔE3(Orf2-6)-EGFP的酶切鉴定结果图。
图13复制缺陷型Ad4载体的生产及纯化结果图。
图14为复制缺陷型Ad4载体在HEK293及A549细胞中的噬斑形成实验结果。
图15为pAd7质粒的构建流程图(A)和酶切鉴定结果图(B)。
图16为pAd7ΔE3质粒的构建流程图(A)和酶切鉴定结果图(B)。
图17为pAd7ΔE1ΔE3质粒的构建流程图(A)和酶切鉴定结果图(B)。
图18为pAd7ΔE1ΔE3(Orf2-6)质粒的构建流程图(A)和酶切鉴定结果图(B)。
图19为pAd7ΔE1ΔE3(Orf2-6)-EGFP质粒的构建流程图(A)和酶切鉴定结果图(B)。
图20为复制缺陷型Ad7载体的生产及纯化结果图。
图21为复制缺陷型Ad7载体在HEK293及A549细胞中的噬斑形成实验结果。
图22为pAd55ΔE1ΔE3-Kana质粒的构建流程及酶切鉴定结果图。
图23为pAd55ΔE1ΔE3质粒的构建流程及酶切鉴定结果图。
图24为pAd55ΔE1ΔE3(Orf2-6)质粒的构建流程及酶切鉴定结果图。
图25为pAd55ΔE1ΔE3(Orf2-6)-EGFP质粒的构建流程及酶切鉴定结果图。
图26为复制缺陷型Ad55载体的生产及纯化结果图。
图27为复制缺陷型Ad55载体在HEK293及A549细胞中的噬斑形成实验结果。
图28为猕猴血清中Ad3、Ad4、Ad7和Ad55中和抗体水平测定结果。
图29为猕猴血清中Ad2、Ad11和Ad14交叉中和抗体水平测定结果。
图30为猕猴PMBC ELISPOT实验结果。
具体实施方式
本发明术语“人3型腺病毒人、人4型腺病毒、人7型腺病毒、人55型腺病毒”指本领域普通技术人员已知的3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒，实施例中用到的腺病毒基因组也来源于这些已知的人腺病毒。本发明所述复制缺陷型人3型腺病毒载体、人4型腺病毒载体、人7型腺病毒载体和人55型腺病毒载体不局限于实施例所采用的特定临床分离株。
为了能够更清楚地理解本发明的技术内容，特举以下实施例结合附图详细说明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
实施例1 Ad3疫苗的制备
一、E1基因的敲除及pAd3ΔE1ΔE3质粒的构建
1.E1基因敲除穿梭质粒pVax-delE1(L+R)的构建
以pAd3ΔE3为模板进行PCR扩增，得到重组臂L-delE1及R-delE1。
L-delE1引物序列：
L-delE1 F：GATTATTGACTAGAGTATACAGTGCCACCTGACGTCTAAGAAA(SEQ ID NO.1)；
L-delE1 R：GATATCGTTTAAACACTAGTCACACCTCATTTTACGTCACCTTT(SEQ ID NO.2)。
PCR程序：95℃，30秒；62℃，30秒；72℃，20秒；25个循环。
R-delE1引物序列：
R-delE1 F，ACTAGTGTTTAAACGATATCAGCCGGTGTGCGTGGATGTG(SEQ ID NO.3)；
R-delE1 R，CCCAGTAGAAGCGCCGGTGCGAGACCGATGGTCCAGGGC(SEQ ID NO.4)。
PCR程序：95℃，30秒；60℃，30秒；72℃，80秒；25个循环。
L-delE1和L-delE1以同源重组酶(Exnase)接至同样酶切的pVax载体得到p3SE1LR。
2.pAd3ΔE1ΔE3质粒的构建
pVax-delE1(L+R)以BstZ17+SgrAI线性化后，与PmeI单酶切线性化的pAd3ΔE3共转化BJ5183感受态细胞(Stratagene)；经氨苄青霉素抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞；手工提取质粒，得到pAd3ΔE1ΔE3质粒。技术流程如图1所示，酶切鉴定结果如图4所示。所得到的pAd3ΔE1ΔE3质粒中的腺病毒基因组原E1区引入PmeI酶切位点，以方便后续克隆。
二、Ad3 E4基因的改造及pAd3ΔE1ΔE3(Orf2-6)质粒的构建
1.Ad3 E4基因的改造穿梭质粒p3△E4-(L+R)的构建
以Ad3基因组为模板进行PCR扩增，得到重组臂3E4L及3E4R。
3E4L引物序列：
3E4R F：GATTATTGACTAGAGTATACTGTCTAATGGTGGTGCGGCTGA(SEQ ID NO.5)；
3E4R R：CGCGTACAGACTAGAATTC AAGGAATTTCAATAAAAAATGTTGAACTTT(SEQ ID NO.6)。
PCR程序：95℃，30秒；60℃，30秒；72℃，20秒；25个循环。
3E4R引物序列：
3E4R F：GAATTCTAGTCTGTACGCG TCATATCATAGTAGCCTGTCGAACA(SEQ ID NO.7)；
3E4R R：CCCAGTAGAAGCGCCGGTG ATGGCTAATGAGGCTTTGTATGTGT(SEQ ID NO.8)。
PCR程序：95℃，30秒；60℃，30秒；72℃，20秒；25个循环。
采用同源重组酶连接至pVax载体得到Ad3E4基因的改造的穿梭质粒p3△E4-(L+R)。
2.Ad3 E4基因的改造穿梭质粒p3SE4-Orf2-6的构建
以Ad5基因组为模板进行PCR扩增得到Ad5腺病毒E4基因的Orf2-6。
Orf2-6引物序列：
Orf2-6 F：TTTTATTGAAATTCCTT CTACATGGGGGTAGAGTCATAATC(SEQ ID NO.9)；
Orf2-6 R：CAGGCTACTATGATATGA ATGCAGAAACCCGCAGACATGTTT(SEQ ID NO.10)。
PCR程序：95℃，30秒；65℃，30秒；72℃，20秒；25个循环。
采用同源重组酶连接至MluI线性化的p3△E4-(L+R)载体，得到Ad3E4基因的改造的穿梭质粒p3SE4-Orf2-6。
3.pAd3ΔE1ΔE3(Orf2-6)质粒的构建
p3SE4-Orf2-6以BstZ17I+SgrAI双酶切线性化后，与MluI酶切线性化的pAd3ΔE1ΔE3共转化BJ5183感受态细胞；经氨苄青霉素抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞(北京斯百汇生物科技有限公司)；手工提取质粒，得到pAd3ΔE1ΔE3(Orf2-6)质粒，技术流程如图2所示，酶切鉴定结果如图4所示。
三、携带外源基因的穿梭质粒及pAd3ΔE1ΔE3(Orf2-6)-EGFP质粒的构建
1.构建携带外源基因表达框的穿梭质粒pGK31-EGFP
以pGK143-EGFP为模板，以下列引物PCR得到CMV-EGFP-BGH表达框。
引物序列：
CMV，ACGCGTTGACATTGATTATTGACTA(SEQ ID NO.11)；
BGH，CCTGCTATTGTCTTCCCAATCCT(SEQ ID NO.12)。
PCR程序：95℃，30秒；66℃，30秒；72℃，30s；25个循环。
采用SoultionI将SpeI和EcoRV双酶切的CMV-EGFP-BGH表达框与SpeI+EcoRV双酶切的p3SE1LR载体进行连接，得到携带重组臂的穿梭质粒pGK31-EGFP。
2.构建基因组质粒pAd3ΔE1ΔE3(Orf2-6)-EGFP
pGK31-EGFP质粒以BstZ17I+SgrAI切，乙醇沉淀回收；pAd3ΔE1ΔE3(Orf2-6)以PmeI线性化后乙醇沉淀回收；共转化BJ5183，同源重组得到携带外源基因表达框的pAd3ΔE1ΔE3(Orf2-6)-EGFP质粒，技术流程如图3所示，酶切鉴定结果如图1-4所示。
四、复制缺陷型Ad3载体的拯救与生产
按照常规方法，pAd3ΔE1ΔE3(Orf2-6)和pAd3ΔE1ΔE3(Orf2-6)-EGFP以AsiSI线性化，乙醇沉淀回收，阳离子脂质体转染法转染293细胞，转染后8小时，加入2毫升含5％胎牛血清的DMEM培养基，孵育7-10天，观察细胞病变；出毒后，收集细胞及培养上清，在37度水浴及液氮中反复冻融3次并离心去除细胞碎片，上清感染10厘米皿；2-3天后，收集细胞及培养上清，反复冻融3次并离心去除细胞碎片，上清感染3-5个15厘米皿；2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；上清感染30个15厘米皿2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；上清加至氯化铯密度梯度离心管；4℃，40000转，离心4小时；吸出病毒条带，脱盐，分装；以OD260吸光度测定病毒粒子滴度，计算公式为：病毒浓度＝OD260×稀释倍数×36/基因组长度(Kb)；病毒储存液于-80℃冻存。复制缺陷型Ad3载体的生产及纯化结果如图5所示。
五、复制缺陷型Ad3病毒在A549及293细胞中的复制能力测定
按照常规实验方法，以噬斑形成实验鉴定复制缺陷型Ad3病毒在辅助细胞HEK293和非辅助细胞A549中的生长能力。六孔板中293或A549细胞长至九成满后，以Ad3ΔE1ΔE3(Orf2-6)-EGFP进行感染，感染滴度为1X10 7Vp/孔。感染后4小时，吸走培养基，并铺上1％的琼脂糖凝胶(含1％琼脂糖，1％BSA，1×MEM培养基)。37℃培养箱内放置9-12天后，在荧光显微镜下观察病毒克隆的形成,并拍照记录。结果如图6所示。复制缺陷型Ad3ΔE1ΔE3(Orf2-6)-EGFP仅能在HEK293细胞中形成噬斑，在A549细胞中则不能形成噬斑。这表明，复制缺陷型Ad3载体可在E1基因互补的HEK293细胞中有效增殖，但在非辅助细胞如A549细胞中不具备复制能力，具有减毒表型。同时，该结果也显示复制缺陷型人3型腺病毒载体可携带报告基因进入靶细胞，因而可应用于报告示踪系统中。
实施例2 Ad4疫苗的制备
一、Ad4基因组环化穿梭载体的构建
1.Ad4基因组环化穿梭载体的构建
以Ad4基因组为模板进行PCR扩增，得到重组臂Ad4-L及Ad4-R。
Ad4-L引物序列：
Ad4-L Fw，ATAGAATTCGGGGTGGAGTGTTTTTGCAAG(SEQ ID NO.13)；
Ad4-L RwR，TTTACTAGTGTTTAAACGTAATCGAAACCTCCACGTAATGG(SEQ ID NO.14)。
PCR程序：95℃，30秒；62℃，30秒；72℃，20秒；25个循环。
Ad4-R引物序列：
Ad4-R Fw，ACTAGTAGCTGGATCCAAGCCTCGAGGCACTACAATG(SEQ ID NO.15)；
Ad4-R Rw，CCTGCCGTTCGACGATGCGATCGCCATCATCAATAATATACCTTATAGATGG(SEQ ID NO.16)。
PCR程序：95℃，30秒；55℃，30秒；72℃，80秒；25个循环。
采用同源重组酶连接至pSIMPLE 19(EcoRV)载体(TaKaRa)得到Ad4基因组环化穿梭质粒pT-Ad4(L+R)。
2.pAd4质粒的构建
pT-Ad4(L+R)以SpeI、BamHI线性化后，与Ad4基因组共转化BJ5183感受态细胞；经氨苄青霉素抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞(北京斯百汇生物科技有限公司)；手工提取质粒，得到pAd4，技术流程如附图7所示，酶切图如图12所示。
二、E3基因的敲除及pAd55ΔE3质粒的构建
1.E3基因敲除穿梭质粒pVax-delE3(L+R)的构建
以Ad4基因组为模板进行PCR扩增，得到重组臂L-delE3及R-delE3。
L-delE3(或称为delE3-4L)引物序列：
L-delE3 F，GACATTGATTATTGACTAGTTTCAACACCTGGACCACTGCC(SEQ ID NO.17)；
L-delE3 R，ATTTAAATTGGAATTCAAGGTCAGAGACTGGTTGAAGGATG(SEQ ID NO.18)。
PCR程序：95℃，30秒；55℃，30秒；72℃，20秒；25个循环。
R-delE3(delE3-4R)引物序列：
R-delE3 F，GAATTCCAATTTAAATAGCAGTCTGGCGATACCAAGG(SEQ ID NO.19)；
R-delE3 R，GTTTAAACGGGCCCTCTAGACATTCTTGGTGGTGACAGGGTC(SEQ ID NO.20)。
PCR程序：95℃，30秒；55℃，30秒；72℃，20秒；25个循环。
采用同源重组酶连接至pVax载体得到到敲除E3基因的穿梭质粒pVax-delE3(L+R)。
2.pAd4ΔE3质粒的构建
pVax-delE3(L+R)以SpeI和XbaI双酶切线性化后，与EcoRI部分酶切线性化的pAd4共转化BJ5183感受态细胞(Stratagene)；经氨苄青霉素抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞(北京斯百汇生物科技有限公司)；手工提取质粒，得到pAd4ΔE3质粒，技术流程如附图8所示，酶切图如图2-6所示。所得到的pAd4ΔE3质粒中的腺病毒基因组原E3区引入1个SwaI酶切位点，以方便后续克隆。
三、E1基因的敲除及pAd4ΔE1ΔE3质粒的构建
1.E1基因敲除穿梭质粒pVax-delE3(L+R)的构建
以Ad4基因组为模板进行PCR扩增，得到重组臂L-delE1及R-delE1。
L-delE1(或称为L-delK)引物序列：
L-delE1 F，CCAGATATACGCGTGTATACCATCATCAATAATATACCTTATAGATGG(SEQ ID NO.21)；
L-delE1 R，GATATCAAGTTAATTAAAATCGAAACCTCCACGTAAAC(SEQ ID NO.22)。
PCR程序：95℃，30秒；50℃，30秒；72℃，20秒；25个循环。
R-delE1(或称为R-delK)引物序列：
R-delE1 F，TTAATTAACTTGATATCGTGTGGATGTGACGGAGGAC(SEQ ID NO.23)；
R-delE1 R，GCCCAGTAGAAGCGCCGGTGCGGGATTATTAGTGGAACTTGAG(SEQ ID NO.24)。
PCR程序：95℃，30秒；55℃，30秒；72℃，20秒；25个循环。
采用同源重组酶连接至pVax载体(Invitrogen)得到敲除E1基因的穿梭质粒pVax-delE1(L+R)。
2.pAd4ΔE1ΔE3质粒的构建
pVax-delE1(L+R)以BstZ17I+SgrAI双酶切线性化后，与PsiI酶切线性化的pAd4ΔE3共转化BJ5183感受态细胞(Stratagene)；经氨苄青霉素抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞(北京斯百汇生物科技有限公司)；手工提取质粒，得到pAd4ΔE1ΔE3质粒，技术流程如附图9所示，酶切图如图12所示。所得到的pAd4ΔE1ΔE3质粒中的腺病毒基因组原E1区引入1个PacI酶切位点，以方便后续克隆。
四、Ad4 E4基因的改造及pAd4ΔE1ΔE3(Orf2-6)质粒的构建
1.Ad4 E4基因的改造穿梭质粒pGK143-(L+R)的构建
以Ad4基因组为模板进行PCR扩增，得到重组臂4E4L及4E4R。
4E4L引物序列：
4E4R F，CATTGATTATTGACTAGAGTATACCATGCTGGCGCGGCTGACCTAGCT(SEQ ID NO.25)；
4E4R R，CGGATCCGCTGTGATTCCAACCACCGAGGACAGCCCTC(SEQ ID NO.26)。
PCR程序：95℃，30秒；60℃，30秒；72℃，20秒；25个循环。
4E4R引物序列：
4E4R F，CGGATCCGTCCAGCATGGTTAGTGTTTTTGGTGATCTGTAGAAC(SEQ ID NO.27)；
4E4R R，TAGAAGCGCCGGTGGGTAAGCTATGGACGCTGAG(SEQ ID NO.28)。
PCR程序：95℃，30秒；60℃，30秒；72℃，20秒；25个循环。
采用同源重组酶连接至pVax载体(Invitrogen)得到到Ad4E4基因的改造的穿梭质粒pGK143-(L+R)。
2.Ad4 E4基因的改造穿梭质粒pGK143-Orf2-6的构建
以Ad5基因组为模板进行PCR扩增得到Ad5腺病毒E4基因的Orf2-6。
Orf2-6引物序列：
Orf2-6 F，TCCTCGGTGGTTGGAATCACAGCTACATGGGGGTAGAGTCATAATCG(SEQ ID NO.29)；
Orf2-6 R，CCAAAAACACTAACCATGCTGGAATGCAGAAACCCGCAGACATGTTTGAG(SEQ ID NO.30)。
PCR程序：95℃，30秒；65℃，30秒；72℃，20秒；25个循环。
采用同源重组酶连接至BamHI线性化的pGK143-(L+R)载体，得到Ad4 E4基因的改造的穿梭质粒pGK143-Orf2-6。
3.pAd4ΔE1ΔE3(Orf2-6)质粒的构建
pGK143-Orf2-6以BstZ17I+SgrAI双酶切线性化后，与SwaI酶切线性化的pAd4ΔE1ΔE3共转化BJ5183感受态细胞(Stratagene)；经氨苄青霉素抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞(北京斯百汇生物科技有限公司)；手工提取质粒，得到pAd4ΔE1ΔE3(Orf2-6)质粒，技术流程如图10所示，酶切图如图12所示。
五、携带外源基因的穿梭质粒及pAd4ΔE1ΔE3(Orf2-6)-EGFP质粒的构建
1.构建携带外源基因表达框的穿梭质粒pGK3-EGFP
1. 1以Ad4基因组为模板PCR扩增得到E1区同源重组臂4SE1L及4SE1R：
SE1L引物序列：
4SE1L Fw，CCAGATATACGCGTGTATACCATCATCAATAATATACCTTATAGATGG(SEQ ID NO.31)；
4SE1R Rw，GATATCAAGTTAATTAAAATCGAAACCTCCACGTAAAC(SEQ ID NO.32)。
PCR程序：95℃，30秒；55℃，30秒；72℃，20秒；25个循环。
4SE1R引物序列：
4SE1R Fw，TTAATTAACTTGATATCGTGTGGATGTGACGGAGGAC(SEQ ID NO.33)；
4SE1R Rw，GCCCAGTAGAAGCGCCGGTGCGGGATTATTAGTGGAACTTGAG(SEQ ID NO.34)。
PCR程序：95℃，30秒；55℃，30秒；72℃，1分钟30秒；25个循环。
1.2构建携带重组臂的穿梭质粒pGK41-(L+R)。
采用同源重组酶(Vazyme)连接将E1区同源重组臂4SE1L和4SE1R连接至pVax载体，得到携带重组臂的穿梭质粒pGK41-(L+R)。
1.3构建携带外源基因表达框的穿梭质粒pGK41-EGFP。
以pGA1-EGFP为模板，以下列引物PCR得到CMV-EGFP-BGH表达框。
引物序列：
CMV，GTCACATCCACACGATACTAGTTATTAATAGTAATCAATTACGGG(SEQ ID NO.35)；
BGH，TTTTAATTAACTTGATCCTGCTATTGTCTTCCCAATC(SEQ ID NO.36)。
PCR程序：95℃，30秒；66℃，30秒；72℃，30s；25个循环。
采用同源重组酶(Vazyme)将CMV-EGFP-BGH表达框连接至pGK41-(L+R)载体，得到携带重组臂的穿梭质粒pGK41-EGFP。
2.构建基因组质粒pAd4ΔE1ΔE3(Orf2-6)-EGFP
pGK41-EGFP质粒以BstZ17I+SgrAI切，乙醇沉淀回收；pAd4ΔE1ΔE3(Orf2-6)以PacI线性化后乙醇沉淀回收；共转化BJ5183，同源重组得到携带外源基因表达框的pAd4ΔE1ΔE3(Orf2-6)-EGFP质粒，技术流程如图11所示。双酶切鉴定结果参见图12。
六、复制缺陷型Ad4载体的拯救与生产
按照常规方法，pAd4ΔE1ΔE3(Orf2-6)和pAd4ΔE1ΔE3(Orf2-6)-EGFP以AsiSI线性化，乙醇沉淀回收，阳离子脂质体转染法转染293细胞，转染后8小时，加入2毫升含5％胎牛血清的DMEM培养基，孵育7-10天，观察细胞病变；出毒后，收集细胞及培养上清，在37度水浴及液氮中反复冻融3次并离心去除细胞碎片，上清感染10厘米皿；2-3天后，收集细胞及培养上清，反复冻融3次并离心去除细胞碎片，上清感染3-5个15厘米皿；2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；上清感染30个15厘米皿2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片；上清加至氯化铯密度梯度离心管；4℃，40000转，离心4小时；吸出病毒条带，脱盐，分装；以OD260吸光度测定病毒粒子滴度，计算公式为：病毒浓度＝OD260×稀释倍数×36/基因组长度(Kb)；病毒储存液于-80℃冻存。复制缺陷型Ad4载体的生产及纯化结果如附图13所示。
七、复制缺陷型Ad4病毒在A549及293细胞中的复制能力测定
按照常规实验方法，以噬斑形成实验鉴定复制缺陷型Ad4病毒在辅助细胞HEK293和非辅助细胞A549中的生长能力。六孔板中293或A549细胞长至九成满后，以Ad4ΔE1ΔE3(Orf2-6)-EGFP进行感染，感染滴度为1X107Vp/孔。感染后4小时，吸走培养基，并铺上1％的琼脂糖凝胶(含1％琼脂糖，1％BSA，1×MEM培养基)。37℃培养箱内放置9-12天后，在荧光显微镜下观察病毒克隆的形成,并拍照记录。结果如图14所示。复制缺陷型Ad4ΔE1ΔE3(Orf2-6)-EGFP仅能在HEK293细胞中形成噬斑，在A549细胞中则不能形成噬斑。这表明，复制缺陷型Ad4载体可在E1基因互补的HEK293细胞中有效增殖，但在非辅助细胞如A549细胞中不具备复制能力，具有减毒表型。同时，该结果也显示复制缺陷型人4型腺病毒载体可携带报告基因进入靶细胞，因而可应用于报告示踪系统中。
实施例3复制缺陷型Ad7疫苗的制备
一、Ad7基因组的环化
1.构建环化Ad7基因组的穿梭质粒pT-Ad7(L+R)
以Ad7的基因组为模板，PCR获得Ad7基因组的左臂(L-Ad7)和右臂(R-Ad7)。
L-Ad7引物：
L-Ad7-F：ACTGCGATCGCCTCTCTATTTAATATACCTTATAGATGG(SEQ ID NO.37)；
L-Ad7-R：ACATGGATCCTCACTGAAGATAATCTCCTGTGG(SEQ ID NO.38)。
PCR条件：95℃，3min；95℃，30s；56℃30s；72℃，40s；cycles 30；72℃，5min；12℃保存。
R-Ad7引物：
R-Ad7-F：AGCTGGATCCGAACCACCAGTAATATCATCAAAG(SEQ ID NO.39)；
R-Ad7-R：TGAGCGATCGCCTCTCTATATAATATACCTTATAGATGGAA(SEQ ID NO.40)。
PCR条件：95℃，3min；95℃，30s；56℃，30s；72℃，1min；cycles 30；72℃，5min；12℃保存。
PCR产物和T载体使用Exnase重组酶进行三片段连接得到pT-Ad7(L+R)。
2.构建pAd7
pT-Ad7(L+R)使用BamHI进行酶切线性化，然后和Ad7的基因组共转化BJ5183感受态细胞进行重组，氨苄抗性平板进行抗性筛选，将筛选得到的单克隆扩增后提取其质粒转化XL-Blue化学感受态细胞，提取质粒得到pAd7，使用不同的酶切方式进行鉴定，pAd7在基因组的两侧引入了两个AsisI酶切位点，方便后续对改造后的Ad7基因组进行线性化进行病毒拯救。具体构建过程如图15所示。
二、E3基因的敲除及pAd7ΔE3质粒的构建
1.构建E3基因敲除的穿梭质粒pVax-ΔE3(L+R)
E3基因敲除穿梭质粒pVax-ΔE3(L+R)的构建。以Ad7的基因组为模板，PCR获得E3基因的左臂(L-ΔE3)和右臂(R-ΔE3)。
L-ΔE3引物：
L-ΔE3-F：CATACTAGTCTGTCTACTTCAACCCCTTCTCCG(SEQ ID NO.41)；
L-ΔE3-R:GCAGAATTCATTTAAATGGAGGAAGGGTCTGGGTCTTCTG(SEQ ID NO.42)。
PCR条件：95℃，3min；95℃，30s；63℃30s；72℃，30s；cycles 30；72℃，5min；12℃保存。
R-ΔE3引物：
R-ΔE3-F:GCAGATATCATTTAAATAGACCCTATGCGGCCTAAGAGAC(SEQ ID NO.43)；
R-ΔE3-R：ACATCTAGAGACAGTTGGCTCTGGTGGGGT(SEQ ID NO.44)。
PCR条件：95℃，3min；95℃，30s；61℃30s；72℃，40s；cycles 30；72℃，5min；12℃保存。
L-ΔE3使用SpeI+EcoRI酶切后，连接至相同酶切的pVax载体上，得到pVax-L-ΔE3。R-ΔE3使用EcoRV+XbaI进行酶切，连接至相同酶切的pVax-L-ΔE3骨架上得到pVax-ΔE3(L+R)。
2.pAd7ΔE3质粒的构建
pVax-ΔE3(L+R)以SpeI+XbaI线性化，pAd7以EcoRI线性化，乙醇沉淀法回收后共转化BJ5183感受态细胞，涂至氨苄抗性平板，手提取质粒后，继续转化XL-Blue感受态细胞，手提取质粒并进行酶切鉴定。得到敲除E3基因并在E3区引入唯一的单酶切位点SwaI的基因组质粒pAd7ΔE3。SwaI酶切位点的插入，方便在E3基因区进行线性化。穿梭质粒及pAd7ΔE3质粒的构建示意图及大质粒的酶切鉴定结果如图16所示。
三、E1基因的敲除及pAd7ΔE1ΔE3质粒的构建
1.构建E1基因敲除的穿梭质粒pT-Ad7ΔE1(L+R)
E1基因敲除穿梭质粒pT-Ad7ΔE1(L+R)的构建。以Ad7的基因组为模板，PCR获得E1基因的左臂(L-ΔE1)和右臂(R-ΔE1)。
L-ΔE1引物：
L-ΔE1-F：ACTCACCGGCGGCGATCGCCTCTCTATTTAATATACCTTATAGATGG(SEQ ID NO.45)；
L-ΔE1-R：ATCACAATTGAATTCGTTTAAACGTAATCGAAACCTCCACGTAA(SEQ ID NO.46)。
PCR条件：95℃，3min；95℃，30s；54℃30s；72℃，30s；cycles 30；72℃，5min；12℃保存。
R-SE1引物：
R-SE1-F：ATAGAATTC ACTAGTGAGGCCCGATCATTTGGTGCT(SEQ ID NO.47)；
R-SE1-R：ACGTATAC CTATCATTATGGATGAGTGCATGG(SEQ ID NO.48)。
PCR条件：95℃，3min；95℃，30s；61℃30s；72℃，1min 10s；cycles 30；72℃，5min；12℃保存。
PCR产物和T载体使用Exnase重组酶进行三片段连接得到pT-Ad7(L+R)。
2.pAd7ΔE1ΔE3质粒的构建
pT-Ad7-ΔE1(L+R)以Bstz17I线性化，pAd7以AatII线性化，乙醇沉淀法回收后共转化BJ5183感受态细胞，涂至氨苄抗性平板，手提取质粒后，继续转化XL-Blue感受态细胞，手提取质粒并进行酶切鉴定。敲除E1基因并在E1区引入单酶切位点PmeI的基因组质粒pAd7ΔE1ΔE3。PmeI酶切位点的插入，方便在E1基因区进行线性化。穿梭质粒及pAd7ΔE1ΔE3质粒的构建示意图及大质粒的酶切鉴定结果如图17所示。
四、构建整合Ad5E4部分序列的质粒pAd7ΔE1ΔE3(Orf2-6)
1.构建E4基因区的穿梭质粒p7SE4。以Ad7的基因组为模板PCR获得E4基因区的左臂(L-SE4)和右臂(R-SE4)
扩增Ad7 L-SE4的引物序列：
L-SE4-F:CGCGGATCTTCCAGAGATGTTTAAACAACCAGTTACTCCTAGAACAGTCAGC(SEQ ID NO.49)；
L-SE4-R:ACGCGTATGGATTTAAAT CGATGCAGGCGAGAGTCTATTC(SEQ ID NO.50)。
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃,45s；cycles 30；72℃,5min；
扩增Ad7 R-SE4的引物序列：
R-SE4-F:ATTTAAATCCATACGCG TGGAGTTCTTATTAAGTGCGGATGG(SEQ ID NO.51)
R-SE4-R:GCCTGCCGTTCGACGATGTTTAAAC CAGCTGGCACGACAGGTTTC(SEQ ID NO.52)。
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃,30s；cycles 30；72℃,5min；
PCR获得的L-SE4、R-SE4片段和平末端的T载体进行三片段连接得到p7SE4。
2.构建携带Ad5E4部分序列的E4基因区的穿梭质粒p7SE4(Orf2-6)。以Ad5的基因组为模板PCR获得Ad5 E4 Orf2-6
Ad5 Orf2-6-F:TCACAGTCCAACTGCT CCTACATGGGGGTAGAGTCATAATCG(SEQ ID NO.53)；
Ad5 Orf2-6-R:GCGCGGTAACCTATTG CATGCAGAAACCCGCAGACATG(SEQ ID NO.54)。
PCR条件：95℃，3min；95℃，30s；65℃30s；72℃,2min；cycles 30；72℃,5min；
以p7SE4为模板PCR获得其骨架序列：
p7SE4-F:CAATAGGTTACCGCGCTGCG(SEQ ID NO.55)；
P7SE4-R:AGCAGTTGGACTGTGAAAGCGC(SEQ ID NO.56)。
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃,6min；cycles 30；72℃,6min；
将上述PCR获得的片段利用Exnase酶进行双片段连接得到p7SE4(Orf2-6)；
3.构建质粒pAd7ΔE1ΔE3(Orf2-6)
p7SE4(Orf2-6)使用PmeI进行酶切线性化，pAd7ΔE1ΔE3使用SwaI进行酶切线性化，利用乙醇沉淀法回收上述两种酶切产物，共转化BJ5183感受态细胞进行重组，将氨苄平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL-Blue感受态细胞，提取质粒得到pAd7ΔE1ΔE3(Orf2-6)，提取质粒进行酶切鉴定，pAd7ΔE1ΔE3(Orf2-6)具体构建过程及大质粒的鉴定结果参见图18。
五、携带外源基因的E1基因区穿梭质粒及pAd7ΔE1ΔE3(Orf2-6)-EGFP质粒的构建
1.构建携带外源基因表达框的E1基因区穿梭质粒pGK71-EGFP
1)以Ad7的基因组为模板PCR获得E1基因区穿梭质粒的左臂SE1L和右臂SE1R。
SE1L的扩增：
SE1L-F:CCAGATATACGCGTGTATACTTAATTAACGGCATCAGAGCAGATTGTACTG(SEQ ID NO.57)；
SE1L-R:GTTTAAACAAGATTTAAATGTAATCGAAACCTCCACGTAAACG(SEQ ID NO.58)。
SE1R的扩增：
SE1R-F:ATTTAAATCTTGTTTAAACGAATTCACTAGTGAGGCCCGATC(SEQ ID NO.59)；
SE1R-R:GCCCAGTAGAAGCGCCGGTGTTAATTAACAAGTAGCTTGTCCTCAGCCAGG(SEQ ID NO.60)。
2)构建携带E1基因区重组臂的穿梭质粒pSE1LR。
使用Bstz17I+SgraI双酶切质粒pVax回收质粒骨架，然后和上述PCR获得SE1L、SE1R使用Exnase酶进行三片段连接得到pSE1LR。
3)构建携带EGFP表达框的穿梭质粒pGK71-EGFP。
以实验室保存的pGA1-EGFP质粒为模板，PCR扩增获得CMV-EGFP-BGH的表达框。
扩增CMV-EGFP-BGH的引物序列：
CMV-EGFP-BGH-F:ACTAGTGAATTCGTTTACTAGTTATTAATAGTAATCAATTACGGG(SEQ ID NO.61)；
CMV-EGFP-BGH-R:CATTTAAATCTTGTTTCCTGCTATTGTCTTCCCAATC(SEQ ID NO.62)；
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃2min；cycles 30；72℃,5min；
pSE1LR使用PmeI酶切进行线性化，然后和PCR扩增获得的CMV-EGFP-BGH表达框使用 Exnase酶进行连接得到pGK71-EGFP。
2.构建插入外源基因EGFP的腺病毒重组质粒pAd7ΔE1ΔE3(Orf2-6)-EGFP
pGK71-EGFP使用PacI进行线性化，pAd7ΔE1ΔE3(Orf2-6)使用PmeI酶切进行线性化，利用乙醇沉淀法回收上述两种酶切产物，共转化BJ5183感受态细胞进行重组，将氨苄平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL-Blue感受态细胞，提取质粒得到pAd7ΔE1ΔE3(Orf2-6)-EGFP，提取质粒进行酶切鉴定，pAd7ΔE1ΔE3(Orf2-6)-EGFP具体构建过程及大质粒的鉴定结果参见图19。
六、复制缺陷型Ad7载体的拯救与生产
按照常规方法，pAd7ΔE1ΔE3(Orf2-6)和pAd7ΔE1ΔE3(Orf2-6)-EGFP以AsiSI线性化，乙醇沉淀回收，阳离子脂质体转染法转染293细胞，转染后4-6小时后，加入2毫升含5％胎牛血清的DMEM培养基，孵育7-10天，观察细胞病变；出毒后，收集细胞及培养上清，在37度水浴及液氮中反复冻融3次并离心去除细胞碎片，上清感染10厘米皿；2-3天后，收集细胞及培养上清，反复冻融3次并离心去除细胞碎片，上清感染10-15个15厘米皿；2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片，上清加至氯化铯密度梯度离心管；4℃,35000转，离心4小时；吸出病毒条带，脱盐，分装；以OD260吸光度测定病毒粒子滴度，计算公式为：病毒浓度＝OD260×稀释倍数×36/基因组长度(Kb)；病毒储存液于-80℃冻存。复制缺陷型Ad7载体的生产及纯化结果如图20所示。
七、复制缺陷型Ad7在293和A549细胞中复制能力测定
采用噬斑实验测定复制缺陷型Ad7载体在辅助细胞293和非辅助细胞A549中的复制能力。在6孔细胞板中接种293或者A549细胞，待到细胞密度接近100％时，将收获的的P1代Ad7ΔE1ΔE3(Orf2-6)-EGFP病毒原液梯度稀释后分别感染293或者A549细胞，每个病毒浓度做两个重复。病毒感染细胞2h后，吸去培养基，每孔铺上2ml左右的琼脂糖凝胶(含1ml 1.4％琼脂糖，1ml 1×MEM培养基，200ul BSA，1×青链霉素抗生素)。培养9-12天左右，利用荧光显微镜观察病毒携带的绿色荧光表达，并寻找病毒克隆的形成，拍照记录。结果如图21所示，Ad14ΔE1ΔE3(Orf2-6)-EGFP同时删除了E1和E3基因，只能在辅助细胞293中形成病毒噬斑，却无法在人正常的A549细胞形成病毒噬斑。以上结果表明复制缺陷型Ad7载体可在辅助细胞293中复制，却无法在人的正常细胞如A549细胞中复制，具有减毒表型。此外，复制缺陷型Ad7载体可在感染的细胞中表达携带的报告基因，运用于生物示踪系统中。
实施例4 Ad55疫苗的制备
一、E1基因的敲除及pAd55ΔE1ΔE3-Kana质粒的构建
1.E1基因敲除穿梭质粒pVax-delE1(L+R)的构建
以Ad55基因组为模板进行PCR扩增，得到重组臂L-delE1及R-delE1。
L-delE1引物序列：
L-delE1 F：ATAGAATTCGGGGTGGAGTGTTTTTGCAAG(SEQ ID NO.63)；
L-delE1 R：TTTACTAGTGTTTAAACGTAATCGAAACCTCCACGTAATGG(SEQ ID NO.64)。
PCR程序：95℃，30秒；62℃，30秒；72℃，20秒；25个循环。
R-arm引物序列：
R-delE1 F：ATTTCTAGAGTTTAAACGAGACCGGATCATTTGGTTATTG(SEQ ID NO.65)；
R-delE1 R：AAAGAATTCGGGAAATGCAAATCTGTGAGGG(SEQ ID NO.66)。
PCR程序：95℃，30秒；60℃，30秒；72℃，80秒；25个循环。
L-delE1以SpeI+EcoRI酶切后连接至同样酶切的pVax载体得到pVax-L-delE1；R-delE1以EcoRI+XbaI双酶切后连接至同样酶切的pVax-L-delE1得到敲除E1基因的穿梭质粒pVax-delE1(L+R)。
2.pAd55ΔE1ΔE3-Kana质粒的构建
pVax-delE1(L+R)以EcoRI线性化后，与PacI单酶切线性化的pAd55ΔE3共转化BJ5183感受态细胞；经氨苄青霉素、卡那霉素双抗性筛选后，手工提取质粒，进一步转化XL-Blue感受态细胞；手工提取质粒，得到pAd55ΔE1ΔE3-Kana质粒。以不同的酶切组合进行酶切鉴定，如图22所示。所得到的pAd55ΔE1ΔE3-Kana质粒中的腺病毒基因组原E1区引入2个PmeI酶切位点，以方便后续克隆。
二、Kana抗性基因的敲除及pAd55ΔE1ΔE3质粒的构建
1.Kana抗性基因敲除穿梭质粒pVax-delK(L+R)的构建
以Ad55基因组为模板进行PCR扩增，得到重组臂L-delK及R-delK。
L-delK引物序列：
L-delK F：ATAACTAGTGGGGTGGAGTGTTTTTGCAAG(SEQ ID NO.67)；
L-delK R：TTTGAATTCGTTTAAACGTAATCGAAACCTCCACGTAATGG(SEQ ID NO.68)。
PCR程序：95℃，30秒；61℃，30秒；72℃，20秒；25个循环。
R-delK引物序列：
R-delK F：ATCGTTTAAACGAGACCGGATCATTTGGTTATTG(SEQ ID NO.69)；
R-delK R：ATCTCTAGAGGGAAATGCAAATCTGTGAGGG(SEQ ID NO.70)。
PCR程序：95℃，30秒；60℃，30秒；72℃，80秒；25个循环。
L-delK以SpeI+EcoRI酶切后连接至同样酶切的pVax载体得到pVax-L-delK；R-delK以EcoRI+XbaI双酶切后连接至同样酶切的pVax-L-delE3得到敲除Kana基因的穿梭质粒pVax-delK(L+R)。
2.pAd55ΔE1ΔE3质粒的构建
pVax-delK(L+R)以SpeI+XbaI线性化，pAd55ΔE1ΔE3-Kana以PmeI线性化，乙醇沉淀法回收后共转化BJ5183感受态细胞，涂至氨苄抗性平板，手工提取质粒后，继续转化XL-Blue感受态细胞，手工提取质粒并进行酶切鉴定。得到去除Kana抗性基因并在E1区引入唯一酶切位点PmeI的基因组 质粒pAd55ΔE1ΔE3。穿梭质粒及pAd55ΔE1ΔE3质粒的构建示意图及酶切鉴定结果如图23所示。
三、Ad55 E4基因的改造及pAd55ΔE1ΔE3(Orf2-6)质粒的构建
1.Ad55 E4基因改造的穿梭质粒构建
(1)以Ad55基因组为模板，以下列引物进行PCR扩增，得到Ad55 E4基因。
R-delE3 Mlu：GATCACGCGTGGACTAAGAGACCTGCTACCCATG(SEQ ID NO.71)；
L-delK R：TGTAGATCTGTTTAAACCTTTAGCCCCATTACGTCAGTTTAG(SEQ ID NO.72)。
PCR程序：95℃，30秒；61℃，30秒；72℃，4分钟；25个循环。
PCR产物末端加磷酸化之后，与平末端T载体(TaKaRa)连接，得到p55E4。
(2)以p55E4质粒为模板，以下列引物进行PCR扩增，得到末端加入SapI酶切位点且去除Ad55 E4 Orf(2-6)基因的线性化p55E4。
Sap-p55E4 R：TTACGCTCTTCCTAGCCGTGATCCAGACTCCGG(SEQ ID NO.73)；
Sap-p55E4orf2 F：ATAGCTCTTCCCATTGTTAGTTTTGAATGAGTCTGCA(SEQ ID NO.74)；
PCR程序：95℃，30秒；61.5℃，30秒；72℃，4分钟；25个循环。
以Ad5基因组为模板，PCR扩增得到末端加入SapI位点的Ad5 E4 Orf(2-6)。
Sap-5ORF2-6 F：AATAGCTCTTCCCTACATGGGGGTAGAGTCATAATCG(SEQ ID NO.75)
Sap-5ORF2-6 R：ATATGCTCTTCCATGCAGAAACCCGCAGACATG(SEQ ID NO.76)
PCR程序：95℃，30秒；61℃，30秒；72℃，2分钟；25个循环。
上述线性化p55E4及Ad5 E4 Orf(2-6)片段以SapI酶切后相连接，得到p55E4(Orf2-6)。
2.pAd55ΔE1ΔE3(Orf2-6)质粒的构建。
p55E4(Orf2-6)以MluI+PmeI线性化，pAd55ΔE1ΔE3以PsiI线性化，共转化BJ5183感受态细胞，重组得到缺失E1、E3基因，且E4基因经过改造的基因组质粒pAd55ΔE1ΔE3(Orf2-6)。具体构建过程及鉴定结果参见图24。
四、携带外源基因的穿梭质粒和pAd55ΔE1ΔE3(Orf2-6)-EGFP质粒的构建
1.构建携带外源基因表达框的穿梭质粒pGK551-EGFP
1)以Ad55基因组为模板PCR扩增得到E1区同源重组臂SE1L及SE1R：
SE1L引物序列：
SE1L F，AATGGTACCGGGGTGGAGTGTTTTTGCAAG(SEQ ID NO.77)；
SE1L R，ATCGTAATCGAAACCTCCACGTAATGG(SEQ ID NO.78)。
PCR程序：95℃，30秒；61℃，30秒；72℃，30秒；25个循环。
SE1R引物序列：
SE1R F，AACACTAGTGAGACCGGATCATTTGGTTATTG(SEQ ID NO.79)；
SE1R R，TTAACGCGTGTATACGGGAAATGCAAATCTGTGAGGG(SEQ ID NO.80)。
PCR程序：95℃，30秒；60℃，30秒；72℃，1分钟30秒；25个循环。
2)构建携带重组臂的穿梭质粒pSE1LR。
pSE3LR以KpnI+EcoRV切，SE1L同样酶切，连接得到pSE1L；pSE1L以SpeI+MluI切，SE1R同样切，连接得到pSE1LR。
3)构建携带外源基因表达框的穿梭质粒pGK551-EGFP等。
以pGA1-EGFP为模板，以下列引物PCR得到CMV-EGFP-BGH表达框。
CMV，AGATATACGCGTTGACATTGATTATTGACTAG(SEQ ID NO.81)；
BGH，GCTGGTTCTTTCCGCCTCAGAAG(SEQ ID NO.82)。
PCR程序：95℃，30秒；66℃，30秒；72℃，1分钟45秒；25个循环。
pSE1LR以SpeI+EcoRV切，CMV-EGFP-BGH以SpeI切，连接得到目标穿梭质粒pGK551-EGFP。
2.构建基因组质粒pAd55ΔE1ΔE3(Orf2-6)-EGFP等
pGK551-EGFP质粒以BstZ17I+SgrAI切，乙醇沉淀回收；pAd55ΔE1ΔE3(Orf2-6)以PmeI线性化后乙醇沉淀回收；共转化BJ5183，同源重组得到携带外源基因表达框的pAd55ΔE1ΔE3(Orf2-6)-EGFP质粒。具体构建过程及鉴定结果参见图25。
五、复制缺陷型Ad55载体的拯救与生产
按照常规方法，pAd55ΔE1ΔE3(Orf2-6)和pAd55ΔE1ΔE3(Orf2-6)-EGFP分别以AsiSI线性化，乙醇沉淀回收，阳离子脂质体转染法转染293细胞，转染后8小时，加入2毫升含5％胎牛血清的DMEM培养基，孵育7-10天，观察细胞病变；出毒后，收集细胞及培养上清，在37度水浴及液氮中反复冻融3次并离心去除细胞碎片，上清感染10厘米皿；2-3天后，收集细胞及培养上清，反复冻融3次并离心去除细胞碎片，上清感染6-10个15厘米皿；2-3天后，收集细胞，反复冻融3次并离心去除细胞碎片，上清加至氯化铯密度梯度离心管；4℃,35000转，离心4小时；吸出病毒条带，脱盐，分装；以OD260吸光度测定病毒粒子滴度，计算公式为：病毒浓度＝OD260×稀释倍数×36/基因组长度(Kb)；病毒储存液于-80℃冻存。复制缺陷型Ad55载体的生产及纯化结果如图26所示。
六、复制缺陷型Ad55病毒在A549及293细胞中的复制能力鉴定
按照常规方法，以噬斑形成实验鉴定复制缺陷型Ad55载体在辅助细胞293以及非辅助细胞A549中的生长能力。六孔板中293或A549细胞长至九成满后，以Ad55ΔE1ΔE3(Orf2-6)-EGFP进行感染，感染滴度为1X10 7Vp/孔。感染后4小时，吸走培养基，并铺上1％的琼脂糖凝胶(含1％琼脂糖，5％胎牛血清，1×MEM培养基)。37℃培养箱内放置10-12天后，在荧光显微镜下观察病毒克隆的形成,并拍照记录。结果如图27所示。复制缺陷型Ad55ΔE1ΔE3(Orf2-6)-EGFP仅能在293细胞中形成噬斑，在A549细胞中则不能形成噬斑。这表明，复制缺陷型Ad55载体可在293细胞中有效增殖，但在正常人体细胞如A549细胞中不具备复制能力，具有减毒表型。同时，该结果也显示复制缺陷型人55型腺病毒载体可携带报告基因进入靶细胞，因而可应用于报告示踪系统中。
实施例5四价疫苗制备
将经氯化铯密度梯度力离心纯化后的复制缺陷型Ad3、Ad4、Ad7和Ad55疫苗分别稀释至8×10 11vp/ml，以500ul/管的量进行独立分装，-80℃保存。Ad3、Ad4、Ad7和Ad55四价疫苗(Ad3：2×10 10vp/ml；Ad4：2×10 10vp/ml，Ad7：2×10 10vp/ml；Ad4：2×10 10vp/ml)：取分装的Ad3、Ad4、Ad7和Ad55疫苗各250ul(1管)转移至15ml离心管中，进行10倍稀释，充分混匀，-80℃保存待用。
实施例6 Ad3、Ad4、Ad7和Ad55四价疫苗在猕猴中免疫原性评价
设计Ad3、Ad4、Ad7和Ad55四价疫苗在猕猴中免疫原性评价方案，如表1所示，按照设计的免疫方案对Ad3、Ad4、Ad7和Ad55四价疫苗的免疫原性进行评价。
表1
Figure PCTCN2021097796-appb-000001
选取成年恒河猴，分为2组，每组4只。采取肌注(手臂)免疫的方式，第1组免疫Ad3、Ad4、Ad7和Ad55四价疫苗(实验组)，第2组为对照组。免疫后第28天，静脉采血分离血清，测定抗Ad3、Ad4、Ad7和Ad55的特异性中和抗体。同时按上述方案第35天加强免疫；第49天(加强免疫2周)，静脉取血并分离血清，测定抗Ad3、Ad4、Ad7和Ad55的特异性中和抗体。结果如图28所示，实验组初免后即可产生高效价抗Ad3、Ad4、Ad7和Ad55的特异性中和抗体，而加强后抗体效价水平显著提升；对照组样品中Ad3、Ad4、Ad7和Ad55的中和抗体呈阴性。此外，我们对该四价疫苗对其他血清型腺病毒的交叉保护进行了分析，结果显示，加强免疫后两周的血清中存在较高效价的Ad2、Ad11和Ad14的抗体，证明该疫苗存在交叉保护作用，结果图29所示。我们分离了加强免疫后猕猴外周血单个核细胞(PBMC)进行了ELISPOT分析，结果如图30所示，该四价疫苗免疫后能够有效刺激猕猴机体产生较强的特异性细胞免疫应答。

Claims (14)

  1. 一种组合物，其包括复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒和55型腺病毒。
  2. 根据权利要求1所述的组合物，其特征在于，所述复制缺陷型人3型腺病毒的E1、E3基因缺失，E4基因的部分编码框置换为人5型腺病毒E4基因的相应编码框。
  3. 根据权利要求1所述的组合物，其特征在于，所述复制缺陷型人4型腺病毒的E1、E3基因缺失，E4基因的部分编码框置换为人5型腺病毒E4基因的相应编码框。
  4. 根据权利要求1所述的组合物，其特征在于，所述复制缺陷型人7型腺病毒的E1、E3基因缺失，E4基因的部分编码框置换为人5型腺病毒E4基因的相应编码框。
  5. 根据权利要求1所述的组合物，其特征在于，所述复制缺陷型人55型腺病毒的E1、E3基因缺失，E4基因的部分编码框置换为人5型腺病毒E4基因的相应编码框。
  6. 根据权利要求2所述的组合物，其特征在于，所述E4基因的部分编码框包括Orf2、Orf3、Orf4和Orf6编码框。
  7. 根据权利要求3所述的组合物，其特征在于，所述E4基因的部分编码框包括Orf2、Orf3、Orf4和Orf6编码框。
  8. 根据权利要求4所述的组合物，其特征在于，所述E4基因的部分编码框包括Orf2、Orf3、Orf4和Orf6编码框。
  9. 根据权利要求5所述的组合物，其特征在于，所述E4基因的部分编码框包括Orf2、Orf3、Orf4和Orf6编码框。
  10. 根据权利要求1-9所述的组合物，其特征在于，所述复制缺陷型人3型腺病毒、4型腺病毒、7型腺病毒、55型腺病毒中至少有一种腺病毒的E1基因区域整合外源基因表达框。
  11. 根据1-10任一权利要求所述的组合物在制备疫苗，检测试剂，调节基因功能的制剂或药物中的应用。
  12. 一种腺病毒四价疫苗制剂，其包括1-10任一权利要求所述的组合物。
  13. 根据权利要求12所述的腺病毒四价疫苗制剂，其特征在于，所述制剂还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
  14. 根据权利要求12所述的腺病毒四价疫苗制剂，其特征在于，所述制剂也对其它血清型腺病毒产生了交叉保护。
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