WO2015024484A1 - 一种新型狂犬病疫苗及其制备方法 - Google Patents

一种新型狂犬病疫苗及其制备方法 Download PDF

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WO2015024484A1
WO2015024484A1 PCT/CN2014/084578 CN2014084578W WO2015024484A1 WO 2015024484 A1 WO2015024484 A1 WO 2015024484A1 CN 2014084578 W CN2014084578 W CN 2014084578W WO 2015024484 A1 WO2015024484 A1 WO 2015024484A1
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vector
rabies
rabies vaccine
adenovirus
adc68
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French (fr)
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周东明
迟喻丹
邓飞
蓝柯
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成都远睿生物技术有限公司
中国科学院上海巴斯德研究所
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • 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
    • A61P31/14—Antivirals for RNA 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
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5256—Virus expressing foreign proteins
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention is in the field of biotechnology and virology; more specifically, the present invention relates to a novel rabies vaccine and a method of preparing the same.
  • Rabies is a lethal zoonotic infection characterized by rabies virus that is characterized by a violation of the central nervous system. Rabies causes more than 50,000 deaths each year worldwide, with a huge impact on life and health and the economy.
  • the rabies virus is a single-stranded negative-strand RNA virus belonging to the genus Rhabdovirus of the Rhabdoviridae. The shape is elastic, the nucleocapsid is helically symmetric, and the surface has an envelope.
  • the glycoprotein (Gp) antigen on the outer membrane of rabies virus is a component of the spine process on the surface of the virus.
  • the glycoprotein can induce the body to produce neutralizing antibodies and induce Cellular immunity, neutralizing antibodies have complete protection against viral infection.
  • WHO World Health Organization
  • the titer of human or animal serum rabies virus neutralizing antibody is higher than 0. 5IU/ml is considered to achieve effective protection level (Nishizono A et al, Evaluation of an improved rapid neutral izing antibody detection test (RAPINA) for qual itative and semiquantitative detection of rabies neutral izing antibody in humans and dogs. Vaccine. 2012; 30 (26): 3891-6).
  • the sick dog is the main source of rabies infection, followed by a variety of wild or domestic animals such as cats, pigs and foxes.
  • the main mode of transmission of rabies virus is through the bite or the infiltration of the dog's saliva through the damaged mucous membrane or skin. It has also been reported that rabies virus can be transmitted by aerosol spread, and the animal enters the olfactory nerve endings through the inhalation route and quickly reaches the central nervous system. Therefore, close contact with humans, such as dogs, is an important factor leading to a high incidence of human rabies. Vaccination against rabies is an effective way to prevent and control the occurrence of rabies.
  • the widely used primary cell culture vaccine and subcultured purified vaccine are inconvenient because of the high production cost, the high frequency of administration, and the need for combined adjuvants. Immunization applied to rabies virus storage hosts. Therefore, in China, the preparation of high-quality, inexpensive, convenient and easy-to-vaccinate rabies vaccine is very important for the prevention and control of animal rabies. Effective control of the source of rabies virus infection and the severance of its transmission depend on the development of a new generation of genetic engineering vaccines.
  • Adenoviral vectors have achieved some results in vaccine research and gene therapy.
  • recombinant adenovirus AdHu5 expression p53 (“Jian Zaisheng”) has played an important role in tumor gene therapy.
  • the use of adenoviral vectors to express foreign genes to prepare recombinant genetic engineering vaccines and the integration of RNA interference technology to block the life cycle of virus-dependent hosts have been widely reported.
  • the application of adenovirus as a vector for vaccine development has the following advantages: 1.
  • the infected host has a wide range of infections, can infect both dividing and non-dividing cells, and is highly efficient in introducing cells.
  • adenovirus is a vaccine vector with great potential to be tested and popularized, and it has shown good application prospects in the research of new genetic engineering vaccines.
  • AdHu5 and AdHu2 Since commonly used human serotypes of adenoviruses such as AdHu5 and AdHu2 are commonly infected, 60-80% of individuals in the population have corresponding neutralizing antibodies, which will impair the immune effect of vaccines based on AdHu5 and AdHu2. To overcome this problem, it is necessary to develop rare human serotypes or adenoviruses from other animal sources as vaccine or gene therapy vectors.
  • a rabies vaccine vector comprising: a replication-deficient recombinant adenoviral vector, and ligated between the vector cleavage sites I-Ceu l and Pl-Sce l a coding sequence for a rabies virus glycoprotein (Gp) antigen;
  • the replication-defective recombinant adenoviral vector comprises: a modified chimpanzee-type adenovirus AdC68 genomic sequence; wherein, a majority of the coding sequence of E1 is replaced by a linker (Linker); Sites I-Ceu I and ⁇ -Sce I.
  • Linker linker
  • the coding sequence of the rabies virus glycoprotein (Gp) antigen is as shown in SEQ ID NO: 1.
  • the linker sequence is set forth in SEQ ID NO: 3.
  • nucleotide sequence of the adenovirus expression vector is as shown in SEQ ID NO: 2.
  • the preparation method of the replication-defective recombinant adenoviral vector is:
  • the chimpanzee-type adenovirus AdC68 gene was divided into four fragments, which were sequentially inserted into the backbone vector, and the coding sequence was substituted for most of the coding sequences of El in the AdC68 genome;
  • amino acid position of each fragment is calculated based on the nucleotide sequence of GenBank accession number AC_000011.
  • the majority of the coding sequences of the adenovirus El are excised by Nde I and SnaB I, and the size is about 3.5 kb, which is replaced by the endonuclease I-Ceu I and Linker fragment of PI-Sce I.
  • the backbone carrier is a PNEB 193 vector.
  • a method of preparing a rabies vaccine comprising:
  • the rabies vaccine vector of (1) is transfected into a virus-producing cell, and the virus is packaged in the cell to obtain an immunogenic rabies vaccine.
  • a rabies vaccine is provided, which is prepared by the method described above.
  • a kit is provided, the kit comprising the rabies vaccine.
  • kits for preparing a vaccine comprising: the rabies vaccine vector described above.
  • the kit further comprises: a virus producing cell.
  • the virus producing cell is a HEK293 cell.
  • A enzyme digestion identification. 1. 1Kb DNA marker; 2. Nhe I and Xba I were digested to identify pUC57_0/Gp vector; 3. Nhe I and Xba I were digested to identify pshuttle-CMV vector.
  • the pAdshuttle-CMV/Gp vector was identified by double digestion with C, Nhe I and EcoR I.
  • Bgl I I, BamH I and Xho I digest the pAdC68_Gp recombinant adenoviral genome.
  • adenovirus pAdC68-Gp to 108 and 10 1Q vps infected HEK293 cells i.e., 293A (24h).
  • Recombinant adenovirus pAdC68-Gp Huh7 cells were infected at 10 8 and 10 10 vps (24 h).
  • Bgl I I, BamH I and Xho I digest the 5th generation recombinant adenoviral genome.
  • the 15th generation recombinant adenovirus pAdC68-Gp was infected with HEK293 and Huh7 cells at 10 1Q vps (24h).
  • the inventors have intensively studied and constructed a novel rabies vaccine vector based on the vaccine vector of the chimpanzee adenovirus AdC68 genome by an improved construction method.
  • the vaccine vector can be used to prepare a viral vaccine that is highly expressed and has good immunogenicity.
  • the present inventors selected a rare serotype or an adenovirus of other species as a vaccine carrier, since the population generally does not have a neutralizing antibody against chimpanzee-type adenovirus, and the chimpanzee-type adenovirus is used as a vaccine vector. A better immune effect can be obtained. Therefore, the inventors constructed the chimpanzee-type adenovirus AdC68 as a replication-deficient recombinant expression vector, which was tested to have good immunogenicity and genetic stability. Based on the AdC68 vector, the inventors further prepared a novel rabies vaccine. When the novel vaccine is immunized to mice, it can effectively induce neutralizing antibodies against rabies virus and has strong resistance to viral infection.
  • the present invention provides an adenovirus expression vector comprising: a replication-deficient recombinant adenoviral vector, and a rabies virus ligated between the vector cleavage sites I-Ceu I and Pl-Sce l a coding sequence of a glycoprotein (Gp) antigen; wherein the replication-defective recombinant adenoviral vector comprises: an engineered chimpanzee-type adenovirus AdC68 genomic sequence; wherein a majority of the coding sequence of E1 is replaced by a linker (Linker)
  • Linker linker
  • the inventors performed a detailed sequence alignment to finally determine the use of the cleavage sites I-Ceu I and Pl-Sce I as insertion sites for foreign genes, thereby not causing expression in adenoviruses. Other locations of the carrier cause shear.
  • adenovirus AdC68 As a vaccine vector, that is, to make full use of certain restriction sites present in the adenoviral genome.
  • adenovirus AdC68 According to the analysis of the entire genome sequence of adenovirus AdC68, it was divided into four fragments of KE, AK, XA and PX, and then gradually cloned into the vector of PNEB193 by restriction enzyme ligation, and finally a replication-defective adenovirus was obtained.
  • adenovirus AdC68 In the instructions, the two parts of Nde l and SnaB I were used to delete the El part associated with adenovirus replication, which was about 3.5 kb, which was replaced with the homing enzymes I-Ceu I and ⁇ - Linker fragment of Sce I.
  • the replication-deficient adenovirus AdC68 constructed by this method was linearized to successfully package and amplify a genetically stable recombinant adenovirus in HEK293 cells.
  • KE, AK, XA and PX can include restriction enzyme sites between the four fragments, which facilitates the organic linkage of the various elements.
  • the expression vector usually also contains an origin of replication and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter (e.g., CMV) in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells.
  • the present invention also provides a coding sequence for a codon-optimized rabies virus glycoprotein (Gp) antigen, as shown in SEQ ID NO: 1, which enables efficient expression.
  • Gp codon-optimized rabies virus glycoprotein
  • the present invention also provides a method for preparing an adenovirus expression vector, the method comprising: dividing a chimpanzee-type adenovirus AdC68 gene into four fragments, sequentially loading into a skeleton vector, and replacing the E1 in the AdC68 genome with a linker sequence
  • the coding sequence; in the ligation sequence, the restriction sites I-Ceu I and Pl-Sce I are set.
  • the invention also provides a method for preparing a rabies vaccine, the method comprising: providing the rabies vaccine vector; transfecting the expression vector into a virus producing cell, and packaging the virus in the cell to obtain an immunogenic rabies vaccine.
  • the virus After obtaining the adenovirus expression vector, it is transfected into a virus producing cell to carry out virus propagation. After a period of time after transfection, the virus can be harvested. As a preferred mode of the present invention, the harvested virus can be repeatedly infected with the virus producing cells for continuous passage.
  • the determination of the virus titer (TCID50) can be carried out according to a conventional method in the art. Accordingly, the present invention also provides a rabies vaccine which is prepared by the method of the present invention.
  • the present invention also provides a kit for preparing a vaccine, the kit comprising the rabies vaccine vector.
  • the kit may also include virus producing cells such as HEK293 cells.
  • instructions for the preparation of the vaccine may also be included in the kit.
  • the shuttle vector pShuttle_CMV was purchased from Clontech Laboratories, Inc.
  • the wild type adenovirus AdC68 (GenBank accession number AC_000011) was purchased from ATCC.
  • the PNEB193 plasmid was purchased from New England Biolabs.
  • the recombinant plasmid carrying the optimized codon Gp (ERA strain) PUC57-0/Gp was synthesized by Nanjing Kingsray Biotechnology Co., Ltd.
  • the competent strain Stbl2 was purchased from Invitrogen.
  • Adenovirus packaging cell line HEK293 (293A) cells were purchased from ATCC.
  • the El-deficient replication-deficient adenoviral vector pAdC68 (pAdC68-El-deleted) was constructed as follows:
  • primers for amplifying the KE fragment were designed (Table 1), and the target product KE fragment was amplified by PCR.
  • the total volume of the PCR amplification system was 50uL, and the reaction cycle parameters were: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for lmin; annealing temperature at 57 °C for 30 s; and 72 °C for 2. 2 min.
  • the KE fragment was amplified by EcoR I and Kpn I, and the KE fragment was purified by agarose gel.
  • the KE fragment was ligated into the PENB193 vector, which was digested with the same restriction enzyme, into DH5 ⁇ competent cells, and the positive clone was picked and digested. And sequencing identified ⁇ 193- ⁇ .
  • Link - F A CmgsmGTlCGGCG 9 ⁇ Underlined restriction endonuclease sites.
  • Asc I and Kpn I were double-digested with AdC68 genome, and the AK fragment was recovered by low melting point agarose gel electrophoresis, ligated into the same digested PNEB193-KE vector, transformed into Stbl2 competent cells, and picked positive clones.
  • Xba I and Asc I double-cut the AdC68 genome, due to the presence of three cleavage sites in the adenovirus AdC68 by Asc I, on Asc I
  • the restriction enzyme digestion was carried out, and the AdC68 genome was digested at 37 °C for 10 s.
  • the largest XA fragment of the low-melting gel was digested and ligated into the pNEB 193-KE-AK vector which was digested with the same restriction enzyme, and the pNEB was identified by restriction enzyme digestion. 193-KE-AK-XA.
  • primers for amplification of the sputum fragment were designed (Table 1), and the target product ⁇ fragment was amplified by PCR.
  • the total volume of the PCR amplification system was 50 uL, and the reaction cycle parameters were: pre-denaturation at 95 ° C for 5 min ; denaturation at 94 ° C for 1 min; annealing temperature at 55 ° C lmin; and extension at 72 ° C for 6 min.
  • the PX fragment was amplified by Pme I and Xba I double-enzyme digestion, and the PX fragment of the target product was recovered by agarose gel, ligated into the same digested PNEB193 vector, transformed into Stbl2 competent cells, and positive clones were picked.
  • the region near the left end of the adenovirus AdC68 ITR was sequenced.
  • the Xba l-encoding adenovirus AdC68 genome was recovered from the low-melting-point agarose gel to obtain the Xba I-Xba I portion of AdC68, and was ligated into the pBB193-PX vector digested with Xba I and transformed into Stbl2 competent cells. , pick positive clones, and identify by endonuclease pNEB193-PX o
  • the Linker primers were designed according to the sequence of pShuttle-EM plasmid (Table 1).
  • the Linker product was amplified by PCR.
  • the total volume of the PCR amplification system was 50 uL.
  • the reaction cycle parameters were: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for 1 min. Annealing temperature 55 ° C lmin; 72 ° C extension lmin.
  • Linker fragment amplified by SnaB I and Nde I double-enzyme-cleaved PCR Linker product was recovered from agarose gel, and ligated into pNEB193_PX vector which was digested with the same restriction enzyme (by sequence analysis of AdC68 genome, using SnaB I and Nde I The site can delete its El part sequence, transform into DH5 ⁇ competent cells, pick positive clones, and identify pNEB193-PX-Linker by restriction enzyme digestion.
  • Linker's sequence was inserted between positions 459-3011 of the AdC68 genome, replacing most of the sequence of E1.
  • the sequence of Linker is SEQ ID NO: 3.
  • the pNEB193_PX_Linker plasmid DNA was digested with Xba I and Pme I, and the PX_Linker fragment was recovered by agarose gel, and ligated into the pNEB 193-KE-AK-XA vector recovered by the same low-melting agarose gel, and transformed into Stb.
  • the competent cells positive clones were picked and identified by enzyme digestion to obtain pAdC68-El-del e t e d.
  • the nucleotide sequence of the replication defective recombinant adenoviral vector pAdC68-El-del e t e d is SEQ ID NO: 2.
  • T4DNA ligase Taq DNA polymerase, DNA gel recovery kit, plasmid extraction kit, etc. are all purchased from Bao Bioengineering (large Description Book)). Restriction enzymes such as Bgl II, BamH I, Nhe I, Pac I were purchased from NEB Corporation.
  • Lipofectamine 2000 DNA transfection reagent was purchased from Invitrogen.
  • the codon-optimized Gp gene (SEQ ID NO: 1) was synthesized according to the nucleic acid sequence of the Gp coding region of the rabies virus ERA strain in GenBank, and EcoR I was sequentially inserted at the 5' end of the target gene of the optimized codon. And the Nhe I restriction site, the 3' end was inserted into the Xba I restriction site (Fig. 1), and the optimized Gp gene was cloned into the commercial vector PUC57 vector by EcoR I and Xba I double digestion, and the recombinant plasmid was obtained. PUC57-0/Gp.
  • the PUC57-0/Gp plasmid was digested with Nhe I and Xba I to form the target gene with cohesive ends, and the adenoviral shuttle vector pshuttle-CMV was digested with Nhe I and Xba I to obtain a linearized vector.
  • the sticky ends of the above products were ligated using T4 DNA ligase.
  • the ligation product was transformed by a conventional method, screened on a kanamycin-resistant agar plate, and cultured overnight at 37 ° C in LB medium to extract plasmid DNA. Identification by restriction enzyme digestion, PCR, etc., and a positive recombinant plasmid was obtained, which was named pAdshuttle-CMV/Gp.
  • the plasmid pshuttle-CMV/Gp was digested with PI_Sce I and I_Ceu I to form the target gene with cohesive ends, and the adenoviral vector pAdC68-El-del e t e d was digested with PI_Sce I and I_Ceu I to obtain a linear form. Carrier.
  • the target fragment was isolated using a low melting point agarose gel, and the gel containing the desired fragment and the vector was incubated at 65 ° C for 5 minutes, and after completely dissolved, the sticky ends of the above products were ligated using T4 DNA ligase.
  • the ligation product was transformed in the competent bacteria Stbl2 according to a conventional method, and an appropriate amount of the transformed product was applied to an ampicillin-resistant agar plate, cultured at 30 for 24 hours, and the ampicillin-resistant clone was selected, and the plasmid DNA was extracted in small amounts with 0. Electrophoretic mobility analysis was performed on an 8% agarose gel, and Bgl II, BamH I, and Xho I digestion profiles were performed. The resulting recombinant adenovirus plasmid broth was expanded (1: 1000) to obtain a large amount of high-quality plasmid DNA. A recombinant adenoviral vector containing the rabies virus gene was obtained and designated pAdC68-Gp.
  • the recombinant adenoviral plasmid pAdC68-Gp was linearized with restriction endonuclease Pac I, and the plasmid was transferred into HEK293 cells (6-well plate) by lipofection. The cells were incubated at 37 ° C for 6-8 days. Plaque. After the cells were rounded and suspended, the cells were collected, and after repeated freezing and thawing, the virus supernatant was taken to infect HEK293 cells (75 ml cell culture flask).
  • virus about 20-40 150 ml cell culture flasks
  • virus was purified by cesium chloride density gradient centrifugation, and the OD value was measured, and glycerin having a final concentration of 10% was added at -80 °C.
  • Viral genomic DNA (viral content 10 12 vps) was extracted and analyzed by Bgl II, BamH I and Nhe I restriction maps. The nucleic acid sequence of the fragment of interest is detected using the CMV promoter universal primer.
  • the purified virus was continuously passaged for 15 generations in HEK293 cells to verify the genetic stability of the recombinant vector.
  • the recombinant adenovirus with a certain titer was intramuscularly immunized with ICR mice, and the concentration of neutralizing antibody in the serum was measured 3 weeks later.
  • the neutralizing antibody titer was determined by Wuhan Institute of Biological Products.
  • mice 10 ICR mice were injected with 10 1Q V ps recombinant adenovirus AdC68-Gp; control group: 10 ICR mice were injected with 10 10 vps adenovirus vector AdC68-3 ⁇ 4)t.
  • PI-Sce I and I-Ceu I were digested with pAdshuttle-CMV/gp and pAdC68, and the above products were ligated with T4 DNA ligase. After selecting the ampicillin-resistant clone, the plasmid pAdC68-Gp was identified by restriction enzyme digestion, as shown in Fig. 3, which was consistent with the expected size. The sequencing results showed that the obtained nucleic acid sequence was identical to the nucleic acid sequence after the optimized fragment of the target fragment.
  • the recombinant adenoviral plasmid DNA was prepared in large amounts, and the recombinant plasmid digested with Pac I was transfected into HEK293 cells. Significant cytopathic changes occurred 6 days later. In the early stage of the lesion, plaques appeared in the cells, gradually becoming larger, and later in the state of pulling the net, and finally the cells all floated. 20 bottles of 150 ml of cells infected with the virus were collected, and the frozen-thawed mixture was purified by cesium chloride density gradient centrifugation, and the recombinant adenovirus 0D value was determined to be 8. 9 X 10 12 vps/ml. The viral genome was extracted, identified by restriction enzyme digestion, and the fragment size was consistent with expectations.
  • Example 3 Determination of recombinant adenovirus infection titer and genetic stability analysis
  • HEK293 cells and the hepatoma cell line Huh7 were infected with different numbers of viruses, as shown in Figure 4.
  • plaques appeared in HEK293 cells (10 s vps); the infected Huh7 cells (10 s vps) were not significantly different from the control group.
  • Huh7 Plaques also appear in the cells.
  • the purified virus was continuously transferred to HEK293 cells for 15 generations, and the virus was collected and purified in small amounts.
  • the genomes of the 5th and 15th generation viruses were identified by enzyme digestion, and the recombinant virus was not mutated, and the infection of the primary virus was maintained.
  • Ability as shown in Figure 5.
  • the recombinant adenovirus vector AdC68-Gp was used to immunize ICR mice, and the neutralizing antibody titer in the serum was measured 3 weeks later, as shown in Fig. 6.
  • the neutralizing antibody titer in the serum of the immunized group (mean 38.3 IU/ml) was significantly higher than that of the control group, and the recombinant chimpanzee adenosis Description Book AdC68-Gp is effective in inducing high titer neutralizing antibodies.
  • the present inventors have completed the construction of a novel recombinant rabies vaccine vector, detection of genetic stability, and monitoring of immunogenicity, and the results indicate that a novel rabies vaccine based on adenovirus vector AdC68 can induce high titer in mice. Specific neutralizing antibodies, well beyond the criteria for WHO-specified protective effects.

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Abstract

本发明涉及一种新型狂犬病疫苗及其制备方法。本发明人基于黑猩猩型腺病毒AdC68基因组的疫苗载体，构建了一种新型狂犬病疫苗载体。本发明还提供了以所述的疫苗载体制备的可高效表达且具有良好免疫原性的病毒疫苗。

Description

说 明 书 一种新型狂犬病疫苗及其制备方法
技术领域
本发明属于生物技术和病毒学领域； 更具体地， 本发明涉及一种新型狂犬病疫苗及其制 备方法。
背景技术
狂犬病是由狂犬病毒引发的以侵犯中枢神经系统为特征的致死性人畜共患传染病。狂犬 病在全球每年导致 5 万多人死亡， 给生命健康和社会经济造成巨大影响。 狂犬病毒是单股负 链 RNA病毒， 属于弹状病毒科弹状病毒属， 外形呈弹状， 核衣壳呈螺旋对称， 表面具有包膜。 狂犬病毒外膜上的糖蛋白（Gp) 抗原是病毒表面棘突的成分， 有凝集细胞的能力， 能与乙酰胆 碱受体结合使病毒具有嗜神经性； 糖蛋白能诱导机体产生中和抗体并诱导细胞免疫， 中和抗 体对于病毒感染具有完全保护作用。 按世界卫生组织 (WHO) 的要求， 人或动物血清狂犬病毒 中和抗体的效价高于 0. 5IU/ml 被认为达到有效保护水平（Nishizono A等， Evaluation of an improved rapid neutral izing antibody detection test (RAPINA) for qual itative and semiquantitative detection of rabies neutral izing antibody in humans and dogs. Vaccine. 2012； 30 (26)： 3891-6)。
病犬是狂犬病主要的传染源， 其次是猫、 猪及狐狸等各种野生或家养动物。 狂犬病毒主 要的传播方式是经咬伤传播或病犬唾液经损伤的粘膜或皮肤而入侵。 另有报道证实狂犬病毒 可通过气溶胶散布传播， 动物通过吸入途径使狂犬病毒进入嗅神经末梢从而迅速到达中枢神 经系统。 因此犬等与人类密切接触是导致人狂犬病发病率居高不下的重要因素。 接种狂犬病 疫苗是预防和控制狂犬病发生的有效途径， 目前广泛采用的原代细胞培养疫苗和传代细胞精 制纯化疫苗由于生产成本昂贵， 给药次数较多， 以及需要联合佐剂等诸多限制因素， 不便应 用于狂犬病毒储存宿主的免疫接种。 因此， 在我国，制备质优、 价廉、 方便、 易接种的狂犬病 疫苗对于预防控制动物狂犬病十分重要。 有效控制狂犬病毒的传染源、 切断其传播途径有赖 于新一代基因工程疫苗的发展。
腺病毒载体在疫苗研究以及基因治疗方面的应用已取得一定成果， 如重组腺病毒 AdHu5 表达 p53 ( "今又生"） 已在肿瘤基因治疗方面发挥重要作用。 在病毒防治的研究中， 利用腺 病毒载体表达外源基因制备重组基因工程疫苗以及通过结合 RNA干扰技术来阻碍病毒依赖 宿主的生活周期等均有广泛报道。 腺病毒作为载体应用于疫苗的开发具有以下优点： ①感染 的宿主范围广， 能感染分裂和非分裂细胞，导入细胞的效率较高。②遗传背景清楚，致病性低， 不整合到染色体中，不会导致插入突变。 ③外源基因表达水平高， 可以容纳约 8-10kb 的外源 说 明 书 基因， 能同时表达多个基因。 ④增殖速度快，易培养，可以产生较高的病毒滴度， 适用多种途 径免疫。⑤免疫原性强，能够协同外源蛋白引起强烈的特异性细胞免疫和体液免疫。基于上述 优点，腺病毒是一种大有潜力值得试验和推广的疫苗载体，在新型基因工程疫苗研究中越来越 显示出良好的应用前景。
由于常用的人血清型腺病毒如 AdHu5、 AdHu2 等普遍感染人， 因此人群中 60-80% 的个体 存在相应的中和抗体，将削弱以 AdHu5、AdHu2 等为载体的疫苗的免疫效果。为克服这个问题， 开发稀有人血清型或来自其它动物来源的腺病毒为疫苗或基因治疗载体是有必要的。
发明内容
本发明的目的在于提供一种狂犬病疫苗及其制备方法。
在本发明的第一方面， 提供一种狂犬病疫苗载体， 所述表达载体包括： 复制缺陷型重组 腺病毒载体，以及连接于该载体酶切位点 I-Ceu l 和 Pl-Sce l 之间的狂犬病毒糖蛋白（Gp) 抗 原的编码序列；
其中， 所述的复制缺陷型重组腺病毒载体包括： 改造的黑猩猩型腺病毒 AdC68 基因组序 列； 其中， E1 的大部份编码序列由连接序列（Linker) 替换； 所述的连接序列中设置酶切位 点 I-Ceu I 和 ΡΙ-Sce I。
在一个优选例中， 所述的狂犬病毒糖蛋白（Gp) 抗原的编码序列如 SEQ ID N0: 1 所示。 在另一优选例中， 所述的连接序列如 SEQ ID NO: 3 所示。
在另一优选例中， 所述的腺病毒表达载体的核苷酸序列如 SEQ ID NO: 2 所示。
在另一优选例中， 所述的复制缺陷型重组腺病毒载体制备方法为：
将黑猩猩型腺病毒 AdC68 基因组分成 4 个片段， 依次装入到骨架载体中， 并且， 用连接 序列替换 AdC68 基因组中 El 的大部份编码序列；
所述的 4 个片段分别是：
黑猩猩型腺病毒 AdC68 基因组第 1-6025 位；
黑猩猩型腺病毒 AdC68 基因组第 6026-17279 位；
黑猩猩型腺病毒 AdC68 基因组第 17280-34196 位； 和
黑猩猩型腺病毒 AdC68 基因组第 34197-36519 位。
在另一优选例中， 各片段的氨基酸位点计算基于 GenBank 登录号 AC_000011 的核苷酸序 列。
在另一优选例中， 利用 Nde I 和 SnaB I 两个酶切位点切除腺病毒 El 的大部份编码序列， 大小约为 3. 5kb， 将其替换为含有内切酶 I-Ceu I 和 PI-Sce I的 Linker片段。 说 明 书 在另一优选例中， 所述的骨架载体是 PNEB193载体。
在本发明的另一方面， 提供一种制备狂犬病疫苗的方法， 所述方法包括：
(1) 提供所述的狂犬病疫苗载体；
(2) 将（1) 的狂犬病疫苗载体转染病毒生产细胞， 病毒在细胞内包装， 从而获得具有免 疫原性的狂犬病疫苗。
在本发明的另一方面， 提供一种狂犬病疫苗， 所述疫苗由上面所述的方法制备获得。 在本发明的另一方面， 提供一种药盒， 所述的药盒中含有所述的狂犬病疫苗。
在本发明的另一方面， 提供一种用于制备疫苗的试剂盒， 所述试剂盒包括： 前面所述的 狂犬病疫苗载体。
在一个优选例中， 所述试剂盒还包括： 病毒生产细胞。
在另一优选例中， 所述的病毒生产细胞是 HEK293 细胞。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。
附图说明
图 1、 新型狂犬病疫苗载体构建策略。
图 2、 pUC57-0/Gp 载体和腺病毒穿梭载体 pAdshuttle-CMV/Gp 酶切鉴定。
A、 酶切鉴定。 1、 1Kb DNA marker ； 2、 Nhe I 和 Xba I 双酶切鉴定 pUC57_0/Gp 载体； 3、 Nhe I 和 Xba I 双酶切鉴定 pshuttle-CMV载体。
B、 Nhe I 和 Xba I 双酶切鉴定 pAdshuttle-CMV/Gp 载体； 1 ： 1Kb DNA ladder
marker ； 2, 3、 反向克隆； 4、 正确克隆。
C、 Nhe I 和 EcoR I 双酶切鉴定 pAdshuttle-CMV/Gp 载体。
图 3、 重组腺病毒载体 pAdC68-Gp 和重组病毒基因组酶切鉴定。
1、 1Kb DNA ladder marker ；
2, 3， 4、 Bgl I I， BamH I 和 Xho I 酶切 pAdC68_Gp 重组腺病毒基因组。
5、 1Kb DNA ladder marker ；
6， 7， 8、 Bgl I I， BamH I 和 Xho I 酶切重组腺病毒质粒 DNA pAdC68_Gp。
图 4、 重组腺病毒感染滴度的检测。
A、 重组腺病毒 pAdC68-Gp 以 108 和 101Qvps 感染 HEK293 细胞 (即 293A) (24h)。
B、 重组腺病毒 pAdC68-Gp 以 108 和 1010vps 感染 Huh7 细胞（24h)。
图 5、 重组腺病毒遗传稳定性的检测。
A、 重组腺病毒 AdC68-Gp 基因组第 5 代和第 15 代的酶切鉴定。 说 明 书
1、 1Kb DNA ladder marker。
2, 3， 4、 Bgl I I， BamH I 和 Xho I 酶切第 5 代重组腺病毒基因组。
5、 1Kb DNA ladder marker。
6， 7， 8、 Bgl I I， BamH I 和 Xho I 酶切第 15 代重组腺病毒基因组。
B、 第 15 代重组腺病毒 pAdC68-Gp 以 101Qvps 分别感染 HEK293 和 Huh7 细胞（24h)。
图 6、 血清中狂犬病毒中和抗体效价检测。 左： 重组腺病毒 AdC68-Gp 免疫组小鼠
中和抗体效价； 右： 腺病毒空载体 AdC68-ept 免疫小鼠中和抗体效价。
具体实施方式
本发明人经过深入的研究， 通过改进构建方法， 基于黑猩猩型腺病毒 AdC68 基因组的疫 苗载体， 构建了一种新型狂犬病疫苗载体。 所述的疫苗载体可应用于制备可高效表达且具有 良好免疫原性的病毒疫苗。
为解决现有技术的问题， 本发明人选择稀有血清型或其他种属来源的腺病毒作为疫苗载 体， 由于人群一般不存在针对黑猩猩型腺病毒的中和抗体， 以黑猩猩型腺病毒为疫苗载体可 获得较好的免疫效果。 因此， 本发明人构建了黑猩猩型腺病毒 AdC68 为复制缺陷型重组表达 载体， 经检验该载体具有良好的免疫原性和遗传稳定性。 在该 AdC68 载体的基础上， 本发明 人进一步制备了新型狂犬病疫苗。 该新型疫苗免疫小鼠后， 能有效地诱导针对狂犬病毒的中 和抗体， 对病毒感染产生强大的抵抗力。
因此， 本发明提供了一种腺病毒表达载体， 所述表达载体包括： 复制缺陷型重组腺病毒 载体， 以及连接于该载体酶切位点 I-Ceu I 和 Pl-Sce l 之间的狂犬病毒糖蛋白（Gp)抗原的编 码序列； 其中， 所述的复制缺陷型重组腺病毒载体包括： 改造的黑猩猩型腺病毒 AdC68 基因 组序列； 其中， E1 的大部份编码序列由连接序列（Linker) 替换； 所述的连接序列中设置酶 切位点 I-Ceu I 和 Pl-Sce I。
针对腺病毒 AdC68 基因组， 本发明人经过细致的序列比对， 最终确定了应用酶切位点 I-Ceu I和 Pl-Sce I作为外源基因的插入位点， 从而不会导致在腺病毒表达载体的其它位置造 成剪切。
由于腺病毒的基因组比较大， 大约有 36kb， 使直接克隆重组腺病毒载体成为技术瓶颈。 因此， 本发明人开发了能通过酶切连接的快速方法直接构建基于黑猩猩型腺病毒 AdC68 作为 疫苗载体， 即充分利用腺病毒基因组中存在的某些酶切位点。 根据对腺病毒 AdC68 整个基因 组序列的分析， 将其分成 KE， AK， XA和 PX 四个片段， 然后通过酶切连接的方法再逐步克隆 到 PNEB193 的载体中， 最终获得一个复制缺陷型的腺病毒克隆。其中在腺病毒 AdC68 的 PX 部 说 明 书 分， 利用 Nde l 和 SnaB I 两个酶切位点删除了与腺病毒复制相关的 El 部分， 大小约为 3. 5kb， 将其替换为含有归位内切酶 I-Ceu I 和 ΡΙ-Sce I 的 Linker 片段。 此方法构建的复制缺陷型 腺病毒 AdC68 经过线性化能够在 HEK293 细胞中成功包装并扩增出遗传稳定的重组腺病毒。 KE， AK， XA和 PX 四个片段之间可包括限制性的酶切位点， 这样有利于各元件的有机连接。
所述的表达载体通常还含有复制起点和 / 或标记基因等。 本领域的技术人员熟知的方法 能用于构建本发明所需的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体内 重组技术等。 所述的 DNA序列可有效连接到表达载体中的适当启动子（ 如 CMV) 上， 以指导 mRNA合成。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外， 表达载体优 选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿主细胞的表型性状。
本发明还提供了密码子优化的狂犬病毒糖蛋白（Gp) 抗原的编码序列， 如 SEQ ID N0: 1 所 示， 其能够实现高效的表达。
本发明还提供了一种制备腺病毒表达载体的方法， 所述方法包括： 将黑猩猩型腺病毒 AdC68 基因组分成 4 个片段， 依次装入到骨架载体中， 并且， 用连接序列替换 AdC68 基因组 中 E1 编码序列； 所述的连接序列中， 设置酶切位点 I-Ceu I 和 Pl-Sce I。
本发明还提供了一种制备狂犬病疫苗的方法， 所述方法包括： 提供所述的狂犬病疫苗载 体； 将该表达载体转染病毒生产细胞， 病毒在细胞内包装， 从而获得具有免疫原性的狂犬病 疫苗。
获得所述腺病毒表达载体后， 将之转染病毒生产细胞， 进行病毒的繁殖。 转染后的一段时间 后， 可以收获病毒。 作为本发明的优选方式， 收获的病毒可反复感染病毒生产细胞， 持续传 代。 病毒滴度 (TCID50) 的测定可以根据本领域常规方法进行。 因此， 本发明还提供了一种 狂犬病疫苗， 所述疫苗由本发明所述的方法制备获得。
基于本发明的新改进， 本发明还提供了一种用于制备疫苗的试剂盒， 所述试剂盒包括所 述的狂犬病疫苗载体。
所述的试剂盒还可包括病毒生产细胞， 如 HEK293 细胞。 此外， 所述的试剂盒中还可包括 说明疫苗制备方法的使用说明书。
下面结合具体实施例， 进一步阐述本发明。 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， 通常按照常规条件如 J. 萨姆布鲁克等编著， 分子克隆实验指南， 第三版， 科学出版社， 2002中所述的条件， 或按照 制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按重量计算。
1、 材料和方法 说 明 书
1. 1 质粒及菌株
穿梭载体 pShuttle_CMV购自 Clontech Laboratories, Inc。
野生型腺病毒 AdC68 (GenBank登录号 AC_000011)购于 ATCC。
PNEB193 质粒购自 New England Biolabs。
携带有优化密码子的目的基因 Gp (ERA株)的重组质粒 PUC57-0/Gp由南京金斯瑞生物科技 有限公司合成。
感受态菌株 Stbl2购自 Invitrogen。
腺病毒包装细胞系 HEK293 (293A)细胞购自 ATCC。
El缺失的复制缺陷型腺病毒载体 pAdC68 (pAdC68-El-deleted)构建方法如下：
(1) 质粒 pNEB193-KE 的构建
根据 PNEB193载体上的多克隆位点和腺病毒 AdC68基因组的序列， 设计扩增 KE 片段的引物 (表 1)， PCR扩增目的产物 KE片段。 PCR扩增体系总体积为 50uL， 反应循环参数为： 95°C预变性 5min; 94°C变性 lmin; 退火温度 57°C 30s ； 72°C延伸 2. 2min。 EcoR I和 Kpn I双酶切 PCR扩增 的 KE片段， 琼脂糖凝胶纯化 KE片段， 连接至经相同酶切的 PNEB193载体， 转化入 DH5 α感受态 细胞中， 挑取阳性克隆， 经酶切和测序鉴定得到 ρΝΕΒ193-ΚΕ。
表 1、 PCR引物序列 f:塑-鍾 麵薩
KE F: (： G ffiCCXI SlM^CW: (SEQ ID NO: 5}
CACCACCTCC OGTACCACA {SEQ ID NO: 6)
FX R TOr A OTWl OAlCiTC m^TO X CI固则 C>: 7)
Link - F: A CmgsmGTlCGGCG 9} 下划线标示限制性内切酶位点。
(2)质粒 pNEB193-KE-AK的构建
Asc I和 Kpn I双酶切 AdC68基因组， 低熔点琼脂糖凝胶电泳回收 AK片段， 连接至经相同酶 切的 PNEB193-KE载体， 转化入 Stbl2感受态细胞中， 挑取阳性克隆， 经酶切鉴定得到
Figure imgf000007_0001
(3) 质粒 pNEB193-KE-AK-XA的构建
Xba I和 Asc I双酶切 AdC68基因组， 由于 Asc I在腺病毒 AdC68存在三个酶切位点， 对 Asc I 说 明 书 进行部分酶切， 置于 37°C酶切 AdC68基因组 10s， 低熔点凝胶回收酶切的最大的 XA片段， 连接 至经相同酶切的 pNEB 193-KE-AK载体， 酶切鉴定得到 pNEB 193-KE-AK-XA。
(4) 质粒 ρΝΕΒ193-ΡΧ 的构建
根据 PNEB193载体上的多克隆位点和腺病毒基因组的序列， 设计扩增 ΡΧ片段的引物 (表 1)， PCR扩增目的产物 ΡΧ片段。 PCR扩增体系总体积为 50uL， 反应循环参数为： 95°C预变性 5min; 94°C变性 lmin; 退火温度 55°C lmin; 72°C延伸 6min。 Pme I和 Xba I双酶切 PCR扩增回收的 PX 片段， 琼脂糖凝胶回收目的产物 PX片段， 连接至经相同酶切的 PNEB193载体， 转化入 Stbl2感 受态细胞中， 挑取阳性克隆， 其中靠近腺病毒 AdC68左端 ITR的区域经测序鉴定。 Xba l酶切腺 病毒 AdC68基因组， 低熔点琼脂糖凝胶回收获得 AdC68中的 Xba I-Xba I部分， 并替换连接至经 Xba I酶切的 pNEB193-PX载体中， 转化入 Stbl2感受态细胞中， 挑取阳性克隆， 酶切鉴定得到 pNEB193-PX o
(5)删除 pNEB 193-PX质粒中 AdC68基因组中 E 1部分
EcoR I和 Mfe I双酶切 pShuttle_CMV质粒， 依据同尾酶的性质将空载体连接， 转化入 DH5a 感受态细胞中得到 pShuttle-EM的质粒。 根据 pShuttle-EM质粒的序列设计扩增 Linker的引物 (表 1)， PCR扩增 Linker产物， PCR扩增体系总体积为 50uL，反应循环参数为： 95°C预变性 5min ； 94°C变性 lmin; 退火温度 55°C lmin ； 72°C延伸 lmin。 SnaB I和 Nde I双酶切 PCR扩增的 Linker 片段， 琼脂糖凝胶回收 Linker 产物， 连接至经相同酶切的 pNEB193_PX载体 (通过对 AdC68基因 组序列分析， 利用 SnaB I和 Nde I两个酶切位点能将其 El部分序列删除）， 转化入 DH5 α感受态 细胞， 挑取阳性克隆， 经酶切鉴定得到 pNEB193-PX-Linker。
Linker 的序列插入在 AdC68 基因组第 459-3011 位之间， 替换 E1 的大部份序列。 Linker 的序列如 SEQ ID NO: 3。
(6) 复制缺陷型腺病毒 pAdC68质粒构建
Xba I和 Pme I双酶切 pNEB193_PX_Linker质粒 DNA， 并利用琼脂糖凝胶回收 PX_Linker片段， 连接至经相同酶切的低熔点琼脂糖凝胶回收的 pNEB 193-KE-AK-XA载体，转化入 Stb 12感受态细 胞中， 挑取阳性克隆， 经酶切鉴定得到 pAdC68-El-deleted。
复制缺陷型重组腺病毒载体 pAdC68-El-deleted 的核苷酸序列如 SEQ ID NO: 2。
1. 2 实验动物
6-8周龄雌性 ICR小鼠购自上海斯莱克实验动物有限责任公司。
1. 3 主要试剂
T4DNA连接酶、 Taq DNA聚合酶、 DNA凝胶回收试剂盒、质粒提取试剂盒等均购自宝生物工程 (大 说 明 书 连）有限公司。 Bgl II、 BamH I、 Nhe I， Pac I 等限制性内切酶购自 NEB公司。
Lipofectamine2000DNA转染试剂购自 Invitrogen。
1. 4 目的基因的获得
根据 GenBank 中狂犬病毒 ERA株中 Gp 编码区的核酸序列，合成经密码子优化后的 Gp 基因 (SEQ ID N0: 1)， 并在已优化密码子的目的基因的 5' 端依次接入 EcoR I 和 Nhe I 酶切位点， 3' 端接入 Xba I 酶切位点（ 图 1)， 通过 EcoR I 和 Xba I 双酶切将优化后的 Gp 基因克隆到商 品化载体 PUC57 载体， 获重组质粒 PUC57-0/Gp。
1. 5 重组腺病毒穿梭载体的获得
将 PUC57-0/Gp 质粒经 Nhe I 和 Xba I 双酶切后形成带有粘性末端的目的基因， 同时将腺病 毒穿梭载体 pshuttle-CMV经 Nhe I和 Xba I双酶切得到线性化的载体， 使用 T4DNA连接酶对上述 产物的粘端进行连接。 按常规方法将连接产物转化， 在含卡那霉素抗性的琼脂平板上筛选， 在 LB 培养基中 37°C过夜培养， 提取质粒 DNA。 采用酶切、 PCR等方法进行鉴定， 得到阳性重 组质粒， 命名为 pAdshuttle-CMV/Gp。
1. 6 获得重组腺病毒质粒
质粒 pshuttle-CMV/Gp 经过 PI_Sce I 和 I_Ceu I 双酶切后形成带有粘性末端的目的基因， 同时将腺病毒载体 pAdC68-El-deleted经过 PI_Sce I 和 I_Ceu I 双酶切得到线形化的载体。 利用低熔点琼脂糖胶分离目的片段， 将含有目的片段和载体的凝胶 65°C孵育 5 分钟， 待完全 溶解后使用 T4DNA连接酶对上述产物的粘端进行连接。在感受态菌 Stbl2中按常规方法将连接 产物转化， 取适量的转化产物涂至含氨苄抗性的琼脂平板上， 于 30 培养 24h， 挑选氨苄抗性 克隆， 小量提取质粒 DNA，用 0. 8%琼脂糖凝胶进行电泳迁移率分析，并分别进行 Bgl II、 BamH I、 Xho I 酶切图谱分析。 将所得的重组腺病毒质粒菌液扩大培养（1 ： 1000) 获得大量高质 量的质粒 DNA。 获得含狂犬病毒基因的重组腺病毒载体命名为 pAdC68-Gp。
1. 7 制备重组腺病毒
用限制性内切酶 Pac I 线性化重组腺病毒质粒 pAdC68-Gp， 应用脂质体转染的方法将质粒 转入 HEK293 细胞 (6 孔板） 中， 37°C孵育 6-8 天， 出现明显的噬斑。 待细胞变圆、 悬浮后收 集细胞， 反复冻融三次后取病毒上清感染 HEK293 细胞 (75ml 细胞培养瓶）。 重复以上步骤直 至收集适量病毒（ 约 20-40 个 150ml 细胞培养瓶）， 利用氯化铯密度梯度离心法纯化病毒， 测 定 0D 值， 加入终浓度为 10% 的甘油存于 -80°C。 提取病毒基因组 DNA (病毒含量 1012vps)， 并 进行 Bgl II、 BamH I , Nhe I 酶切图谱分析。 利用 CMV promoter 通用引物检测目的片段的核 酸序列。 纯化后的病毒在 HEK293 细胞中连续传 15 代， 验证重组载体的遗传稳定性。 说 明 书
1. 8 免疫原性分析
取一定滴度的重组腺病毒经肌注免疫 ICR小鼠， 3 周后检测血清中中和抗体的浓度， 中和 抗体滴度由武汉生物制品研究所测定。
实验组： 10 只 ICR小鼠注射 101Q Vps 重组腺病毒 AdC68-Gp ； 对照组： 10 只 ICR小鼠注射 1010vps 腺病毒空载体 AdC68-¾)t。
2、 实施例
实施例 1、 狂犬病毒 Gp 编码区的克隆
琼脂糖凝胶电泳显示酶切 PUC57-0/Gp 与预期大小（bp) 相符， 如图 2 ； 由于 Nhe I和 Xba I 为同尾酶，在筛选重组穿梭质粒 pAdshuttle-CMV/gp 时可能出现反向克隆，首先应用 Nhe I 和 Xba I 双酶切鉴定阳性克隆， 再利用 pAdshuttle-CMV/gp 目的片段以外区域的单酶切位点 EcoR I 再次鉴定， 酶切重组腺病毒穿梭载体 pAdshuttle-CMV/Gp, 结果与预期一致。 测序结 果表明， 得到的核酸序列与目的片段优化密码子后的核酸序列相同。
实施例 2、 重组腺病毒的鉴定
PI-Sce I 和 I-Ceu I 双酶切 pAdshuttle-CMV/gp 和 pAdC68， 用 T4DNA连接酶对上述产 物进行连接。 挑选氨苄抗性的克隆后， 酶切鉴定质粒 pAdC68-Gp， 如图 3， 与预期大小一致。 测序结果表明， 得到的核酸序列与目的片段优化密码子后的核酸序列相同。
大量制备重组腺病毒质粒 DNA， 将 Pac I 酶切后的重组质粒转染 HEK293 细胞。 6_8天后 出现明显的细胞病变。 病变初期细胞中出现噬斑， 逐渐变大， 后期呈拉网状态， 最终细胞全 部漂浮。将已感染病毒的 20 瓶 150ml 的细胞收集起来， 反复冻融的混合物用氯化铯密度梯度 离心法纯化病毒， 测定重组腺病毒 0D 值为 8. 9 X 1012vps/ml。 提取病毒基因组， 酶切鉴定， 片 段大小与预期一致。
实施例 3、 重组腺病毒感染滴度的测定及遗传稳定性分析
用不同数量的病毒感染 HEK293 细胞和肝癌细胞系 Huh7，如图 4。感染 24h 后，在 HEK293 细 胞中出现噬斑（ 病毒量为 10svps) ； 而感染后的 Huh7 细胞（ 病毒量为 10svps) 与对照组无明 显差别， 当病毒量达到 li^ ps, Huh7 细胞中也出现噬斑。 将纯化后的病毒在 HEK293 细胞中 连续传 15 代， 小量收集病毒并且纯化， 酶切分别鉴定第 5 代和第 15 代病毒的基因组， 结果 证明重组病毒没有突变， 并保持原代病毒的感染能力， 如图 5。
实施例 4、 血清中中和抗体滴度的测定
将重组腺病毒载体 AdC68-Gp 免疫 ICR小鼠， 3 周后测定血清中的中和抗体效价， 如图 6。 免疫组小鼠血清中中和抗体效价（ 平均值 38. 3IU/ml) 明显高于对照组，该重组黑猩猩型腺病 说 明 书 毒 AdC68-Gp 能够有效地诱导高效价中和抗体。
综上， 本发明人已经完成新型重组狂犬病疫苗载体的构建、 遗传稳定性的检测以及免疫 原性的监测， 结果表明新型基于腺病毒载体 AdC68 的狂犬病疫苗在小鼠体内能诱导出高滴度 的特异性中和抗体， 远超出 WHO指定的保护性效果的标准。
在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引用作 为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本 发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims

权利要求书
1. 一种狂犬病疫苗载体， 其特征在于， 所述表达载体包括： 复制缺陷型重组腺病毒载 体， 以及连接于该载体酶切位点 I-Ceu I 和 Pl-Sce l 之间的狂犬病毒糖蛋白抗原的编码序 列；
其中， 所述的复制缺陷型重组腺病毒载体包括： 改造的黑猩猩型腺病毒 AdC68基因组 序列；其中， E1 的大部份编码序列由连接序列替换；所述的连接序列中设置酶切位点 I-Ceul 和 PI- See I。
2. 如权利要求 1 所述的狂犬病疫苗载体 ， 其特征在于， 所述的狂犬病毒糖蛋白抗原的 编码序列如 SEQ ID N0 : 1 所示。
3. 如权利要求 1 所述的狂犬病疫苗载体， 其特征在于， 所述的连接序列如 SEQ ID NO : 3 所示。
4. 如权利要求 1 所述的狂犬病疫苗载体， 其特征在于， 所述的腺病毒表达载体的核苷 酸序列如 SEQ ID NO : 2 所示。
5. 如权利要求 1 所述的狂犬病疫苗载体， 其特征在于， 所述的复制缺陷型重组腺病毒 载体制备方法为：
将黑猩猩型腺病毒 AdC68基因组分成 4 个片段， 依次装入到骨架载体中， 并且， 用连 接序列替换 AdC68 基因组中 El 的大部份编码序列；
所述的 4个片段分别是：
黑猩猩型腺病毒 AdC68 基因组第 1-6025 位；
黑猩猩型腺病毒 AdC68 基因组第 6026-17279位；
黑猩猩型腺病毒 AdC68基因组第 17280-34196 位； 和
黑猩猩型腺病毒 AdC68基因组第 34197-36519 位。
6. 一种制备狂犬病疫苗的方法， 其特征在于， 所述方法包括：
(1) 提供权利要求 1-5任一项所述的狂犬病疫苗载体；
(2) 将 (1) 的狂犬病疫苗载体转染病毒生产细胞， 病毒在细胞内包装， 从而获得具有 免疫原性的狂犬病疫苗。
7. —种狂犬病疫苗， 其特征在于， 所述疫苗由权利要求 6 所述的方法制备获得。
8. 一种药盒， 其特征在于， 所述的药盒中含有权利要求 7所述的狂犬病疫苗。
9. 一种用于制备疫苗的试剂盒， 其特征在于， 所述试剂盒包括：
权利要求 1-5 任一项所述的狂犬病疫苗载体。
10. 如权利要求 9 所述的试剂盒， 其特征在于， 所述试剂盒还包括： 病毒生产细胞。
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CN103468743B (zh) * 2013-08-19 2015-07-29 中国科学院上海巴斯德研究所 一种狂犬病疫苗及其制备方法
CN107574175A (zh) * 2017-09-11 2018-01-12 南方医科大学 一种基于重组腺病毒的表达载体及其构建方法
CN107686843A (zh) * 2017-09-11 2018-02-13 南方医科大学 一种基于腺病毒AdC6的表达载体及其构建方法
CN107723279B (zh) * 2017-10-18 2020-11-10 成都远睿生物技术有限公司 一种缺陷型腺病毒AdC68-GP的培养方法
CN108588038A (zh) * 2018-04-10 2018-09-28 华中农业大学 一种基于糖蛋白密码子优化的狂犬病新型重组疫苗
CN114591922A (zh) * 2022-03-30 2022-06-07 长睿生物技术(成都)有限公司 一种表达狂犬病毒g蛋白的重组黑猩猩源腺病毒及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468743A (zh) * 2013-08-19 2013-12-25 中国科学院上海巴斯德研究所 一种新型狂犬病疫苗及其制备方法
CN103937835A (zh) * 2013-08-19 2014-07-23 中国科学院上海巴斯德研究所 一种基于腺病毒AdC68的表达载体及其构建方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2451864C (en) * 2001-06-22 2013-01-15 The Wistar Institute Of Anatomy And Biology Methods of inducing a cytotoxic immune response and recombinant simian adenovirus compositions useful therein
WO2005123930A1 (en) * 2004-06-21 2005-12-29 Medizinische Hochschule Hannover Lentiviral vector
US20130209512A1 (en) * 2010-08-16 2013-08-15 Hildegund C. J. Ertl Universal influenza a vaccines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468743A (zh) * 2013-08-19 2013-12-25 中国科学院上海巴斯德研究所 一种新型狂犬病疫苗及其制备方法
CN103937835A (zh) * 2013-08-19 2014-07-23 中国科学院上海巴斯德研究所 一种基于腺病毒AdC68的表达载体及其构建方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 1 July 2008 (2008-07-01), GEUE, L. ET AL., accession no. BN11294.1 *
FARINA, S.F. ET AL.: "Replication-Defective Vector Based on a Chimpanzee Adenovirus", JOURNAL OF VIROLOGY, vol. 75, no. 23, 31 December 2001 (2001-12-31), pages 11603 - 11613 *
XIANG, Z.Q. ET AL.: "Novel, Chimpanzee Serotype 68-Based Adenoviral Vaccine Carrier for Induction of Antibodies to a Transgene Product", JOURNAL OF VIROLOGY, vol. 76, no. 6, 31 March 2002 (2002-03-31), pages 2667 - 2675 *
ZHOU, D.M. ET AL.: "A Universal Influenza A Vaccine Based on Adenovirus Expressing Matrix-2 Ectodomain and Nucleoprotein Protects Mice from Lethal Challenge", MOLECULAR THERAPY, vol. 18, no. 12, 28 September 2010 (2010-09-28), pages 2182 - 2189 *

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