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WO2006002594A1 - Adenovirus canin de type 2 recombinant, procede d'elaboration et utilisation - Google Patents

Adenovirus canin de type 2 recombinant, procede d'elaboration et utilisation Download PDF

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WO2006002594A1
WO2006002594A1 PCT/CN2005/000951 CN2005000951W WO2006002594A1 WO 2006002594 A1 WO2006002594 A1 WO 2006002594A1 CN 2005000951 W CN2005000951 W CN 2005000951W WO 2006002594 A1 WO2006002594 A1 WO 2006002594A1
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recombinant
adenovirus
canine
cav
rabies
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Shoufeng Zhang
Rongliang Hu
Haitao Li
Yongzhi Wang
Changchun Tu
Xianzhu Xia
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Institute of Military Veterinary Academy of Military Medical Sciences PLA
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Institute of Military Veterinary Academy of Military Medical Sciences PLA
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Definitions

  • the invention relates to a recombinant canine type 2 adenovirus and a obtaining method and application thereof, in particular to a recombinant canine type 2 adenovirus carrying a canine type 2 adenovirus vaccine strain as a carrier and a method for obtaining the same, and a canine adenovirus and rabies The application of vaccines.
  • Rabies is a zoonotic disease that poses a serious threat to human and animal health. There are no effective treatments available today, causing 100% death in the event of an episode. Therefore, to date, only preventive measures have been taken for the disease.
  • Vaccines currently used for the prevention of canine rabies mainly include concentrated purified inactivated vaccines and freeze-dried attenuated live vaccines. The preparation process of concentrated and inactivated vaccine is complicated and costly, and is more suitable for developed countries; the preparation process of attenuated live vaccine is relatively simple, and the immunization period is also long, and it is widely used in developing countries. However, the live attenuated vaccine has the potential to be toxic to the ancestors in sensitive animals. There have been reports of rabies after vaccination.
  • Rabies Virus consists of five structural proteins: glycoprotein (G), nuclear protein (N), matrix protein (M), phosphorylated protein (NS) and transcriptase large protein (L).
  • Glycoprotein is the only protective antigen component that can induce the production of neutralizing antibodies in the body; nuclear proteins mainly induce specific cellular immunity, and can also promote the production of neutralizing antibodies; other structural components have not been found to be related to immune function.
  • vaccines with glycoproteins and nuclear proteins as target antigens have been actively studied. Subunit vaccines, genetic engineering subunit vaccines, anti-idiotypic antibody vaccines, live vector vaccines, and DNA vaccines have been reported.
  • Virus and human type 5 adenovirus are recombinant live vector vaccines for expressing glycoprotein or nuclear protein. They have been tested by wild animals in North America and Western Europe, and have proved to have good immunoprotective effects. However, the former has re-immunization. . The latter played an important role in rabies prevention trials in European wildlife, but there were certain safety issues when used as a vector for human gene therapy, and thus were not formally approved for use. Research reports on DNA vaccines have been reported in recent years. However, in general, the expression levels of antigenic proteins in the current rabies DNA vaccine and their induced antibody levels are very low, which is not effective for rabies. Preventive effect.
  • the vaccine for rabies prevention in China is a live attenuated vaccine.
  • This kind of vaccine has been banned in developed countries.
  • the US and European countries mainly use inactivated vaccines, but the cost is high and the immunization period is short.
  • the source of rabies is controlled.
  • the recombinant human type 5 adenovirus-rabies vaccine is mainly used, but since the vector virus in the recombinant vaccine has certain safety problems in gene therapy for humans, it has not been approved for use in domestic animals. .
  • rabies inactivated vaccines are mainly distributed in western countries such as France, the Netherlands, Spain, and the United States.
  • the use of rabies inactivated vaccines in China is mainly based on imports, and the safe and effective rabies recombinant live vector vaccine has not yet been listed.
  • Canine adenovirus is a member of the adenoviral mammalian adenovirus genus. It is divided into two types. Canine type 1 adenovirus (CAV-1) is a canine infectious hepatitis virus, canine type 2 adenovirus (CAV). - 2) Dog infectious laryngotracheitis virus, canine adenovirus is the most pathogenic class of mammalian adenovirus.
  • the adenoviral genome is a single-molecule linear double-stranded DNA of about 30 - 38 Kb in length, including nine major structural genes and four major early genes, encoding the structural and non-structural proteins of the virus, respectively.
  • the E3 region is an in vitro replication non-essential region of the adenovirus, and the adenovirus that lacks or replaces the E3 region can still grow on the cell.
  • the E3 region is used as an insertion site for a foreign gene, if the E3 region is deleted by 2 kb, it will accommodate an exogenous gene fragment of about 4 kb.
  • the maximum limit of DNA fragments that can be removed from the E3 region is not well understood, but it is clearly not possible to extend to the structural protein genes flanking the E3 region.
  • the adenoviral vector constructed by inserting a foreign gene in the E3 region is a non-dependent replicable vector, which can autonomously replicate and express foreign proteins in various organs and cells in the body, and is widely used. It is applied to protein characterization studies, diagnostic antigens and subunit vaccines, and preparation of recombinant live vector vaccines.
  • CAV-2 and CAV-1 have cross-immunogenicity
  • CAV-2 attenuated strains have been widely used for immunoprophylaxis of canine infectious hepatitis and infectious laryngotracheitis, so the deletion and modification of the cloned CAV-2 E3 region It will lay the foundation for the construction of expression vectors based on the CAV-2 E3 region.
  • the recombinant canine type 2 adenovirus provided by the invention is a canine type 2 adenovirus as a carrier, in which A recombinant virus obtained by inserting a foreign gene or an expression cassette containing a foreign gene after partial deletion in the E3 region of the genome; the foreign gene is a rabies virus sugar and an I or nuclear protein gene.
  • the rabies virus glycoprotein or nucleoprotein gene is derived from the rabies virus vaccine strain SRV 9 (Hou Shikuan, Yue Junming, Zhang Maolin et al. Screening, identification and experimental immunization of rabies oral vaccine strains. Chinese Journal of Zoonoses, 1995, 11 (6): 145-148), the registration number of the rabies virus glycoprotein gene in Genbank is: AF499686, and the registration number of the rabies virus nucleoprotein gene in Genbank is: AF499686.
  • a second object of the present invention is to provide a method for obtaining the above recombinant canine type 2 adenovirus.
  • the recombinant canine type 2 adenovirus provided by the invention is obtained by partially deleting the E3 region of the canine type 2 adenovirus genome at the plasmid level, and inserting a foreign gene or an expression cassette containing the foreign gene at the deletion site. Thus, the recombinant transformation of the canine type 2 adenovirus genome is completed.
  • the recombinant canine type 2 adenovirus genome sequence is then excised from the vector plasmid, and after transfecting the host cell, the recombinant canine type 2 adenovirus is obtained; the foreign gene is a rabies virus sugar and/or nuclear protein gene. .
  • the modification of the canine type 2 adenovirus genome is carried out on a plasmid vector in which the canine type 2 adenovirus genome gene is cloned, and the plasmid vector used can be any copy number in Escherichia coli (such as JM109).
  • High plasmid vectors such as pBlueScriptII, pUC18, pcDNA3 or pGEM-3Zf, allow large amounts of foreign genes to be replicated.
  • Partial deletion of the E3 region of the canine type 2 adenovirus genome is achieved by digesting the natural cleavage site in the E3 region and/or the cleavage site generated by the artificial mutation to remove the sequence between the cleavage sites.
  • the first 13 nucleotides of the E3 region are the 3' end sequence of the pVIII gene and are not in the deletion region.
  • the expression cassette carrying the rabies virus glycoprotein or nucleoprotein gene may be the cytomegalovirus (CMV) early (IE) promoter immediately sugar rabies virus and / or nucleoprotein cDNA and SV 4.
  • CMV cytomegalovirus
  • IE cytomegalovirus
  • the poly A (poly A) termination signal of the virus or bovine growth hormone gene is composed.
  • the promoter in the expression cassette of the rabies virus sugar and/or nucleoprotein gene needs to be inserted positively into the canine type 2 adenovirus genome, so that the transcription directions of both are identical.
  • the host cell may be any susceptible cell of canine type 2 adenovirus, such as MDCK cells, canine testis primary cells or PK cells, etc., preferably MDCK cells.
  • the recombinant canine type 2 adenovirus genome can be transfected into a host cell by a liposome method or a calcium phosphate coprecipitation method.
  • a third object of the present invention is to provide a recombinant live vaccine capable of simultaneously preventing canine rabies and adenovirus infection.
  • a fourth object of the present invention is to provide a genetically recombinant live vaccine capable of simultaneously preventing canine rabies and canine infectious hepatitis.
  • the genetic recombinant live vaccine provided by the present invention has an active component which is a recombinant canine type 2 adenovirus which stimulates the body to generate a specific immune response.
  • one or more immunologically acceptable immunoadjuvants such as canine interleukin 2, canine interleukin 18, etc., may be added to the above vaccine.
  • the vaccine of the present invention can be prepared as an injection, and the vaccine of this dosage form can be prepared according to a conventional method in the field of biological products.
  • the above vaccine is generally used in an amount of 0.5 - 2 mL, and the virus content is 10 6 - 10 7 TCID 50 , and the immunization once every six months to one year can effectively prevent the above two diseases.
  • Figure 1 is a schematic diagram showing the arrangement of the upstream fragment (1013 bp) and the downstream fragment (972 bp) of the canine type 2 adenovirus genome gene in the plasmid vector pPoly-CAV53end.
  • Example 1 Obtainment of recombinant canine type 2 adenovirus
  • CAV-2 canine type 2 adenovirus
  • R5 downstream primer: 5 ' -GCGGCCGCTTCGGCAAGGGCCTTTAGATAGC-3 ' (underlined base is the restriction endonuclease Notl recognition site)
  • F3 upstream primer: 5, -GCGGCCGCACTCATAGAAGTAGGCAGCTCCG-3 ' (underlined base is the restriction endonuclease Notl recognition site)
  • R3 downstream primer: 5 ' -CGGCGCGCCATCATCAATAATATACAGGACAAAG-3 ' (underlined base is the restriction endonuclease s ⁇ I recognition site)
  • CAV-2 canine type 2 adenovirus
  • YCA-18 strain canine type 2 adenovirus
  • PCR amplification was carried out under the guidance of primer pair 1 (primer F5 and primer R5) and primer pair 2 (primer F3 and primer R3).
  • the reaction system was : 10 ⁇ L of 10X PCR buffer, 4 ⁇ L of 25 mM MgCl 2 , 1 ⁇ L of CAV 2 DNA (200 ng), 5 L of lOmM upstream and downstream primers, 3 ⁇ L of 2.5 mM dNTPs, Pf u DNA polymerase (Promega) lwL ( lU), add deionized water to supplement the reaction system to 50 L.
  • the PCR reaction conditions were: 96 ⁇ 5 min, then 95 ° C for 40 sec, 55 ° C for 40 sec, 72 ° C for 120 sec, for a total of 25 cycles.
  • the three digested products were ligated at 12 ° C for 12-24 hours.
  • the upper and lower end fragments of the canine type 2 adenovirus genome were forwardly and ligated into the multiple cloning site of plasmid pPolyll.
  • the ligation system was : 10 ⁇ L of 2X ligation buffer, about 100 ng of each of the three DNA fragments, 1 ⁇ L of T 4 DNA ligase (TaKaRa), and the reaction system was added to 20 L with deionized water. After the reaction, 5 L of ligation product was taken and transformed into E.
  • coli JM109 plated on LB-resistant agar plates containing 50 ⁇ g/mL ampicillin, and recombinant colonies were identified, which contained the canine type 2 adenovirus genome gene.
  • the recombinant plasmid of about 1. Okb DNA fragment at both ends of the downstream was named pPoly-CAV53end (4. lkb).
  • the upstream part of pPoly-CAV53end has a restriction endonuclease Clal recognition site at the front end, and a restriction endonuclease/cl recognition site at the back end of the downstream fragment, and a single restriction endonuclease Notl recognition is formed between the two fragments.
  • the arrangement of the two fragments in the plasmid is shown in Figure 1.
  • the pPoly-Cav53end can be digested with restriction endonuclease/fcil, and the digested product can be detected by 1% agarose gel electrophoresis.
  • CAV-2 genomic DNA lg was taken and digested with restriction endonuclease Kpnl. After the reaction was completed, the digested product was subjected to 1% agarose gel electrophoresis, and cut under ultraviolet light. A band containing the digested product, and a DNA fragment containing the E3 region of 4. 8 kb was recovered by a DNA gel recovery kit, and the position of K in the CAV-2 genome was from the 23'-position of the 5' end to the 28,164th base.
  • the plasmid pVAX1 (Invitrogen) was digested with restriction endonuclease Kpnl and dephosphorylated with alkaline phosphatase (CIAP, Promega) to recover the linearized fragment of 3. Okb.
  • CIP alkaline phosphatase
  • a 4.8 kb fragment containing the E3 region and pVAX1 of 3. Okb were ligated, and the recombinant plasmid was named P VAX_E3 (7.8 kb).
  • Plasmid pTG containing rabies virus glycoprotein gene was digested with ⁇ (Yuan Huijun, Zhang Shoufeng, Zhang Maolin et al., Glycoprotein nucleic acid sequence and antigenic characteristics of rabies virus SRV9 vaccine strain Research, Chinese Virology, 2003, 18 (1): 63-67), Klenow recovers a 1.6 kb rabies virus glycoprotein gene fragment (GenBank No.: AF499686), which is linearized by EcoI digestion and recovery.
  • the plasmid pIRESlneo (Clontech) was transformed into ⁇ COUM109, and plated on LB-resistant agar plates containing 50 ⁇ g/mL ampicillin to identify recombinant colonies and recombinants containing the rabies virus glycoprotein gene.
  • Named pIG-neo (6.8 kb); pIG- neo, Klenow was double-digested with TV il and 3 ⁇ 4sl, 5.4 kb fragment was recovered, and self-ligated with T 4 DNA ligase, transformed into E.
  • coli JM109 coated on Recombinant colonies were identified on LB-resistant agar plates containing 50 ⁇ g/mL ampicillin, and the obtained expression plasmid for removing elements such as IVS, IRES, neo and the like was named pIG- neo ⁇ (5.4 kb).
  • the plasmid pIG- neoA was digested with Nrul and Xhol, and the 2.5 kb fragment (designated E) was recovered after Klenow was filled in.
  • the 6.76 kb fragment G recovered in step 1 was ligated with T 4 DNA ligase to transform E. coli JM109, coated.
  • Plasmid pTN containing rabies virus nucleoprotein gene was digested with S a ll and 3 ⁇ 4al (Yuan Huijun, Zhai Rongliang, Zhang Shoufeng et al. Cloning, expression and characterization of nucleoprotein gene of rabies virus SRV9 clone, Chinese Journal of Preventive Veterinary Medicine, 2003, 25 (1) : 5-8), after the Klenow was filled, the 1.4 kb rabies virus nucleoprotein gene fragment (GenBank: AF499686) was recovered, and the linearized plasmid pIRESlneo (Clontech), which was digested and recovered by EcoIN, was ligated.
  • the recovered ligation product was transformed into ⁇ coli JM109, plated on LB-resistant agar plates containing 50 ⁇ g/mL ampicillin, and recombinant colonies were identified.
  • the recombinant plasmid containing the rabies virus nucleoprotein gene was named pIN- neo (6.6). Kb); pIN- neo, Klenow was digested with ⁇ and Jbal, and the 5.2 kb fragment was recovered and self-ligated with T 4 DNA ligase to transform A coZi JM109, which was applied to 50 ⁇ g/mL ampicillin.
  • pIN- neo ⁇ (5.2 kb).
  • the plasmid pIN-neo ⁇ was digested with Nrul and 3 ⁇ 4oI, and the 2.4 kb fragment (designated N) was recovered after Klenow was filled in.
  • the 6.76 kb fragment G recovered in step 1 was ligated with T 4 DNA ligase to transform ⁇ : coii JM109, Applying to LB-resistant agar plates containing 50 ⁇ g/mL ampicillin, identifying recombinant colonies, obtaining a forward recombinant plasmid containing the rabies virus nucleoprotein gene expression cassette, named PVAX-E3-N ( 9. lkb ).
  • the recombinant plasmids pVAX-E3-G, pVAX-E3-N and the pPolyll-CAV-2 constructed in the first step were digested with the restriction enzymes Nrul and Sedl, respectively, and the lengths were respectively recovered.
  • the CAV-2 recombinant genomic plasmid vector of the viral glycoprotein gene expression cassette was named P PolyII-CAV-G (34.7 kb), and the CAV-2 recombinant genomic plasmid vector having the rabies virus nucleoprotein gene expression cassette inserted into the E3 region was named.
  • P PolyII-CAV-G 34.7 kb
  • CAV-2 recombinant genomic plasmid vector having the rabies virus nucleoprotein gene expression cassette inserted into the E3 region was named.
  • pPolyll- CAV-N 34. 5kb).
  • the cells and culture supernatants were frozen and thawed three times and collected and dispensed. Under electron microscopy, a large number of typical adenoviral particles were obtained, and recombinant canine type 2 adenovirus was obtained.
  • Example 2 Identification of recombinant canine type 2 adenovirus
  • a single layer of MDCK cells transfected with pPolyl l-CAV-G and pPolyll-CAV-N in a 50 raL flask was used to identify the genome of the recombinant virus as follows: Wash twice with PBS, add 800 ⁇ L of freshly prepared cell lysate (0.6% SDS, 10 mM EDTA, 100 ⁇ g/mL proteinase K (Promega), incubate for 1 hour at 37 °C, add 200 w L 5M NaCl, gently mix, ice bath for 1 hour.
  • the rabies virus glycoprotein recombinant adenovirus genome was digested by Sad to obtain a map of the fragment of 9412 bp + 5095 bp + 4715 bp + 4235 bp + 4230 bp + 3392 bp + 1624 bp.
  • the rabies virus nucleoprotein recombinant adenovirus genome was ⁇ 72/ Enzyme digestion
  • the 11964 bp + 6394 bp + 3716 bp + 3142 bp + 2928 bp + 2362 bp + 851 bp + 765 bp + 734 bp restriction fragment map indicates that the complete expression cassette of the rabies virus sugar or nuclear protein gene was correctly inserted into the recombinant virus genome. And the expression cassette is in a partially deleted E3 region, and the promoter orientation is consistent with the viral genome transcription direction. ' 2. TCID 5 . Determination
  • lmL containing the rabies virus nucleoprotein gene canine adenovirus type 2 recombinant virus TCID 5 to 10- 6 ⁇ ° - 10- 7 ⁇ °, indicating that the recombinant canine adenovirus type 2 can proliferate in the MDCK cell line, its production viral vector virus strains yield YCA- 18 quite.
  • the protein sample on the gel was electrotransferred to the nitrocellulose membrane, and it was sequentially reacted with rabbit anti-rabies virus (prepared by the conventional method, purified rabbit porcine virus SRV9 strain immunized adult rabbit), HRP labeled goat anti-rabbit two Anti-(Sigma) reaction, finally DAB/H color development, canine virus type 2 adenovirus recombinant virus containing rabies virus glycoprotein gene expressed rabies virus glycoprotein, its molecular weight is about 66kD, dog 2 containing rabies virus and protein gene The adenovirus recombinant virus expresses a rabies virus nucleoprotein with a molecular weight of about 55 kD, which is consistent with the molecular weight of the corresponding protein of the rabies virus SRV 9 strain.
  • rabbit anti-rabies virus prepared by the conventional method, purified rabbit porcine virus SRV9 strain immunized adult rabbit
  • the virulence test showed that the injected dogs did not show any abnormalities such as diet and mental performance, and the body temperature was fluctuating within the normal range (37. 5 °C - 39. 0 °C).
  • the white blood cell count in the body showed that the total number of white blood cells (7. 5-16. 0 X 10VD classification counts were within the normal range, and the injection dogs did not show clinical manifestations such as blue eyes or anaesthesia.
  • No abnormal changes were observed in the main tissues and organs. There was no significant difference between the recombinant liver injection of canine liver and kidney tissue sections and the control tissue of the control dogs. It indicated that the normal vaccination dose and 10 times the amount of recombinant virus were safe for dogs, no disease, no pathological damage to the main organs.
  • the stability identification results showed that after 40 passages of the recombinant virus, the size and direction of the expression cassette of the recombinant virus genome, including rabies sugar or nuclear protein, remained unchanged, and the rabies virus sugar or nuclear protein DNA sequence did not change.
  • Example 3 Recombinant canine type 2 adenovirus expressing rabies glycoprotein-immunized canine test experimental animals: 40 unincorporated 4- 5 month old puppies, randomly divided into groups A and B, 20 in each group .
  • mice immunization The TCID 5Q 1 (T 6 ' D CAV-2 attenuated vaccine (ie, the vector virus of the present invention) and the recombinant canine type 2 adenovirus expressing rabies glycoprotein were immunized to group A and B, respectively. For each strip of 0.5 mL, the neck was injected subcutaneously.
  • T 6 ' D CAV-2 attenuated vaccine ie, the vector virus of the present invention
  • canine type 2 adenovirus expressing rabies glycoprotein were immunized to group A and B, respectively.
  • the neck was injected subcutaneously.
  • Immunodetection A small amount of venous blood was collected before and 7 days, 14 days, 21 days, 28 days, 35 days and 42 days after immunization, and serum was separated.
  • the indirect ELISA method was used to detect the main antigenic region of canine adenovirus and rabies virus glycoprotein. Antibody level.
  • the color reaction was carried out, and the OD value at 490 nm was measured to evaluate and compare the antibody levels of the test dogs before and after immunization and between the different groups.
  • Adenovirus antibody rabies virus antibody adenovirus antibody rabies virus antibody before immunization 0.087 ⁇ 0.016 0.064 ⁇ 0.011 0.082 ⁇ 0.019 0.070 ⁇ 0.013 7d after immunization 0.327 ⁇ 0.044 0.074 ⁇ 0.009 0.334 ⁇ 0.034 0.195 ⁇ 0.016 14d after immunization 0.501 ⁇ 0.035 0.069 ⁇ 0.013 0.550 ⁇ 0.045 0.304 ⁇ 0.029 21d after immunization 0.782 ⁇ 0.030 0.089 ⁇ 0.021 0.769 ⁇ 0.020 0.466 ⁇ 0.042 28d after immunization 0.803 ⁇ 0.039 0.059 ⁇ 0.010 0.815 ⁇ 0.029 0.487 ⁇ 0.031 35d after immunization 0.836 ⁇ 0.047 0.062 ⁇ 0.008 0.833 ⁇ 0.036 0.524 ⁇ 0.050.
  • Virulent challenge test Group A dogs 6 weeks (42 days) after immunization were randomly divided into two groups: hyperthyroidism and hyperthyroidism. Group B dogs were randomly divided into two groups, acetaminophen and acetaminophen.
  • Example 4 Recombinant canine expressing rabies nucleoprotein The test of immunized type 2 adenovirus-immunized dogs Experimental animals: 40 puppies of 4-5 months old, which were not immunized, were randomly divided into two groups of A and B, 20 in each group.
  • Animal immunization TCID 5 respectively.
  • a 1 ( ⁇ 6 '° CAV- 2. attenuated vaccine and recombinant canine type 2 adenovirus expressing rabies nucleoprotein were immunized against group A and B, each dose was 0.5 mL, and the neck was injected subcutaneously.
  • Immunodetection A small amount of intravenous anticoagulation was taken before and 7 days, 14 days, 21 days, 28 days, 35 days and 42 days after immunization, and the leukocytes were separated and suspended in PBS. Fluorescently labeled goat anti-canine CD4+ and CD8+ antiserum were compared with suspended leukocytes at 4 °C for 1 h, washed with PBS and then subjected to FACS. The ratio of CD4+ cells to total lymphocyte ratio and CD4+/CD8+ ratio in A and B groups were compared. To evaluate changes in cellular immune levels in the test dogs after immunization with the rabies virus nucleoprotein recombinant CAV-2 vaccine.
  • Group A and B were inoculated with a virulent mixture of CAV-2 and CAV-1; Groups A and B were inoculated with the rabies virulent strain CVS-11. Each dog was given a dose of 100 LD 5 . . The dogs were isolated and observed 40 days after inoculation, and the incidence and mortality of each group of dogs were recorded in detail.
  • the recombinant canine type 2 adenovirus provided by the invention not only has strict host specificity, is susceptible to canines but does not cause disease, is not susceptible to other species of animals, and has good immunogenicity and complete protection. It can effectively express the glycoprotein and/or nuclear protein of rabies virus during replication and proliferation in the host, and stimulate the body to produce humoral and cellular immune responses against the two proteins.
  • the invention can immunize animals by oral or injection, induces the body to produce neutralizing antibodies against the adenovirus and rabies virus, and has a cellular immune response, and has protection against the attack of the virulent strains of adenovirus and rabies virus, and has the ability to replace the current weakly sterilized vaccine and destroy Live seedlings have become a major rabies vaccine for immunized canines on the market, and thus play an important role in the prevention of canine adenovirus and rabies worldwide.
  • more than 90% of human rabies is caused by It is transmitted by canines, and therefore, the present invention will also play an active role in the control of human rabies.
  • the invention has excellent application prospects in the medical field and will have great social benefits.

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Abstract

L'invention concerne un adénovirus canin de type 2 (CAV-2) rmbinant, son procéd?d'obtention, et son utilisation. Ce viu ecombinant est ?base d'un CAV-2 vecteur. La région E3 dugivr des attaques fatales du CAV et du virus e la rage. Ce CAV-2 recombinant, qui est un remplaçant potentiel es vaccins courants,est usceptible, d'une part de devenir le principal vaccin contre les rages animales et les maladies ?CAV, et d'autre part de jouer un rôle important, au plan mondil, dans la préventione la ge canine et des maladies ?CAV. En utre, ce CAV-2 recombinant pourrait également jouer un rôle actifans la prophylaxie et lthérapintirabique chez l'humain.
PCT/CN2005/000951 2004-07-07 2005-06-30 Adenovirus canin de type 2 recombinant, procede d'elaboration et utilisation Ceased WO2006002594A1 (fr)

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CN112813103A (zh) * 2021-01-28 2021-05-18 云舟生物科技（广州）有限公司 一种标准品载体及其在测定重组腺相关病毒滴度中的应用

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CN101358202B (zh) * 2007-07-30 2011-02-09 中国农业科学院哈尔滨兽医研究所 重组犬2型腺病毒转移载体、其构建方法及应用
CN101406698B (zh) * 2007-08-22 2012-05-23 中国人民解放军军事医学科学院军事兽医研究所 展示狂犬病病毒保护性抗原的犬ⅱ型腺病毒活载体重组疫苗
CN102335424B (zh) * 2007-08-22 2013-12-04 中国人民解放军军事医学科学院军事兽医研究所 利用ix蛋白展示狂犬病病毒保护性抗原的犬ii型腺病毒活载体重组疫苗
CN102335423B (zh) * 2007-08-22 2014-01-01 中国人民解放军军事医学科学院军事兽医研究所 利用纤突展示狂犬病病毒保护性抗原的犬ii型腺病毒活载体重组疫苗
CN102178947A (zh) * 2011-03-16 2011-09-14 中国人民解放军军事医学科学院军事兽医研究所 一种表达小反刍兽疫病毒h基因的活载体疫苗及制备方法
CN102964433A (zh) * 2012-06-29 2013-03-13 中国人民解放军军事医学科学院军事兽医研究所 一种高免疫原性狂犬病毒糖蛋白及其制备方法与应用
CN108872572B (zh) * 2018-05-30 2021-06-01 广州优迪生物科技股份有限公司 一种用于检测狂犬病毒抗体的试剂盒
CN112725288B (zh) * 2021-01-15 2022-07-26 北京华夏兴洋生物科技有限公司 犬腺病毒2型弱毒疫苗株及其应用

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WO1991002804A1 (fr) * 1989-08-22 1991-03-07 Ov Limited Sequence d'adenovirus recombinant exprimant l'antigene du rhabdovirus et son utilisation comme vaccin
WO1996039178A1 (fr) * 1995-06-05 1996-12-12 The Wistar Institute Of Anatomy And Biology Recombinant humain du type 5 de l'adenovirus a deficience de replication, utilise comme porteur de vaccins
WO1998000166A1 (fr) * 1996-07-03 1998-01-08 Merial, Inc. ADENOVIRUS CANIN DE RECOMBINAISON (ACR) CONTENANT DE l'ADN EXOGENE

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WO1991002804A1 (fr) * 1989-08-22 1991-03-07 Ov Limited Sequence d'adenovirus recombinant exprimant l'antigene du rhabdovirus et son utilisation comme vaccin
WO1996039178A1 (fr) * 1995-06-05 1996-12-12 The Wistar Institute Of Anatomy And Biology Recombinant humain du type 5 de l'adenovirus a deficience de replication, utilise comme porteur de vaccins
WO1998000166A1 (fr) * 1996-07-03 1998-01-08 Merial, Inc. ADENOVIRUS CANIN DE RECOMBINAISON (ACR) CONTENANT DE l'ADN EXOGENE

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Publication number Priority date Publication date Assignee Title
CN112813103A (zh) * 2021-01-28 2021-05-18 云舟生物科技（广州）有限公司 一种标准品载体及其在测定重组腺相关病毒滴度中的应用

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