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Asia1 type foot-and-mouth disease recombinant virus and preparation method and application thereof

A foot-and-mouth disease virus and recombinant virus technology, applied in biochemical equipment and methods, antiviral agents, viruses/phages, etc., can solve the problem that GV cannot be effectively protected

Active Publication Date: 2013-08-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Asia1 type foot-and-mouth disease can be divided into seven groups. Among them, the GV and GII strains are prevalent in my country. The immune cross-challenge experiment shows that the GV strain can effectively protect the GV and GII epidemic strains, and the GII strain vaccine is effective against the GII epidemic virus. strains are well protected, but not effective against GV

Method used

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  • Asia1 type foot-and-mouth disease recombinant virus and preparation method and application thereof
  • Asia1 type foot-and-mouth disease recombinant virus and preparation method and application thereof
  • Asia1 type foot-and-mouth disease recombinant virus and preparation method and application thereof

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preparation example Construction

[0052] A preparation method of Asia1 type foot-and-mouth disease recombinant virus, specifically as follows:

[0053] 1, prepare the reverse genetic operating system of Asia1 type foot-and-mouth disease recombinant virus strain, this system comprises: (1) eukaryotic transcription plasmid, this transcription plasmid can express described recombination Asia1 type foot-and-mouth disease virus full-length gene cDNA sequence, plasmid is in virus whole Ribozymes (hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz)) are embedded on both sides of the long cDNA, and polymerase (pol) I and pol II transcription promoters are added at the 5′ end of the outer side, and at the 3′ There is a terminator at the end;

[0054] (2) Asia1 type foot-and-mouth disease recombinant plasmid prAsia1-FMDV transfects sensitive cells, preferably BHK-21 cells or IBRS-2 cells. The Asia1 type foot-and-mouth disease recombinant virus is rescued by the above-mentioned rescue system, and the plasmi...

Embodiment 1

[0061] Example 1. Obtainment of partial L and P12A gene sequences of Asia1 type foot-and-mouth disease virus Asia1 / HN / 06 strain.

[0062] The Asia1 / HN / 06 virus strain used by the inventor is preserved by the National Foot-and-Mouth Disease Reference Laboratory designated by the Veterinary Bureau of the Ministry of Agriculture, and the public can obtain it through a letter of entrustment issued by the Veterinary Bureau of the Ministry of Agriculture. Use the RNAeasy Mini Kit (Qiagen Company) to extract the total RNA of the Asia1 / HN / 06 strain, reverse-transcribe the first-strand cDNA of the virus with primer oligNot I, use the first-strand cDNA of synthesis as a template, and use the primers Asia1P12A-F and The gene sequence of Asia1 / HN / 06 strain was amplified from Asia1P12A-R. The above three specific primers for the Asia1 / HN / 06 strain are oligNot I (5'-ttttctaga gcggccgc t38-3'), Asia1P12A-F(5'-ttttc cttaag ggacaagaacatgctgtgtttgcctgtgt-3′) and Asia1P12A-R (5′-actcacatcgatg...

Embodiment 2

[0063] Example 2. Construction of Asia1 type foot-and-mouth disease recombinant virus infectious clone.

[0064] Based on the framework of the O-type FMD virus O / CHA / 99 strain rescue system prO / CHA / 99, the O-type FMD virus rescue system prO / CHA / 99 is as follows: figure 2 Shown: the viral genome contains the human cytomegalovirus RNA polymerase II promoter (Human cytomegalovirus RNA polymerase II promoter, PII) and the modified splicing sequence encoding the bovine auxin polynucleotides signal at the upstream of the 5' end of the viral genome. There is a murine RNA polymerase I promoter (Mouse RNA polymerase I promoter, PI) among them; a murine polymerase terminator I (Murine terminator I, T I ) and polymerase terminator II (Murine terminator II, TII) sequences; and Hammerhead ribozyme (HamRz) and hepatitis D enzyme (Hammerhead ribozyme, HamRz) and hepatitis D enzyme ( The core sequence of Hepatitis delta ribozyme, HdvRz), wherein the hepatitis D enzyme has 88 ribonucleic aci...

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Abstract

The invention relates to an Asia1 type foot-and-mouth disease recombinant virus without pathogenicity for a host and a preparation method and application thereof. A saving system is efficient eukaryotic plasmids which are constructed by gene engineering and can express exact foot-and-mouth disease virus genome RNA (Ribonucleic Acid), and therefore the foot-and-mouth disease recombinant virus can be constructed and prepared; vaccine strains with high titer and good antigen matching property can be prepared by using the plasmids, can be prepared into live vaccines or inactivated vaccines and can effectively stimulate bodies to produce immune response after being used for immunizing pigs and cattle, provide an immune protective effect on the pigs and the cattle and effectively protect GV and GII prevalent strains, the immune protection rate can reach 100 percent, and the median protective dose (PD50) is 6.34 to 13.59; and the recombinant virus has the advantages of high titer, high antigen matching property with the prevalent strains, wide antigen spectrum and high immune protection rate, does not have pathogenicity for pig and cattle hosts, does not form toxemia or expel toxin, and can be applied to prevention and control of Asia1 type foot-and-mouth disease viruses of China and neighboring countries.

Description

technical field [0001] The invention relates to an Asia1 type foot-and-mouth disease recombinant virus characterized by high titer, good antigen matching, no pathogenicity to host pigs and cattle, enhanced immune response in the host body, and high immune protection rate by gene recombination technology and its preparation The method and the method for developing the Asia1 type foot-and-mouth disease vaccine by using the foot-and-mouth disease recombinant virus belong to the field of biotechnology and biological products. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infectious disease caused by FMD virus (Foot-and-mouth disease virus, FMDV) infecting cloven-hoofed animals such as pigs, cattle and sheep. . FMDV is a single-stranded positive-sense RNA virus belonging to the family Picornaviridae and the genus Aphthovirus. The virus is divided into seven serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3 types). Asia1 type f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63A61K39/135A61P31/14C12R1/93
Inventor 郑海学杨帆刘湘涛靳野郭建宏曹伟军何继军张克山才学鹏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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