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Recombinant vaccine strain for foot-and-mouth disease type A as well as preparation method thereof and application thereof

A recombinant vaccine and foot-and-mouth disease technology, applied in the field of biotechnology and biological products, can solve the problems of poor production performance and lack of antigen matching, and achieve the effects of reducing production costs, high titers, and improving production performance

Inactive Publication Date: 2012-10-31
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] The purpose of the present invention is to solve the current technical problems of lack of antigen matching and poor production performance of type A foot-and-mouth disease vaccine strains, and provide a type A foot-and-mouth disease recombinant vaccine strain and its preparation method and application, which are prepared by inactivating the vaccine strain Vaccine, 28 days after immunizing cattle, use 10000 times BID 50 Toxic doses were used to challenge the virus, and the observation was continued for 12 days. The results showed that: the full dose was 100% protective

Method used

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  • Recombinant vaccine strain for foot-and-mouth disease type A as well as preparation method thereof and application thereof
  • Recombinant vaccine strain for foot-and-mouth disease type A as well as preparation method thereof and application thereof
  • Recombinant vaccine strain for foot-and-mouth disease type A as well as preparation method thereof and application thereof

Examples

Experimental program
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preparation example Construction

[0041] A method for preparing a type A foot-and-mouth disease recombinant virus, using the recombinant plasmid to directly transfect foot-and-mouth disease virus-sensitive cells, preferably BHK-21 cells or IBRS-2 cells, to obtain a type A foot-and-mouth disease recombinant virus that matches the epidemic strain antigen , the type A foot-and-mouth disease recombinant virus prepared by the method is named as rA-FMDV.

[0042] The construction method of the above-mentioned type A foot-and-mouth disease virus recombinant plasmid prA-FMDV is to use specific primers to amplify the genome of the type A foot-and-mouth disease virus strain A / WH / CHA / 09 to obtain part of the leader protein L and structural protein P1 genes. use specific Afl II and SgrA The plasmid prO / CHA / 99 of type O foot-and-mouth disease virus was replaced by the restriction site I, and the recombinant plasmid of type A foot-and-mouth disease virus was constructed and named prA-FMDV. Cytopathic pathology (CPE) appe...

Embodiment 1

[0046] Example 1. Obtainment of partial L and P1 gene sequences of type A foot-and-mouth disease virus A / WH / CHA / 09 strain.

[0047] The A / WH / CHA / 09 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 RNAeasy Mini Kit (Qiagen Company) to extract the total RNA of A / WH / CHA / 09 strain, and use primer olig not I reverse-transcribed and synthesized the first-strand cDNA of the virus, and used the synthesized first-strand cDNA as a template to amplify the gene sequence of the A / WH / CHA / 09 strain with primers AP1-F and AP1-R. The above three specific primers for A / WH / CHA / 09 strain are olig not I(5`-ttttctaga gcggccgc t 38 -3'), AP1-F (5'-ttttc cttaag ggacaggaacatgctgtgtttgcctgcgt-3') and AP1-R (5'-tatttt caccggtg caataattttctgcttgt...

Embodiment 2

[0048] Example 2. Construction of infectious clones of type A foot-and-mouth disease recombinant virus.

[0049] 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: Human cytomegalovirus RNA polymerase II promoter (Human cytomegalovirus RNA polymerase II promoter, P II ) and a modified splice sequence encoding the bovine auxin polynucleotides signal, with a mouse RNA polymerase I promoter (Mouse RNA polymerase I promoter, P I ); contains murine polymerase terminator I (Murine terminator I, T) downstream of the 3' end of the viral genome I ) and polymerase terminator II (Murine terminator II, T II ) sequence; and the core sequences of Hammerhead ribozyme (HamRz) and Hepatitis delta ribozyme (HdvRz) chimeric at both ends of the full-length cDNA genome of type O foot-and-mouth disease virus O / CHA / 99, of which type E The hepatitis enzyme has 88 ribonucleic acids in...

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Abstract

The invention relates to a recombinant vaccine strain for foot-and-mouth disease type A, which is constructed by a gene recombination technology and featured with high valence, high antigen compatibility and high immune protective rate, as well as a preparation method thereof and an application thereof. An antigen nucleotide sequence of the vaccine strain is shown by SEQ ID NO. 1; a rescuing system is an efficient eukaryotic plasmid, which is manually constructed and can express an accurate foot-and-mouth disease virus genome RNA; therefore, a foot-and-mouth disease recombinant virus can be constructed and prepared; through the adoption of the plasmid, the vaccine strain with high titer and good antigen compatibility can be prepared so as to prepare inactivated vaccines; after pigs and cattle are immunized, bodies can be effectively stimulated to generate immune responses and immune protective effects on the pig and cattle bodies can be provided; an A type AISA lineage of strain is challenged with 10000 times of 50% cattle infective dose (BID50), so that the immune protective rate reaches 100% and 50% protective dose (PD50) is 10.81-13.59; and the vaccine can be used for preventing and controlling foot-and-mouth diseases type A in China and neighboring countries.

Description

technical field [0001] The present invention relates to a method for constructing a type A foot-and-mouth disease recombinant vaccine strain with high efficiency (high titer, antigen matching and high immune protection rate) with gene recombination technology, its preparation method and application, and using the vaccine strain to produce type A foot-and-mouth disease vaccine The method belongs 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 pigs, cattle and sheep and other cloven-hoofed animals. . FMDV is a single-stranded positive-sense RNA virus belonging to the family Picornaviridae and the genus Aphthovirus. The virus is divided into 7 serotypes (A, O, C, Asian, SAT1, SAT2, and SAT3), among which type A foot-and-mouth disease virus is more prevalent and is the main epidemic strai...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85A61K39/135A61P31/14C12R1/93
Inventor 刘湘涛郑海学靳野杨帆郭建宏曹伟军
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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