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A type foot-and-mouth disease recombinant vaccine strains and preparation method and application thereof

A technology for recombinant vaccines and foot-and-mouth disease, applied in the field of biotechnology and methods in the Ming Dynasty, can solve problems such as poor production performance and lack of antigen matching

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] The purpose of the present invention is to solve the technical problem of current lack of antigen matching and poor production performance of type A foot-and-mouth disease vaccine strains, provide a type A foot-and-mouth disease recombinant vaccine strain and its preparation method and application, use the vaccine strain inactivation to prepare vaccines, 28 days after immunizing cattle, use 10000 times BID 50 Toxic dose was 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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  • A type foot-and-mouth disease recombinant vaccine strains and preparation method and application thereof
  • A type foot-and-mouth disease recombinant vaccine strains and preparation method and application thereof
  • A type foot-and-mouth disease recombinant vaccine strains and preparation method and application thereof

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preparation example Construction

[0051]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.

[0052] 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 leading protein L and structural protein P1 genes. The plasmid prO / CHA / 99 of type O foot-and-mouth disease virus was replaced by specific restriction sites of AflII and SgrAI, and the recombinant plasmid of type A foot-and-mouth disease virus was constructed and named prA-FMDV. Cytopathic pathology (CPE) appeared in t...

Embodiment 1

[0057] Example 1. Obtainment of partial L and P1 gene sequences of type A foot-and-mouth disease virus A / WH / CHA / 09 strain.

[0058] The A / WH / CHA / 09 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 A / WH / CHA / 09 strain, use primer oligNot I to reverse transcribe the first-strand cDNA of the virus, use the synthesized first-strand cDNA as a template, and use the primer AP1- The gene sequence of A / WH / CHA / 09 strain was amplified by F and AP1-R. The above three specific primers for the A / WH / CHA / 09 strain are oligNot I (5'-ttttctaga gcggccgc t 38 -3'), AP1-F(5'-ttttc cttaag ggacaggaacatgctgtgtttgcctgcgt-3′) and AP1-R (5′-tatttt caccggtg caataattttctgcttgtgtctg...

Embodiment 2

[0059] Example 2. Construction of infectious clones of type A foot-and-mouth disease recombinant virus.

[0060] On the basis of the framework of the O-type FMD virus O / CHA / 99 strain rescue system prO / CHA / 99, the O-type FMD virus strain 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 the modified splicing sequence encoding the bovine auxin polynucleotides signal, containing the mouse RNA polymerase I promoter (Mouse RNA polymerase I promoter, P I ); contains murine polymerase terminator I (Murine terminator I, T I ) and polymerase terminator II (Murine terminator II, T II ) sequence; and the core sequences of Hammerhead ribozyme (HamRz) and hepatitis E enzyme (Hepatitis delta ribozyme, HdvRz) chimeric at both ends of the full-length cDNA genome of O-type foot-and-mouth disease virus O / CHA / 99, wherein the type E The hepatitis enzyme has 88 ribonucleic acids...

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Abstract

The invention relates to A type foot-and-mouth disease recombinant vaccine strains prepared by using a reverse genetic manipulation technology and a preparation method and application of the strains. One strain is an A type foot-and-mouth disease recombinant vaccine strain with high titer, antigen matching property and immune protection rate, and the other strain is an A type foot-and-mouth disease recombinant non-pathogenic vaccine strain with high titer, antigen matching property and immune protection rate and without pathogenicity for a host; an antigen nucleotide sequence of each of the vaccine strains is shown as SEQ ID NO: 1; eukaryotic plasmids of viruses can be saved by using a reverse genetic manipulation system; after pigs and cattle are immunized by using the inactivated vaccines prepared from the prepared recombinant vaccine strains, the bodies can be effectively stimulated to produce immune response, and an immune protection effect is provided for the bodies of the pigs and the cattle; through a 10,000-times cattle median infectious dose (BID50) challenge assay of A type AISA topological strains, the immune protection rate reaches 100 percent, and the median protective dose (PD50) is 10.81 to 13.59; and the recombinant vaccine strains can be applied to prevention and control of A type foot-and-mouth disease viruses of China and neighboring countries.

Description

technical field [0001] The invention belongs to the field of biotechnology and biological products, and specifically relates to a type A foot-and-mouth disease recombinant vaccine strain prepared by reverse genetic manipulation technology and its preparation method and application. The method is used to prepare two vaccine strains, one of which has a high titer , A recombinant foot-and-mouth disease vaccine strain with high antigen matching and immune protection rate, and another type A foot-and-mouth disease recombinant non-pathogenic vaccine strain with high titer, antigen matching, high immune protection rate and no pathogenicity to the host strain, and the above two vaccine strains can be used to produce two kinds of vaccines at the same time. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/86A61K39/135A61P31/14C12R1/93
Inventor 刘湘涛郑海学杨帆靳野郭建宏曹伟军张克山田宏何继军董海聚才学鹏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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