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Brucellosis and foot-and-mouth disease bivalent vaccine and preparation method and application thereof

A technology of brucellosis and dual vaccine, which is applied in the field of brucellosis and foot-and-mouth disease dual vaccine and its preparation, can solve problems such as loss of appetite, decreased milk production, infection of humans and animals, etc., and achieves Reduced side effects, convenient operation, and the effect of resisting attacks

Active Publication Date: 2020-10-16
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] With the intensification of the dairy farming industry and the increase in breeding density, brucellosis and foot-and-mouth disease have become major diseases that endanger the development of the dairy industry. As the main source of infection, there is no vaccine available for humans, but the live vaccines currently used have infection risks for both humans and animals; the foot-and-mouth disease vaccine contains a large amount of adjuvant ingredients during use, and there are certain side effects after immunization of cows. Such as increased body temperature, loss of appetite, decreased milk production, etc.

Method used

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  • Brucellosis and foot-and-mouth disease bivalent vaccine and preparation method and application thereof
  • Brucellosis and foot-and-mouth disease bivalent vaccine and preparation method and application thereof
  • Brucellosis and foot-and-mouth disease bivalent vaccine and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Preparation and cultivation of embodiment 1 Brucella ghost bacterial strain

[0063] 1. Preparation of Brucella ghost strains

[0064] 1.1 Construction of the Brucella ghost strain is constructed by transforming the plasmid containing the phage cleavage protein E gene, Staphylococcus aureus nuclease A gene (SNA), and temperature control elements into the A19 strain by electroporation.

[0065] 1.2 Amplification and cloning of phage E gene

[0066] 1.2.1 E gene amplification

[0067] Using the pUC57-E plasmid as a template, use the E gene amplification primers E-F, E-R for amplification. The reaction conditions are: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 56°C annealing for 30 seconds, and 72°C extension for 30 seconds. 30 cycles, 72°C extension for 10 minutes. A 50 μl reaction system is used, and the specific components are as follows:

[0068] E gene amplification PCR reaction system table

[0069]

[0070]

[0071] 1.2.2 E gen...

Embodiment 2

[0098] Embodiment 2 Preparation of foot-and-mouth disease virus-like particles

[0099] 1 Recombinant plasmid construction

[0100] 1.1 VP1, VP2, VP4 gene amplification

[0101] Using the O-type foot-and-mouth disease virus genome as a template, use primers VP1-F / VP1-R (enzyme cutting sites EcoRI, NdeI), VP2-F / VP2-R (enzyme cutting sites NdeI, SacI) containing restriction sites , VP4-F / VP4-R (enzyme cutting sites SacⅠ, BamHI) were respectively amplified to obtain VP1, VP2, VP4 gene sequences.

[0102] 1.2 VP1, VP2, VP4 gene cloning

[0103] Recover the VP1, VP2, and VP4 genes with the agarose gel recovery kit, connect the gene fragments to the pMD18-T Simple vector overnight at 4°C, and transform them into DH5α competent cells. Take 100 μl of the competent cell and vector mixture and spread it on Take the plate containing Amp (100 μg / ml) on TSA, and incubate at 36-38°C for 24 hours.

[0104] Connection System Information

[0105]

[0106] 1.3 Identification of positive...

Embodiment 3

[0115] Embodiment 3 Preparation of brucellosis and foot-and-mouth disease dual vaccine

[0116] Concentrate the FMD virus-like particles 10 times with a hollow fiber column, mix them with Brucella bacterial shadows at a mass ratio of 1:2.5, mix them at 115 rpm at 4-6°C for 26 minutes, and the resulting mixture is cloth Secondary immunization vaccines for rucellosis and foot-and-mouth disease, in which the content of Brucella ghost antigens is not less than 28 μg per serving, and the content of foot-and-mouth disease virus-like particles is not less than 11.2 μg per serving.

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Abstract

The invention provides a brucellosis and foot-and-mouth disease bivalent vaccine and a preparation method and application thereof, and relates to the field of biotechnology. According to the brucellosis and foot-and-mouth disease bivalent vaccine provided by the invention, a brucella bacterial ghost antigen is used as a carrier to load foot-and-mouth disease virus-like particles, so as to achievethe purpose that one vaccine can prevent two diseases; additionally, biological safety risks in the use of brucellosis live vaccines are eliminated, and the problem of side effects during the use of foot-and-mouth disease vaccines is overcame.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a dual vaccine against brucellosis and foot-and-mouth disease, a preparation method and application. Background technique [0002] With the intensification of the dairy farming industry and the increase in breeding density, brucellosis and foot-and-mouth disease have become major diseases that endanger the development of the dairy industry. As the main source of infection, there is no vaccine available for humans, but the live vaccines currently used have infection risks for both humans and animals; the foot-and-mouth disease vaccine contains a large amount of adjuvant ingredients during use, and there are certain side effects after immunization of cows. Such as increased body temperature, loss of appetite, decreased milk production, etc. [0003] In view of this, the present invention is proposed. Contents of the invention [0004] The first object of the present invention is to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/295A61K39/135A61K39/10A61P31/14A61P31/04C12N1/21C12N15/55C12N15/34C12R1/01
CPCA61K39/098A61K39/12A61P31/14A61P31/04C07K14/005C12N9/22C12N2770/32134A61K2039/70A61K2039/5258A61K2039/521A61K2039/523C12N2795/00022Y02A50/30
Inventor 贺笋何传雨刘梦志赵海龙任立松吴冬玲李明启李延涛
Owner 天康生物制药有限公司
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