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Foot-and-mouth disease O type subunit vaccine, and preparation method and application thereof

A subunit vaccine and foot-and-mouth disease technology, applied in the field of molecular biology, can solve problems such as low expression efficiency, limiting value and prospects, and achieve high immune efficacy, improved protection efficacy, and good antigen matching effects

Active Publication Date: 2020-10-16
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

For example, the patent (CN101914501B) discloses the introduction of SUMO into the foot-and-mouth disease structural proteins VPO, VP1, and VP3, and the expression of three plasmids respectively to construct the Asia1 type of foot-and-mouth disease virus-like particles; and the patent (CN104404074B) introduces a small ubiquitin-like fusion protein (SUMO), the co-expression plasmids of SUMO-VP0, SUMO-VP1 and SUMO-VP3 were constructed, and Asia1 type FMD virus-like particles were obtained, but the expression efficiency was low
The highest yield of the purified Asia1 type foot-and-mouth disease virus structural protein prepared by the Escherichia coli expression system is only about 20 mg / L, and at least 50 μg / head is required to produce a fully protected immune antigen, which greatly limits its commercial application value and prospect

Method used

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  • Foot-and-mouth disease O type subunit vaccine, and preparation method and application thereof
  • Foot-and-mouth disease O type subunit vaccine, and preparation method and application thereof
  • Foot-and-mouth disease O type subunit vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1O type foot-and-mouth disease virus strain O / 17002 serum cross-neutralization test

[0053] The present invention unexpectedly found a strain O that has extensive cross-neutralization effect on the O-type foot-and-mouth disease epidemic virus in my country among the epidemic strains of foot-and-mouth disease preserved in the National Foot-and-Mouth Disease Reference Laboratory (the supporting unit is the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences). / 17002.

[0054] The strain O / 17002 was amplified and multiplied by BHK21 cells cultured in suspension, and the virus was harvested to measure the virulence, inactivated by BEI, and emulsified with ISA 206 adjuvant to prepare a vaccine; the vaccine was used to immunize pigs to obtain the strain O / 17002 17002 hyperimmune positive sera. The obtained hyperimmune sera were tested for virus neutralization with four main circulating mutant strains of O / Mya98, O / PanAsia, O / Ind-200...

Embodiment 2

[0059] The preparation of embodiment 2 foot-and-mouth disease virus structural protein composition

[0060] 1. Construction of recombinant expression vector for structural protein of foot-and-mouth disease virus

[0061] (1) Design the gene sequence THS encoding the fusion tag protein composed of the following elements in series, wherein T is the nucleotide sequence of the translation initiation region, H is the nucleotide sequence encoding the histidine tag, and S is the nucleotide sequence encoding the tag containing The nucleotide sequence of Saccharomyces cerevisiae small ubiquitin-like modification protein (SUMO); the nucleotide sequence of THS is shown in SEQ ID NO.11.

[0062] (2) The above-mentioned THS gene sequence was concatenated in sequence with the genes VPO, VP3, and VP1 encoding the structural proteins of the O / 17002 strain, respectively, to form three fusion gene sequences THS-VPO, THS-VP3, and THS-VP1. Wherein said coding O / 17002 strain structural protein ge...

Embodiment 3

[0085] The expression efficiency of embodiment 3 foot-and-mouth disease virus structural protein composition

[0086] According to the method described in Example 2, four kinds of foot-and-mouth disease virus structural protein compositions O / 17002, Re / O / 17002 / VP3, Re / O / 17002 / VP1, Re / O / 17002 / VP3+VP1 were respectively subjected to prokaryotic Expression identification, the expression efficiency of different foot-and-mouth disease virus structural protein compositions was analyzed by SDS-PAGE, and the experimental results were as follows Figure 5 As shown, neither the single gene mutation of the FMDV structural protein VP3 or VP1, nor the simultaneous mutation of the structural proteins VP3 and VP1 will significantly affect the expression efficiency of the FMDV structural protein composition.

[0087] According to the method described in Example 2, four kinds of foot-and-mouth disease virus structural protein compositions O / 17002, Re / O / 17002 / VP3, Re / O / 17002 / VP1, Re / O / 17002 / VP3+...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a foot-and-mouth disease O type subunit vaccine with a cross protection function, and a preparation method and application thereof. Firstly, through a serum cross neutralization experiment, during screening, a result accidently shows that the foot-and-mouth disease O type subunit vaccine in China is provided with astrain with a wide cross neutralization function; on the basis of the amino acid sequences of three structural proteins, including VP0, VP3 and VP1, optimal design is carried out, in virtue of a small ubiquitin-related modifier (SUMO), a single plasmid is screened to simultaneously efficiently, uniform and solubly express the three structural proteins in escherichia coli, and in addition, the three virus structural proteins are successfully self-assembled in vitro; and a target protein which is finally obtained occupies about 30-35% of bacterial cell total proteins, and after purification iscarried out, the maximum yield of the target protein can be 150 mg / L. The minimum complete-protection immunizing dose of the foot-and-mouth disease vaccine prepared by the three foot-and-mouth disease virus structural proteins can be as low as 20 [mu] g / piece, and in addition, the foot-and-mouth disease O type subunit vaccine disclosed by the invention has a wide cross protection function for various foot-and-mouth disease O type prevalent strains in China.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an O-type foot-and-mouth disease subunit vaccine and its preparation method and application. Background technique [0002] Foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV) belongs to Picornaviridae, foot and mouth disease virus genus, virion has no envelope, is icosahedral symmetry, is composed of structural proteins VP0, VP3 and VP1, wherein VP0 protein is composed of VP4 and VP2 composition. The virus is a worldwide severe infectious disease that mainly infects pigs, cattle, sheep and other cloven-hoofed animals. It has the characteristics of rapid replication, high contact infection and strong pathogenicity, and can cause large-scale epidemics especially in pigs. The outbreak of foot-and-mouth disease will restrict the trade of animals and their products, causing significant economic losses and social impacts. The World Organization for Animal Healt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/135A61K39/39A61P31/14C07K19/00C07K1/22C12N15/62C12N15/70
CPCA61K39/12A61K39/39A61K2039/552A61P31/14C07K14/005C07K2319/21C12N15/70C12N2770/32122C12N2770/32134
Inventor 茹毅刘华南张贵财杨帆李丹郭建宏何继军张娇燕李亚军马坤伍春平郝荣增田宏张克山曹伟军马旭升党文靳野
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI